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Sample records for 12s rrna c1494t

  1. Molecular genetic characterization of two pedigrees with mitochondrial 12S rRNA C1494T mutation and aminoglycoside-induced hearing loss%两个线粒体12S rRNA C1494T突变及药物性耳聋家系的分子遗传学研究

    Institute of Scientific and Technical Information of China (English)

    李海峰; 陈智斌; 邢光前

    2011-01-01

    目的:探讨2个氨基糖甙类药物性耳聋及非综合征型耳聋家系的分子遗传学特征.方法:收集家系成员外周血样,常规方法提取基因组DNA.首先,利用基因芯片对中国人4个常见耳聋基因的9个突变热点进行分子筛查,9个位点分别为:CJB2基因的35 delG、176 de116、235 delC和299 delAT;GJB3基因的538 C>T;PDS基因的IVS7-2 A>G和2168 A>G以及mtDNA 12S rRNA基因的1494 C>T和1555 A>G.然后,对两家系的先证者分别进行线粒体DNA全序列及核基因TRMU和MTO1编码区的PCR扩增和测序分析.结果:芯片检测发现两家系的7名母系成员均存在同质性mtDNA 12S rRNA C1494T突变.与修正的剑桥参考序列相比,2名先证者的mtDNA全序列分析共检测到53个碱基变异,但除已知的12S rRNA C1494T突变外,其余52个碱基变异均为已报道的多态性位点;两家系先证者线粒体单体型分别是D4和D5a;TRMU和MTO1基因序列分析无异常发现.结论:线粒体DNA 12SrRNA C1494T突变是两个家系耳聋发生的主要分子基础,而氨基糖甙类抗生素的应用增强了该突变的表型表达:未能证实线粒体单体型以及核基因TRMU和MTO1对家系成员C1494T突变的表型具有修饰作用.%Objective:To explore the molecular genetic characterization of two families with aminoglycoside-induced and nonsyndromie sensofineural hearing loss. Methods:Blood samples were obtained from 7 maternal members and I married-in spouse of the two families. Genomic DNA was extracted with conventional method. Firstly, 9 hot spots for mutations in four most common pathologic genes, GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA, were screened with the DNA mieroarray to detect the deafness-associated mutations. The whole mitochondrial genomes and nuclear modifier genes TRMU and MTO1 of two probands were then PCR amplified and submitted for sequence analysis. Results:Mitochondrial 12S rRNA C1494T mutation was detected in all 7 maternal members of

  2. 碱基淬灭探针技术单管检测线粒体DNA 12S rRNA A1555G及C1494T突变%A novel method of detecting mitochondrial C1494T and A1555G mutations by using the base-quenched probe technique in a single PCR assay

    Institute of Scientific and Technical Information of China (English)

    郑璐; 罗光华; 张俊; 张晓膺; 徐宁

    2012-01-01

    目的 应用碱基淬灭探针技术建立单管同时检测线粒体DNA 12S rRNA A1555G和C1494T突变的方法.方法 探针的一端标记6-羧基荧光素,同时构建4个载体做为扩增模板,分别代表可能的4种基因型组合.对117例耳聋患者外周血标本进行线粒体DNA 12S rRNA A1555G和C1494T突变的检测,同时选取15例样本进行DNA测序,验证所建立方法的可靠性和准确性.结果 根据熔解曲线可以清晰地对线粒体DNA 12S rRNA 1555位点和1494位点的基因型做出判断.117例耳聋患者中3例携带A1555G突变(2.56%),1494位点未检出突变;基于碱基淬灭探针技术的单管检测方法与DNA测序的结果一致.结论 碱基淬灭探针单管检测技术可用于临床线粒体DNA突变耳聋基因检测.%Objective In the present study,we described a new method to detect the mitochondrial DNA (mtDNA) A1555G and C1494T mutations by using the base-quenched probe technique in a single PCR reaction.Methods 6-carboxyfluoresceint (FAM) was directly conjugated to the 3' end of the probe.Four vectors,representing the four possible genotype combinations,were constructed as the amplification templates for the methodology established. In the present study A1555G and C1494T mutations in 117 individuals with hearing loss were detected by the base-quenched probe method and were further validated by the direct DNA sequencing analysis.Results From the melting curve,we could distinguish the four haplotypes accurately.And there were complete concordance between the base-quenched probe method and direct DNA sequencing.Conclusion This method is suitable for clinical test of mtDNA A1555G and C1494T mutations in individuals with hearing loss.

  3. Prevalence of Mitochondrial 12S rRNA Mutations Associated with Aminoglycoside Ototoxicity

    Science.gov (United States)

    Guan, Min-Xin

    2005-01-01

    The mitochondrial DNA (mtDNA) 12S rRNA is a hot spot for mutations associated with both aminoglycoside-induced and nonsyndromic hearing loss. Of those, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region of the 12S rRNA have been associated with hearing loss. These two mutations account for a significant number of…

  4. Sequence analysis of mitochondrial 12S rRNA and tRNASer(UCN) genes in patients with nonsyndromic hearing loss%非综合征耳聋患者线粒体DNA12S rRNA及tRNAser(UCN)基因序列分析

    Institute of Scientific and Technical Information of China (English)

    戴大春; 鲁雅洁; 陈智斌; 魏钦俊; 曹新; 邢光前; 卜行宽

    2012-01-01

    目的:探讨线粒体DNA (mitochondrial DNA,mtDNA) 12S rRNA基因及tRNASer(UCN)基因突变与非综合征耳聋的相关性,以及常见致聋突变携带频率.方法:收集非综合征耳聋患者135例和听力正常对照人群126例的外周血样本,常规方法提取基因组DNA,PCR扩增mtDNA 12S rRNA基因及tRNASer(UCN)基因,扩增产物经直接测序进行耳聋相关突变的鉴定.结果:135例非综合征耳聋患者中共检测到11种mtDNA 12S rRNA碱基变异,其中4种已知致病突变的携带率分别为:A827G,4.4%;T961C,1.5%;T1095C,0.7%;A1555G,1.5%.两组人群均未检测到12S rRNA C1494T突变以及tRNASer(UCN)基因任何形式的致聋突变.结论:mtDNA 12S rRNA是南京地区汉族非综合征耳聋人群的突变热点基因,其常见致聋突变总携带率约为8.1%.%Objective; To determine the prevalence and characteristics of mitochondrial DNA (mtDNA) 12S rRNA gene and tR-NASer(ucn) gene mutations in Chinese subjects with non-syndromic hearing impairment. Methods; The peripheral blood samples were obtained from 135 patients with non-syndromic hearing loss and 126 normal hearing controls. Genomic DNA was isolated from the peripheral leukocytes of all participants using the Puregene DNA Isolation Kits. The subject' s DNA fragments spanning the entire mitochondrial 12S rRNA gene and tRNASer(UCN) gene were PCR amplified. Each fragment was purified and subsequently analyzed by direct sequencing to identify deafness-associated mutations. Results; There were totally eleven 12S rRNA sequence variants detected in 135 hearing-impaired subjects. Of those,4 variants are known deafness-associated mutations with variable frequencies of 4.4% in A827G, 1.5% in T961C,0.7% in T1095C and 1.5% in A1555G. Other variants,such as T1005C,C1048T,T1119C,G709A,C752T and A1382C,seem to be polymorphisms rather than causes of disease. On the other hand,we did not find C1494T mutation in the 12S rRNA gene and any of the known deafness

  5. Mutations of mitochondrial 12S rRNA in gastric carcinoma and their significance

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    Cheng-Bo Han; Jia-Ming Ma; Yan Xin; Xiao-Yun Mao; Yu-Jie Zhao; Dong-Ying Wu; Su-Min Zhang; Yu-Kui Zhang

    2005-01-01

    AIM: To detect the variations of mitochondrial 12S rRNA in patients with gastric carcinoma, and to study their significance and the relationship between these variations and the genesis of gastric carcinoma.METHODS: PCR amplified mitochondrial 12S rRNA of 44 samples including 22 from gastric carcinoma tissues and 22 from adjacent normal tissues, was detected by direct DNA sequencing. Then laser capture microdissection technique (LCM) was used to separate the cancerous cells and dysplasia cells with specific mutations. Denaturing high performance liquid chromatography (DHPLC) plus allele-specific PCR (ASPCR), nest-PCR and polyacrylamide gel electrophoresis (PAGE)were used to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplasia cells. Finally, RNAdraw biosoft was used to analyze the RNA secondary structure of mutant-type 12S rRNA.RESULTS: Compared with Mitomap database, some new variations were found, among which np652 G insertion and np716 T-G transversion were found only in cancerous tissues.There was a statistic difference in the frequency of 12S rRNA variation between intestinal type (12/17, 70.59%) and diffusive type (5/17, 29.41%) of gastric carcinoma (P<0.05).DHPLC analysis showed that 12S rRNA np652 G insertion and np716 T-G transversion were heteroplasmic mutations.The frequency of 12S rRNA variation in cancerous cells was higher than that in dysplasia cells (P<0.01). 12S rRNA np652 G insertion showed obviously negative effects on the stability of 12S rRNA secondary structure, while others such as T-G transversion did not.CONCLUSION: The mutations of mitochondrial 12S rRNA may be associated with the occurrence of intestinal-type gastric carcinoma. Most variations exist both in gastric carcinomas and in normal tissues, and they might not be the characteristics of tumors. However, np652 G insertion and np716 T-G transversion may possess some molecular significance in gastric carcinogenesis. During

  6. Molecular evolution of the mitochondrial 12S rRNA in Ungulata (mammalia).

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    Douzery, E; Catzeflis, F M

    1995-11-01

    The complete 12S rRNA gene has been sequenced in 4 Ungulata (hoofed eutherians) and 1 marsupial and compared to 38 available mammalian sequences in order to investigate the molecular evolution of the mitochondrial small-subunit ribosomal RNA molecule. Ungulata were represented by one artiodactyl (the collared peccary, Tayassu tajacu, suborder Suiformes), two perissodactyls (the Grevy's zebra, Equus grevyi, suborder Hippomorpha; the white rhinoceros, Ceratotherium simum, suborder Ceratomorpha), and one hyracoid (the tree hyrax, Dendrohyrax dorsalis). The fifth species was a marsupial, the eastern gray kangaroo (Macropus giganteus). Several transition/transversion biases characterized the pattern of changes between mammalian 12S rRNA molecules. A bias toward transitions was found among 12S rRNA sequences of Ungulata, illustrating the general bias exhibited by ribosomal and protein-encoding genes of the mitochondrial genome. The derivation of a mammalian 12S rRNA secondary structure model from the comparison of 43 eutherian and marsupial sequences evidenced a pronounced bias against transversions in stems. Moreover, transversional compensatory changes were rare events within double-stranded regions of the ribosomal RNA. Evolutionary characteristics of the 12S rRNA were compared with those of the nuclear 18S and 28S rRNAs. From a phylogenetic point of view, transitions, transversions and indels in stems as well as transversional and indels events in loops gave congruent results for comparisons within orders. Some compensatory changes in double-stranded regions and some indels in single-stranded regions also constituted diagnostic events. The 12S rRNA molecule confirmed the monophyly of infraorder Pecora and order Cetacea and demonstrated the monophyly of the suborder Ruminantia was not supported and the branching pattern between Cetacea and the artiodacytyl suborders Ruminantia and Suiformes was not established. The monophyly of the order Perissodactyla was evidenced

  7. GJB2 and mitochondrial 12S rRNA susceptibility mutations in sudden deafness.

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    Chen, Kaitian; Sun, Liang; Zong, Ling; Wu, Xuan; Zhan, Yuan; Dong, Chang; Cao, Hui; Tang, Haocheng; Jiang, Hongyan

    2016-06-01

    Genetic susceptibility may play an important role in the pathogenesis of sudden deafness. However, the specific genes involved are largely unknown. We sought to explore the frequency of GJB2 and mitochondrial 12S rRNA susceptibility mutations in patients with sudden deafness. Between September 2011 and May 2012, 62 consecutive patients with sudden deafness were seen. In 50 of these, no etiological factors for sudden deafness were found. We detected GJB2 and mitochondrial 12S rRNA variants by direct sequencing in these 50 patients and in 53-aged matched controls with normal hearing. In addition, we undertook functional analyses of the mitochondrial mutations which we detected, applying structural and phylogenetic analysis. GJB2 sequencing identified six mutations, including three pathogenic mutations (c.235delC, c.299-300delAT, c.109G>A) and three polymorphisms, in the study participants, giving an allele frequency of 15.0 %. A homozygous c.109G>A mutation was detected in two participants. A total of 16 variants in mitochondrial 12S rRNA gene were identified in the participants. No significant differences were found in GJB2 heterozygosity or in mitochondrial 12S rRNA variants between patients with sudden deafness and in controls. Our results suggest that the homozygous GJB2 c.109G>A mutation may be a cause of sudden deafness involving both ears. This finding should increase awareness of the likely role of genetic factors in the etiology of sudden deafness in general.

  8. Molecular detection of adulteration in chicken products based on mitochondrial 12S rRNA gene.

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    Abuzinadah, Osama H A; Yacoub, Haitham Ahmed; El Ashmaoui, Hassan M; Ramadan, Hassan A I

    2015-06-01

    The aim of this study is to detect the fraudulent in chicken products constitutes in order to protect consumers in Saudi Arabia from illegal substitutions. Two different approaches were used in this study, direct sequencing of specific fragments of amplified mitochondrial 12S rRNA gene in addition to species-specific PCR primers for confirmation of the obtained Blast search results. The results showed that all processed chicken products were identified as chicken (Gallus gallus) by 90-98% homology depending on obtained sequence quality. Samples labeled with chicken luncheon (samples tested in this study) were identified as turkey meat (Meleagris gallopavo) by 98% homology, suggesting adulteration with inedible parts of turkey in chicken luncheon ingredients. The results showed also that not only chicken luncheon was mixed with inedible parts of turkey but also all chicken products tested in this study (chicken balls, chicken burger, chicken sausage and chicken mined meat) contained this turkey meat. Applying methods used in this study could be useful for accurate and rapid identification of commercial processed meat.

  9. The phylogenetic relationship of the family Lutjanidae based on analyses of AFLP and mitochondrial 12S rRNA sequences

    Institute of Scientific and Technical Information of China (English)

    ZHANG Junbin; LIU Xin

    2006-01-01

    Fishes of the family Lutjanidae are commercially important in South China Sea. However,the phylogeny of Lutjanids is still unclear and there are many controversies over it. Herein, studies about the phylogeny of Lutjanids were performed based on Amplified Fragment Length Polymorphism (AFLP) analysis of genome DNA and sequence analysis of mitochondrial 12S rRNA gene, and 10 Lutjanidae species and 1 Lethrinidae species were employed.The topologies of minimum evolution (ME) trees based on the two analyses respectively were congruent except for positions of genera Pristipomoides and Caesio. The optimal substitution model TrN + G for DNA sequences of 12S rRNA genes in Lutjanids was obtained using MODELTEST 3.6 software and maximum likelihood (ML) analysis supports the topology displayed by the ME tree. The test of log-likelihood suggests that the use of molecular clock calibrations to estimate species divergence time appeared valid. Phylogenetic analyses using AFLP data and DNA sequences of mitochondrial 12S rRNA genes indicated the monophyly of Lutjanus genra. However, further studies are required to reveal the phylogenetic relationship among other genera. In addition, the results demonstrated that AFLP genetic marker was suitable for the phylogenetic analysis of Lutjanids.

  10. Interordinal mammalian relationships: evidence for paenungulate monophyly is provided by complete mitochondrial 12S rRNA sequences.

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    Lavergne, A; Douzery, E; Stichler, T; Catzeflis, F M; Springer, M S

    1996-10-01

    The complete mitochondrial 12S rRNA sequences of 5 placental mammals belonging to the 3 orders Sirenia, Proboscidea, and Hyracoidea are reported together with phylogenetic analyses (distance and parsimony) of a total of 51 mammalian orthologues. This 12S rRNA database now includes the 2 extant proboscideans (the African and Asiatic elephants Loxodonta africana and Elephas maximus), 2 of the 3 extant sirenian genera (the sea cow Dugong dugon and the West Indian manatee Trichechus manatus), and 2 of the 3 extant hyracoid genera (the rock and tree hyraxes Procavia capensis and Dendrohyrax dorsalis). The monophyly of the 3 orders Sirenia, Proboscidea, and Hyracoidea is supported by all kinds of analysis. There are 23 and 3 diagnostic subsitutions shared by the 2 proboscideans and the 2 hyracoids, respectively, but none by the 2 sirenians. The 2 proboscideans exhibit the fastest rates of 12S rRNA evolution among the 11 placental orders studied. Based on various taxonomic sampling methods among eutherian orders and marsupial outgroups, the most strongly supported clade in our comparisons clusters together the 3 orders Sirenia, Proboscidea, and Hyracoidea in the superorder Paenungulata. Within paenungulates, the grouping of sirenians and proboscideans within the mirorder Tethytheria is observed. This branching pattern is supported by all analyses by high bootstrap percentages (BPs) and decay indices. When only one species is selected per order or suborder, the taxonomic sampling leads to a relative variation in bootstrap support of 53% for Tethytheria (BPs ranging from 44 to 93%) and 7% for Paernungulata (92-99%). When each order or suborder is represented by two species, this relative variation decreased to 10% for Tethytheria (78-87%) and 3% for Paenungulata (96-99%). Two nearly exclusive synapomorphies for paenungulates are identified in the form of one transitional compensatory change, but none were detected for tethytherians. Such a robust and reliable resolution of

  11. 线粒体DNA 1555/1494突变耳聋文献回顾%The Meta Analysis of Carrying mtDNA A1555G/C1494T Mutation

    Institute of Scientific and Technical Information of China (English)

    丁志伟; 刘玉和

    2015-01-01

    Objective Clinical characteristics of deaf patients caused by mitochondrial DNA (mtDNA) 1555 /1494 mutations are not clearly defined. Here we review the relative literature to summarize the audiological characteristics of these patients and to discuss the factors affecting hearing loss degree.Methods We have selected “mtDNA 1555/1494 mutation” and “sensorineural hearing loss” as keywords to search for the data in the databases of CNKI , WanFang, VIP, and PubMed. From the resources, we collected the data, including the basic information, audiological records, genetic information, and medication. The literature review was conducted with the software of EXCEL and SPSS 19.0.Results Among the collected 324 cases from 39 publications, 285 cases carrying mtDNA 1555 mutation include 254 Chinese,18 other Asians and 13 non-Asians, and all the 39 cases carrying mtDNA C1494T mutation are Chinese. 230 cases had the specific history of using aminoglycoside antibiotic and 92 cases did not. However, two of them can hear normally one year later. A statistical analysis suggested that the degree of hearing loss in patients with treatment was significantly severer than patients without(X2=25.414,P0.05). In addition, the heterogeneous mutation rate and the degree of deafness had a significant positive correlation (R=0.823,P0.05)。另外,线粒体DNA 1555/1494突变异质率与耳聋程度显著正相关(R=0.823,P<0.05)。结论多种因素影响mtDNA A1555G/C1494T携带者的临床表现。对于线粒体DNA A1555G/C1494T相关耳聋进行早期耳聋基因诊断有重要的临床意义。对突变携带者,严禁使用氨基糖甙类抗生素。

  12. Sequence Characterization of Mitochondrial 12S rRNA Gene in Mouse Deer (Moschiola indica for PCR-RFLP Based Species Identification

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    Chandra Mohan Siddappa

    2013-01-01

    Full Text Available Mitochondrial 12S rRNA has proven to be a useful molecular marker for better conservation and management of the endangered species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP of the mitochondrial 12S rRNA gene has proven to be a reliable and efficient tool for the identification of different Indian deer species of family cervidae. In the present study, mitochondrial 12S rRNA gene sequence of mouse deer (Moschiola indica belonging to the family Tragulidae was characterized and analysed in silico for its use in species identification. Genomic DNA was isolated from the hair follicles and mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was cloned and sequenced for the first time. The sequence of mouse deer showed 90.04, 90.08, 90.04, 91.2, 90.04, and 90.08% identities with sika deer, sambar, hog deer, musk deer, chital, and barking deer, respectively. Restriction mapping in Lasergene (DNAstar Inc., Madison, WI, USA revealed that mouse deer mitochondrial 12S rRNA gene sequence can be differentiated from the other deer species in PCR-RFLP using RsaI, DdeI, BsrI, and BstSFI. With the help of predicted pattern, mouse deer can be identified using genomic DNA from a variety of biomaterials, thereby providing molecular aid in wildlife forensics and conservation of the species.

  13. Frequency and spectrum of mitochondrial 12S rRNA variants in 440 Han Chinese hearing impaired pediatric subjects from two otology clinics

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    Zhou Jianjin

    2011-01-01

    Full Text Available Abstract Background Aminoglycoside ototoxicity is one of the common health problems. Mitochondrial 12S rRNA mutations are one of the important causes of aminoglycoside ototoxicity. However, the incidences of 12S rRNA mutations associated with aminoglycoside ototoxicity are less known. Methods A total of 440 Chinese pediatric hearing-impaired subjects were recruited from two otology clinics in the Ningbo and Wenzhou cities of Zhejiang Province, China. These subjects underwent clinical, genetic evaluation and molecular analysis of mitochondrial 12S rRNA. Resultant mtDNA variants were evaluated by structural and phylogenetic analysis. Results The study samples consisted of 227 males and 213 females. The age of all participants ranged from 1 years old to 18 years, with the median age of 9 years. Ninety-eight subjects (58 males and 40 females had a history of exposure to aminoglycosides, accounting for 22.3% cases of hearing loss in this cohort. Molecular analysis of 12S rRNA gene identified 41 (39 known and 2 novel variants. The incidences of the known deafness-associated 1555A > G, 1494C > T and 1095T > C mutations were 7.5%, 0.45% and 0.91% in this entire hearing-impaired subjects, respectively, and 21.4%, 2% and 2% among 98 subjects with aminoglycoside ototoxicity, respectively. The structural and phylogenetic evaluations showed that a novel 747A > G variant and known 839A > G, 1027A > G, 1310C > T and 1413T > C variants conferred increased sensitivity to aminoglycosides or nonsyndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this rRNA. However, other variants were polymorphisms. Of 44 subjects carrying one of definite or putative deafness-related 12S rRNA variants, only one subject carrying the 1413T > C variant harbored the 235DelC/299DelAT mutations in the GJB2 gene, while none of mutations in GJB2 gene was detected in other 43 subjects. Conclusions Mutations in mitochondrial 12S rRNA

  14. Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations.

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    Ding, Yu; Xia, Bo-Hou; Liu, Qi; Li, Mei-Ya; Huang, Shui-Xian; Zhuo, Guang-Chao

    2016-10-10

    Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1

  15. The Rapid Screening for the Mitochondrial DNA C1494T Mutation in a Deaf Population in China Using Real-time Quantitative PCR%母系遗传非综合征型聋相关线粒体DNA C1494T突变的全国性调查

    Institute of Scientific and Technical Information of China (English)

    李琦; 袁永一; 黄德亮; 韩东一; 戴朴

    2013-01-01

    目的 应用实时荧光定量PCR(real-time-qPCR)对全国范围内的耳聋患者进行mtDNA C1494T的筛查,为进一步研究该突变和聋病的临床基因诊断提供帮助.方法收集国内25个不同省市的特教学校和聋哑学校的3 133例双侧重度或极重度非综合征型聋患者,提取外周血DNA,设计TaqMan-MGB的引物和探针进行real-time-qPCR,对所有阳性样本和随机抽取的495例阴性样本进行直接测序验证.结果 3 133例中,发现C1494T突变14例(14/3 133,0.45%),经测序验证全部为C1494T突变,495例阴性样本中均无C1494T突变.结论 Real-time-qPCR 是进行mtDNA C1494T突变筛查的有效方法,中国耳聋人群中mtDNA C1494T的突变率较低.%Objective To develop a simple, rapid, and reliable real-time quantitative polymerase chain reaction (real-time qPCR) assay based on TaqMan technology using a new minor groove binding (MGB) probe for directly detec ting the mitochondrial DNA (mtDNA) C1494T mutation, and to investigate the carrier frequency in nonsyndromic deaf Chinese subjects. Methods A TaqMan - MGB probe was constructed. Peripheral blood samples were collected from 3 133 nonsyndromic deaf patients and genomic DNA was extracted. A real- time qPCR using MGB probes (wild- type) in a sin gle tube was used to detect the mtDNA C1494T mutation. The results were then compared to the DNA sequence of the PCR products. Results A total of 13 of 3 133 (0. 45%) Chinese nonsyndromic hearing loss patients were C1494T -posi tive. The results of the TaqMan -MGB probe method were consistent with those of sequencing. Conclusion Real-time qPCR with a TaqMan MGB probe is useful for large-scale screening for screening of the C1494T mutation. The mtDNA C1494T mutation has a low carrier frequency in Chinese nonsyndromic hearing loss patients.

  16. Frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among children with sensorineural deafness in China

    Institute of Scientific and Technical Information of China (English)

    Xia Xu; Guangqian Xing; Qinjun Wei; Zhibin Chen; Hongbo Cheng; Xin Cao; Xingkuan Bu

    2006-01-01

    Objective: To investigate the frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among Chinese with sensorineural deafness. Methods: Blood samples from 78 sporadic cases with sensorineural deafness were obtained and DNA was extracted from the leukocytes, then the mitochondrial DNA target fragments were amplified by polymerase chain reaction(PCR). The 1555G mutations were detected by BsmA I restriction endonuclease digestion, every fragment was analyzed by sequencing; All the 961 insC mutation were detected by direct sequencing. Results: The percent age of A1555G mutation and mt961C insertion were 6.4% and 2.6% in the hearing-impaired Chinese subjects respectively. Conclusion: A1555G and 96linsC mutations in mitochondrial DNA 12S rRNA gene regions may play a role in the pathogenesis of hearing loss in the sporadic cases.

  17. Further involvement of the mitochondrial 12S rRNA gene in aminoglycoside-induced deafness: A novel type of heteroplasmy

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    Bacino, C.; Prezant, T.R.; Bu, X. [Cedars-Sinai Medical Center, Los Angeles, CA (United States)] [and others

    1994-09-01

    Aminoglycoside-induced deafness has been linked recently to a predisposing mutation in the 3{prime} end of the small ribosomal RNA (rRNA) gene of human mitochondria (1555 A{yields}G) that makes the mitochondrial rRNA structurally more similar to its bacterial counterpart. This mutation was found in Chinese families in which the susceptibility to develop ototoxic deafness was inherited through the maternal lineage. However, the 1555 A{yields}G mutation was rarely found in sporadic patients in China, where aminoglycosides are commonly used. To further characterize the mutations predisposing to aminoglycoside ototoxicity, we analyzed the 12S rRNA gene in 35 sporadic patients without the 1555 mutation. Using single stranded conformational polymorphism (SSCP) analysis, heteroduplex (HD) analysis, sequencing, and allele-specific oligonucleotide hybridization, we found that 3 of 35 sporadic patients had unique sequence changes in the 12S rRNA gene. Two of these changes were homoplasmic. One of the patients displayed a novel type of heteroplasmy, which we term multiplasmy, with one base deletion at nt 961 and different populations of mitochondrial DNA with varying numbers of inserted cytosines at that site.

  18. Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA.

    Science.gov (United States)

    Stern, S; Powers, T; Changchien, L M; Noller, H F

    1988-06-20

    We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.

  19. Relationships between parasitoid wasps (Hymenoptera: Braconidae: Opiinae), fruit flies (Diptera: Tephritidae) and their host plants based on 16S rRNA, 12S rRNA, and ND1 gene sequences

    Science.gov (United States)

    Ibrahim, N. J.; Md-Zain, B. M.; Yaakop, S.

    2013-11-01

    Opiinae is among the l0 largest subfamilies under the family Braconidae. Opiines species have great potential as natural enemies against fruit fly pests. Before using them as a biological control agent, construction of the phylogenetic trees could facilitate in the molecular identification of individual species and their relationships among members of the Opiines, as well as between Opiines and their host plants. Larval specimens of tephritids were collected from four crop species at five localities throughout the Peninsular Malaysia. A total of 44 specimens of opiines had successfully emerged from the hosts, fruit fly larvae. The DNA sequences of 12S and 16S rRNA were obtained for the braconids while the mitochondrial ND1 sequences were obtained for the tephritids species through polymerase chain reaction. Maximum Parsimony and Bayesian trees were constructed by using PAUP 4.0b10 and MrBayes 3.1.2 to identify the relationships among the taxa. This study illustrates the phylogenetic relationships among parasitoid opiines collected and reared from parasitized fruit flies. The phylogenetic trees constructed based on the mitochondrial 12S and 16S rRNA sequences exhibited similar topology and sequence divergence. The opiines were divided into several clades and subclades according to the genus and species. Each clade also was supported by the similar host plants with high support values. However, their pests were not specific, except for Bactrocera cucurbitae. This study has reconfirmed the associations between Opiinae, tephritids, and host plants based on molecular data.

  20. Mitochondrial tRNAArg T10454C variant may not influence the clinical expression of deafness associated 12S rRNA A1555G mutation.

    Science.gov (United States)

    Luo, Zhiyi

    2016-01-01

    In this study, we examined the "pathogenic" role of the T10454C mutation in mitochondrial tRNA(Arg) gene in deafness expression as increasing reports provided an active role of this mutation in clinical manifestation of deafness associated 12S rRNA A1555G mutation. For this purpose, we reanalyzed the complete mitochondrial DNA (mtDNA) sequence data containing the T10454C mutation. Moreover, we analyzed the reported "polymorphisms" of mtDNA in the proband using the phylogentic approach. To our surprise, other mutations which occurred at protein-coding genes played more important roles in resulting mitochondrial dysfunctions by using the bioinformatic tool. In addition, evolutionary conservation analysis of the T10454C mutation indicated that this mutation was not conserved between different species. To our knowledge, this is the first report that the T10454C variant may not modulate the phenotypic expression of the deafness associated A1555G mutation.

  1. Characterization of 12S rRNA gene for meat identification of common wild and domestic small herbivores as an aid to wildlife forensic

    Directory of Open Access Journals (Sweden)

    E. Joseph

    2013-10-01

    Full Text Available Aim: Chital and sambar are the common wild small herbivores, which are vulnerable to poaching for their meat. Many times poachers claim the wild meat to be that of goat or sheep. Hence, authentic evidences are required to stop such wildlife crime. The present investigation was carried out to study the species specific PCR-RFLP patterns for meat identification of chital and sambar and then differentiation from the meat of goat and sheep. Materials and Methods: Extracted DNA from meat samples were subjected to PCR using the universal primers of 12S rRNA gene. The PCR products were subjected to RFLP and sequencing. Results: The size of amplified PCR products was similar (440 bp in each species and sequence alignment showed more than 89 % similarities among these species. However, phylogenetic analysis revealed that Chital and Sambar are in one cluster while Goat and sheep are in other cluster. To differentiate between species, restriction digestion of the PCR products was carried out to produce characteristic PCR-RFLP patterns for each species. Restriction digestion with RsaI and AluI enzymes produced distinct PCR-RFLP patterns that differentiated the meat of wild species (chital-sambar from that of domestic species (goat-sheep. BsrI restriction digestion revealed unique PCR-RFLP pattern in chital differentiating it from the meat of other three species. Restriction digestion with DdeI enzyme led to the production of distinct PCR-RFLP patterns for chital and sambar to identify their meat individually. Conclusion: This study showed the effectiveness of 12S rRNA gene polymorphism in meat identification. The data can be used as evidence against the poachers to convict the wildlife crime in the court of law [Vet World 2013; 6(5.000: 254-259

  2. Phylogenetic reconstruction of the wolf spiders (Araneae: Lycosidae) using sequences from the 12S rRNA, 28S rRNA, and NADH1 genes: implications for classification, biogeography, and the evolution of web building behavior.

    Science.gov (United States)

    Murphy, Nicholas P; Framenau, Volker W; Donnellan, Stephen C; Harvey, Mark S; Park, Yung-Chul; Austin, Andrew D

    2006-03-01

    Current knowledge of the evolutionary relationships amongst the wolf spiders (Araneae: Lycosidae) is based on assessment of morphological similarity or phylogenetic analysis of a small number of taxa. In order to enhance the current understanding of lycosid relationships, phylogenies of 70 lycosid species were reconstructed by parsimony and Bayesian methods using three molecular markers; the mitochondrial genes 12S rRNA, NADH1, and the nuclear gene 28S rRNA. The resultant trees from the mitochondrial markers were used to assess the current taxonomic status of the Lycosidae and to assess the evolutionary history of sheet-web construction in the group. The results suggest that a number of genera are not monophyletic, including Lycosa, Arctosa, Alopecosa, and Artoria. At the subfamilial level, the status of Pardosinae needs to be re-assessed, and the position of a number of genera within their respective subfamilies is in doubt (e.g., Hippasa and Arctosa in Lycosinae and Xerolycosa, Aulonia and Hygrolycosa in Venoniinae). In addition, a major clade of strictly Australasian taxa may require the creation of a new subfamily. The analysis of sheet-web building in Lycosidae revealed that the interpretation of this trait as an ancestral state relies on two factors: (1) an asymmetrical model favoring the loss of sheet-webs and (2) that the suspended silken tube of Pirata is directly descended from sheet-web building. Paralogous copies of the nuclear 28S rRNA gene were sequenced, confounding the interpretation of the phylogenetic analysis and suggesting that a cautionary approach should be taken to the further use of this gene for lycosid phylogenetic analysis.

  3. Nuclear modifier MTO2 modulates the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Xiangyu He

    Full Text Available The phenotypic manifestations of mitochondrial DNA (mtDNA mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R or P(R 454 mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R, the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S, mto2(P(S and MTO2(P(R. The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

  4. Prevalence of the A1555G (12S rRNA and tRNA Ser(UCN mitochondrial mutations in hearing-impaired Brazilian patients

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    Abreu-Silva R.S.

    2006-01-01

    Full Text Available Mitochondrial mutations are responsible for at least 1% of the cases of hereditary deafness, but the contribution of each mutation has not yet been defined in African-derived or native American genetic backgrounds. A total of 203 unselected hearing-impaired patients were screened for the presence of the mitochondrial mutation A1555G in the 12S rRNA gene and mutations in the tRNA Ser(UCN gene in order to assess their frequency in the ethnically admixed Brazilian population. We found four individuals with A1555G mutation (2%, which is a frequency similar to those reported for European-derived populations in unselected samples. On the other hand, complete sequencing of the tRNA Ser(UCN did not reveal reported pathogenic substitutions, namely A7445G, 7472insC, T7510C, or T7511C. Instead, other rare substitutions were found such as T1291C, A7569G, and G7444A. To evaluate the significance of these findings, 110 "European-Brazilians" and 190 "African-Brazilians" unrelated hearing controls were screened. The T1291C, A7569G and G7444A substitutions were each found in about 1% (2/190 of individuals of African ancestry, suggesting that they are probably polymorphic. Our results indicate that screening for the A1555G mutation is recommended among all Brazilian deaf patients, while testing for mutations in the tRNA Ser(UCN gene should be considered only when other frequent deafness-causing mutations have been excluded or in the presence of a maternal transmission pattern.

  5. Prevalence of the A1555G (12S rRNA and tRNA Ser(UCN mitochondrial mutations in hearing-impaired Brazilian patients

    Directory of Open Access Journals (Sweden)

    R.S. Abreu-Silva

    2006-02-01

    Full Text Available Mitochondrial mutations are responsible for at least 1% of the cases of hereditary deafness, but the contribution of each mutation has not yet been defined in African-derived or native American genetic backgrounds. A total of 203 unselected hearing-impaired patients were screened for the presence of the mitochondrial mutation A1555G in the 12S rRNA gene and mutations in the tRNA Ser(UCN gene in order to assess their frequency in the ethnically admixed Brazilian population. We found four individuals with A1555G mutation (2%, which is a frequency similar to those reported for European-derived populations in unselected samples. On the other hand, complete sequencing of the tRNA Ser(UCN did not reveal reported pathogenic substitutions, namely A7445G, 7472insC, T7510C, or T7511C. Instead, other rare substitutions were found such as T1291C, A7569G, and G7444A. To evaluate the significance of these findings, 110 "European-Brazilians" and 190 "African-Brazilians" unrelated hearing controls were screened. The T1291C, A7569G and G7444A substitutions were each found in about 1% (2/190 of individuals of African ancestry, suggesting that they are probably polymorphic. Our results indicate that screening for the A1555G mutation is recommended among all Brazilian deaf patients, while testing for mutations in the tRNA Ser(UCN gene should be considered only when other frequent deafness-causing mutations have been excluded or in the presence of a maternal transmission pattern.

  6. 两个母系遗传氨基糖苷类抗生素致聋家系线粒体DNA 12S rRNA A1555G突变的分子遗传学研究%Molecular Genetic Study of Two Chinese Pedigrees with Maternally Inherited Hearing Loss Mitochondrial 12S RRNA A1555G Mutation

    Institute of Scientific and Technical Information of China (English)

    孙艺; 杨长亮; 罗燕云; 阳光; 杨慧; 朱丽; 姚行齐

    2014-01-01

    目的 探讨两个氨基糖苷类药物性耳聋大家系的分子遗传学特征.方法 对两个非综合征型感音神经耳聋大家系的成员进行病史调查、全身体检、纯音听阈、声导抗检查;收集、整理临床听力学资料;进行家系遗传学特征的分析并绘制系谱图.收集两个大家系成员的外周静脉血样本,从白细胞中提取基因组DNA,聚合酶链反应扩增,应用直接测序法检测中国人常见的药物性耳聋相关基因——线粒体DNA12S rRNA A1555G突变.结果 根据调查两个家系均来自湖北省,所有母系受检者存在线粒体DNA 12S rRNA 1555位点A→G的突变.结论 线粒体DNA A1555G点突变是导致两个大家系致聋的主要因素之一,具有母系遗传耳聋特点.

  7. 两个携带线粒体12S rRNA 1494C>T突变的耳聋家系的遗传学特征%Characterization of two Chinese families with aminoglycoside-induced and nonsyndromic hearing loss both carrying a mitochondrial 12S rRNA 1494C>T mutation

    Institute of Scientific and Technical Information of China (English)

    龚莎莎; 管敏鑫; 陈波蓓; 彭光华; 郑静; 张婷; 郑斌娇; 方芳; 张初琴; 吕建新

    2012-01-01

    Objective To evaluate the effect of mitochondrial DNA(mtDNA) secondary mutations,haplotypes,GJB2 gene mutations on phenotype of 1494C > T mutation,and to study the molecular pathogenic mechanism of maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss.Methods Two Chinese Han pedigrees of maternally transmitted aminoglycoside induced and nonsyndromic hearing loss were collected.The two probands and their family members underwent clinical,genetic and molecular evaluations including audiological examinations and mutational analysis of mitochondrial genome and GJB2 gene.Results Clinical evaluation revealed wide range of severity,age-at-onset and audiometric configuration of hearing impairment in matrilineal relatives in both families,for which the penetrance of hearing loss was respectively 42.9 % and 28.6% when aminoglycoside-induced deafness was included.When the effect of aminoglycosides was excluded,the penetrances of hearing loss were 14.3% and 14.3%.Sequence analysis of mitochondrial genomes identified a known 12S rRNA 1494C>T mutation,in addition with distinct sets of mtDNA polymorphisms belonging to Eastern Asian haplogroups C4a1a and B4b1c,respectively.Conclusion Mitochondrial 12S rRNA 1494C>T mutation probably underlie the deafness in both families.Lack of significant mutation in the GJB2 gene ruled out involvement of GJB2 in the phenotypic expression.However,aminoglycosides and other nuclear modifier genes may still modify the phenotype of the 1494C>T mutation in these families.The B4b1c is a newly identified haplogroup in aminoglycoside-induced and nonsyndromic hearing loss family carrying the 1494C>T mutation.The 1494C>T mutation seems to have occurred sporadically through evolution.

  8. Species Identification of Beef Based on the Mitochondrial 12S rRNA Gene Sequence%基于线粒体12S rRNA基因序列鉴别牛肉的种源

    Institute of Scientific and Technical Information of China (English)

    王兰萍; 耿荣庆; 王伟; 邓乔文; 徐永新; 陈钥

    2013-01-01

    在提取黄牛肉、牦牛肉和水牛肉总DNA的基础上,设计通用引物进行PCR扩增,电泳回收PCR产物后双向测序,再通过构建系统进化树鉴别牛肉的物种来源.PCR扩增获得的牦牛、水牛12S rDNA基因片段大小都为440 bp,黄牛12S rDNA基因片段大小都为439bp.参照引用的不同牛种12S rDNA基因序列,构建的系统进化树能够清晰地鉴别测序样品的牛种来源.因此,结合运用PCR扩增和DNA测序技术是一种精确可靠的方法,能够有效地运用于牛肉的种源鉴别.%The DNA samples were extracted from cattle, yak and buffalo meat for PCR amplification u-sing universal primers. PCR products were extracted by electrophoresis and sequenced by direct bidirectional sequencing. The phylogenetic tree was constructed for species identification of beef. PCR amplified fragments of yak and buffalo were 440 bp, while 439 bp of cattle. The constructed phylogenetic tree could clearly identify the bovine species of sequenced samples by referencing 12S rDNA gene sequences. Hence, it was a accurate and reliable method to combine PCR amplication with DNA sequencing technology in the species identification of beef.

  9. Analysis on hot spot mutations of GJB2 gene and mitochondrial DNA 12S rRNA among consanguineous induced deafness families%近亲婚配致聋家系中患者GJB2和线粒体DNA 12S rRNA基因突变热点分析

    Institute of Scientific and Technical Information of China (English)

    杨威; 付四清; 董家曙; 王春芳; 陈观明

    2011-01-01

    Objective: To analyze the frequencies of hot spot mutations of the two common deafness gene - GJB2 and mitochondrial DNA 12S rRNA in the consanguineous induced deafness families. Methods: 25 consanguineous induced deafness families received comprehensive physical examination and pure tone test, 48 cases were found, 3 mi venous blood samples were obtained to extract DNA, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) targeted to specific genes were performed, the cases with mutation were confirmed by gene sequencing. Results: 1 case was found with 235delC homozygous mutation of GJB2 gene, 3 cases were found with heterozygous mutation; no A 1555G mutation was found in mitochondrial DNA 12S rRNA. Conclusion: The most of consanguineous induced deafness are autosomal recessive inheritance, but the incidence of 235delC mutation of GJB2 gene is low.%目的:分析GJB2和线粒体DNA 12S rRNA两种常见耳聋致病基因突变热点在近亲婚配致聋家系患者中的发生频率.方法:对25个近亲结婚致聋核心家系的后代进行全面的体格检查和纯音测试,共发现患者48例,采静脉血3 ml,提取DNA,针对特定基因,进行聚合酶链式反应(Polymerase chain reaction,PCR)及限制性片段长度多态性(Restriction fragmentlength polymorphism,RFLP)分析,发现突变者测序验证.结果:发现GJB2基因235delC纯合突变1例,杂合突变3例;未发现线粒体DNA 12S rRNA A1555G突变.结论:近亲结婚家系中遗传性聋多为常染色体隐性遗传,但GJB2基因235delc突变的发生率较低.

  10. Mitochondrial DNA Mutations Associated with Aminoglycoside Ototoxicity

    Institute of Scientific and Technical Information of China (English)

    GUAN Min-Xin

    2006-01-01

    The mitochondrial 12S rRNA has been shown to be the hot spot for mutations associated with both aminoglycoside-induced and non-syndromic hearing loss. Of all the mutations, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region in the 12S rRNA have been associated with aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. The A1555G or C1494T mutation is expected to form novel 1494C-G1555 or 1494U-A1555 base-pair at the highly conserved A-site of 12S rRNA. These transitions make the secondary structure of this RNA more closely resemble the corresponding region of bacterial 16S rRNA. Thus, the new U - A or G-C pair in 12S rRNA created by the C1494T or A1555G transition facilitates the binding of aminoglycosides, thereby accounting for the fact that the exposure to aminoglycosides can induce or worsen hearing loss in individuals carrying these mutations. Furthermore, the growth defect and impairment of mitochondrial translation were observed in cell lines carrying the A1555G or C1494T mutation in the presence of high concentration of aminoglycosides. In addition, nuclear modifier genes and mitochondrial haplotypes modulate the phenotypic manifestation of the A1555G and C1494T mutations. These observations provide the direct genetic and biochemical evidences that the A1555G or C1494T mutation is a pathogenic mtDNA mutation associated with aminoglycoside-induced and nonsyndromic hearing loss. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

  11. Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements.

    Science.gov (United States)

    Gillespie, J J; Johnston, J S; Cannone, J J; Gutell, R R

    2006-10-01

    As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome

  12. 6种水蛭的COⅠ、12S rRNA和16S rRNA基因及分子进化分析%Molecular evolution analysis of COⅠ, 12S rRNA and 16S rRNA gene in six species of leech

    Institute of Scientific and Technical Information of China (English)

    徐云玲; 聂晶; 肖凌

    2013-01-01

    水蛭是一种常见的传统中药,为了解常见蛭类细胞色素氧化酶亚基Ⅰ(COⅠ)、12S rRNA和16S rRNA基因特征和水蛭分子系统进化关系.对常用的入药品种日本医蛭(Hirudo nipponia)、宽体金线蛭(Whitmania pigra)、尖细金线蛭(Whitmania acranulate)和相近物种菲牛蛭(Poecilobdella manillensis)、光润金线蛭(Whitmania laevis)及八目石蛭(Erpobdella octoculata)的COⅠ、12S rRNA和16S rRNA基因进行扩增、测序,利用Mega 5.0分析基因特征、颠换率、分化年代,利用PAUP* 4.10b和MrBayes 3.1.2构建分子系统树.结果表明6种水蛭的COⅠ、12S rRNA和16S rRNA基因全长分别为1534~1536 bp、709~744 bp、1129~1173 bp,GC含量分别为32.35%~34.79%、24.42%~28.49%、24.82%~27.02%,总体颠换率为0.002%~0.760%,分化年代为3.55×106a~9.85×106a;每种水蛭为单系群的支持值均≥82.说明COⅠ、12S rRNA和16S rRNA基因具有种间特异性,可用于6种水蛭的分类鉴别.

  13. Screening of GJB2 235delC mutation and mtDNA 12S rRNA A1555G mutation in Chongqing children with non-syndromic hearing impairment%非综合征性耳聋儿童GJB2 235delC及线粒体DNA 12S rRNA A1555G突变分析

    Institute of Scientific and Technical Information of China (English)

    王冰; 姚红兵; 徐洁; 周媛; 汪武

    2009-01-01

    目的 对耳聋患儿进行GJB2基因、线粒体DNA A1555G位点突变检测,为遗传性耳聋提供诊断依据.方法 对195例耳聋患儿进行遗传性耳聋问卷调查、全面的体格检查、耳鼻咽喉专科检查以及听力学评估(包括纯音测听、脑干诱发电位和耳声发射).对195例非综合征性感音神经性耳聋患儿及100例健康对照个体分别进行GJB2基因235delC突变、线粒体DNA 12S rRNA基因A1555G点突变的限制性内切酶分析.结果 195例患儿者中发现GJB2基因235delc纯合突变、235delC与176-191del16复合及235delC与299-300delAT复合突变等共46例耳聋患儿与GJB2基因突变有关,占23.58%.病患组235delC等位基因频率为18.44%,对照组为2.00%(P<0.01).同时在患儿组还发现了7例线粒体DNA A1555G突变,对照组未发现线粒体DNA A1555G突变.结论 重庆市非综合征型耳聋患儿存在较高的GJB2基因235delC和线粒体DNA 12S rRNA基因A1555G突变发生率,高于全国平均水平.耳聋基因诊断技术可以应用在地区性耳聋病因调查中有重要的意义.对基因型-表现型相关性的研究对遗传性耳聋的治疗及预期疗效判断、遗传咨询、产前诊断等具有重要意义.%Objective To detect the mutations of GJB2 and mtDNA A1555G in Chongqing children with hereditary hearing loss. Methods Totally 195 deaf children were included to identify their medical history of hearing loss, use of aminoglycosides, and other clinical abnormalities through filling a questionnaire by their parents, with 100 normal children as control. The audiological and neurological examinations of these children were conducted, including otoseopy, pure-tone audiometry, acoustic brainstem evoked response (ABR) and otoaeoustic emission. GJB2 gene 235delC mutation and mtDNA A1555G mutation were detected with specific restriction enzyme digestion. Results Homozygous deletion C at position 233-235 of GJB2 (235delC) resul-ted in frameshifi mutation. GJB2

  14. Identification of sika deer and red deer using partial cytochrome b and 12s ribosomal RNA genes%运用Cytb 和12s rRNA 基因鉴别梅花鹿(Cervus nippon)和马鹿(Cervus elaphus)的研究

    Institute of Scientific and Technical Information of China (English)

    李波; 白素英; 徐艳春; 张伟; 马建章

    2006-01-01

    A study was conducted on the identifications of the degraded samples of sika deer (Cervus nippon) and red deer (Cervus elaphus) by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical (0.026±0.006), which was smaller than the smallest nucleotide distance between eastern red deer and sika deer (0.036). Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.%本研究介绍了运用细胞色素b基因和12s核糖体RNA基因部分序列的系统学和核甘酸距离分析来鉴别降解的梅花鹿和马鹿样品.采用PCR和直接测序技术获得了8份嫌疑样品402 bp细胞色素b基因序列,并与来自GenBank数据库 27份同源的细胞色素b基因序列进行比对.3份嫌疑样品与梅花鹿的核甘酸距离值相同(0.026±0.006),小于梅花鹿与东部马鹿间最小的核甘酸距离值(0.036).并且梅花鹿和马鹿的系统学分析表明这些样品与梅花鹿聚为一枝,因此可以推测它们来源于梅花鹿.同样的方法得出另3份嫌疑样品来源于马鹿.该结果被387 bp12s核糖体RNA基因序列的系统学和核甘酸距离分析进一步证实.该方法是有效的,花费的时间少,能帮助减少同类野生动物案件的发生.

  15. Phylogeny of eight snappers (Lutjanidae) in Chinese coastal waters inferred from partial mtDNA 12S rRNA Sequences%基于12S rRNA部分序列分析的中国8种笛鲷科鱼类系统发育初探

    Institute of Scientific and Technical Information of China (English)

    唐优良; 章群; 余帆洋; 周佳怡; 司丛利; 黄小彧; 马奔

    2011-01-01

    Partial sequences of 12S rRNA gene of eight snappers collected in coastal waters of china determined in the present study and two lutjanid sequences downloaded from GenBank were combined to reconstruct Maximum likelihood and Bayesian inference trees with Siganus unimaculatus, S. fuscescens, Monotaxis grandocu/is, Lethrinus obsoletus (perciformes), Myripristis berndti, Beryx splendens, Sargocentron rubrum (Beryciformes), and Danio rerio(Cypriniformes) as outgroups. Totally 170 variable sites and 126 parsimony-informative sites existed in the 895 bp sequences, and the ts/tv ratio was 2.39. Phylogenetic trees showed that the Caesioninae were nested within the Lutjaninae, supporting the recent opinion that the Caesionidae shoud be regarded as a synonym of the Lutjanidae, but their phylogenetic relationships to other lutjanid species remained to be further studied. L. malabarius and L.sebae formed a well-supported clade, supporting the taxonomical treatment of L. malabarius and L. sebae based on morphological characters; however morphologically similar species L. johni and L. argentimaculatus were genetically quite different, indicating that morphologically similar species were not necessarily genetically closely related.%测定了中国近海8种笛鲷鱼类线粒体12s rRNA基因部分序列,结合GenBank中下载的2种笛鲷鱼类序列,以鲈形目(Perciformes)中的单列齿鲷(Monotaxis grandoculis)、黄带裸颊鲷(Lethrinus obsoletus)、尖嘴蓝子鱼(Siganus unimaculatus)、褐蓝子鱼(Siganusfuscescens)和金眼鲷目(Peryciformes)中的点鳍棘鳍鱼(Sargocentron rubrum)、大鳞锯鳞鱼(Myripristis berndti)、红金眼鲷(Beryx splendens)以及鲤形目(Cypriniformes)中的印度斑马鱼(Danio rerio)为外类群,构建最大似然树和贝叶斯推断树,发现在895bp的同源序列中,共有170个变异位点,其中简约信息位点126个,转换/颠换值为2.39;最大似然树和贝叶斯推断树显示梅鲷鱼类(Caesioninae)镶嵌在笛鲷

  16. Phylogeny Relationship among Eleven Members in Cyprinidae in Ertix River Based on 12S rRNA and S7 Intron 1 Gene%利用12S rRNA与S7基因序列分析额尔齐斯河中11种鲤科鱼类的系统发育关系

    Institute of Scientific and Technical Information of China (English)

    孟玮; 杨天燕; 海萨; 蔡林钢; 牛建功

    2015-01-01

    根据线粒体12S rRNA基因和S7核糖体蛋白基因内含子1序列,构建额尔齐斯河中11种鲤科鱼类的系统发育树.结果表明:12S基因AT含量与GC含量相当,而S7基因AT含量明显高于GC含量.12S基因保守区分散在整个序列中,而S7基因保守区均集中在前300bp内.12S rRNA和S7基因系统发育树中有三枝聚在一起,即贝加尔雅罗鱼Leuciscus baicalensis与高体雅罗鱼Leuciscus idus,尖鳍鮈Gobio gobio与麦穗鱼Pseudorasbora parva,金鲫Carassius carassius、银鲫Carassius auratus和鲤Cyprinus carpio.12S基因的聚类树中,丁鱥Tinca tinca与雅罗鱼亚科鱼类聚类,表明两者关系密切;而S7基因的聚类树中丁鱥以较高的支持率与尖鳍鮈、麦穗鱼聚在一起,与鮈亚科关系紧密;分子证据支持将丁鱥列入雅罗鱼系.12S基因构建的三种系统发育树拓扑结构有一定差异,而S7基因构建的三种系统发育树基本一致,支持率也高于前者,表明S7构建鲤科系统发育树的优势高于12S.

  17. Heteroplasmy Levels of Mitochondrial 12S rRNA A1555G Mutation in Pedigrees with Aminoglycoside-Induced and Non-Syndromic Hearing Loss as Detected Using SNaPshot Technique%线粒体12SrRNAA1555G突变耳聋家系的异质率研究

    Institute of Scientific and Technical Information of China (English)

    朱玉华; 翟所强; 戴朴

    2014-01-01

    Objective To study heteroplasmy levels and inheritance patterns of the mitochondrial 12S rRNA A1555G mutation in a K-11 pedigree with aminoglycoside-induced and non-syndromic hearing loss using the SNaPshot technique. Methods Comprehensive history and physical examination were obtained, including history of aminoglycoside use and genetic factors related to the hearing impairment among the pedigree members. Age-appropriate audiological tests were performed and the PTA calculated. Genomic DNA was isolated from whole blood and the level of heteroplasmy in peripheral blood leuko-cytes was determined using SNaPshot technology. Results There were four generations in this K-11 pedigree. Deafness was the only clinical phenotype. The onset age and extent of hearing loss were different among maternal members. The average het-eroplasmy rate of this K-11 pedigree was 87.99%(ranging from 82.32%to 94.65%), and the average heteroplasmy rates of generations II to IV were 88.27%, 86.78%, and 90.31%, respectively. Conclusion There are random shifts in the heteroplasmy level between mothers and offspring with the A1555G mutation in K-11 pedigrees. However, there seems a trend of gradual increase over generations.%目的:利用SNaPshot技术检测线粒体12S rRNA A1555G异质性突变耳聋家系(K-11)母系成员的突变异质率,探讨家系中异质率的分布状况和遗传规律。方法通过家系调查,对线粒体12S rRNA A1555G异质性突变耳聋家系母系成员进行全身系统检查及临床听力学检测;提取基因组和线粒体DNA,针对12S rRNA A1555G突变,设计SnaPshot引物和探针,并对K-11家系母系成员进行异质率检测,分析K-11家系母系成员突变异质率的分布特点和遗传规律。结果 K-11家系共四代,耳聋是此家系的唯一临床表型,家系母系耳聋成员的听力下降时间、程度和听力曲线类型具有明显的个体差异;家系中每位家系母系成员的12S rRNA A1555G突变

  18. 12S rRNA和Cyt b基因部分序列研究13种猫科动物的分子系统关系%Phylogeny of 13 Felidae species from China based on partial 12S rRNA and Cyt b genes sequences

    Institute of Scientific and Technical Information of China (English)

    郑涛; 费荣梅; 吴孝兵

    2005-01-01

    为探讨中国猫科动物(Felidae)的系统发生关系,本文对中国产13种猫科动物的12S rRNA基因(约371 bp)和细胞色素b基因(Cyt b) 部分序列(约355 bp)进行了分析,并采用"最大简约法"和"最大似然法"构建了分子系统树.结果表明:在Cyt b基因序列中,有113个位点存在变异(约为总位点数的31.8%),高于12S rRNA基因序列的44个变异位点(约为总位点数的11.9%);构建的分子系统树显示,猞猁(Lynx lynx)可能是中国最早起源的猫科动物,与其它猫科动物之间的亲缘关系较远,支持将其立为猞猁属(Lynx)的观点;草原斑猫(Felis libyca)、丛林猫(Felis chaus)、兔狲(Otocolobus manul)和荒漠猫(Felis bieti)具有较近的亲缘关系,支持将兔狲划归于猫属(Felis)的观点;金猫(Caopuma temminckii)、云猫(Pardofelis marmorata)具有较近的亲缘关系,但它们与猫属物种之间的亲缘关系可能较远,不支持将它们划归于猫属;豹猫(Ponailurus ribengalensis)、渔猫(Prionailurus viverrinus)具有较近的亲缘关系,支持将它们同归于豹猫属(Ponailurus);云豹(Neofelis nebulosa)、豹(Panthera pardus)、雪豹(Uncia uncia)、虎(Panthera tigris)具有较近的亲缘关系,支持将它们同归于豹属(Panthera)的观点

  19. 淡水豚类线粒体DNA 12S rRNA基因的序列变异及其分子系统学研究%SEQUENCE VARIATION OF MITOCHONDRIAL 12S rRNA GENE AND INFERRED MOLECULAR SYSTEMATICS OF RIVER DOLPHINS

    Institute of Scientific and Technical Information of China (English)

    杨光; 刘珊

    2000-01-01

    淡水豚类4个代表属[白暨豚(Lipotes)、恒河豚(Platanista)、弗西豚(Pontoporia)和亚河豚(Inia)]mtDNA 12S rRNA基因的序列差异水平,高于其他齿鲸类科间的差异,特别是远远高于海豚总科内的科间差异.研究结果支持它们应归属于不同的科,即白暨豚科(Lipotiidae)、恒河豚科(Platanistidae)、弗西豚科(Pontoporidae)和亚河豚科(Iniidae).恒河豚科是鲸类中最早分化出来的独立支系,而其余3个淡水豚科组成一个单系,作为海豚总科的姊妹群.淡水豚类的发生是多系的.白暨豚科、亚河豚科和弗西豚科是否隶属于同一个总科尚需进一步研究.

  20. 四川黑熊线粒体12S和16S rRNA基因的克隆及序列分析%Clon and Sequence Analysis of mtDNA 12S rRNA and 16S rRNA Genes of Sichuan Black Bear (Ursus thibetanus mupinensis)

    Institute of Scientific and Technical Information of China (English)

    吴夏; 陈瑜; 彭正松; 杨军; 侯万儒

    2007-01-01

    利用哺乳动物线粒体基因的保守序列设计引物,采用PCR方法,首次从亚洲黑熊四川亚种(Ursus thibetanus mupinensis)的肌肉组织总DNA中扩增出了线粒体12S rRNA和16S rRNA基因并进行了序列测定及分析.结果表明,四川黑熊12S rRNA基因长965 bp;16S rRNA基因长1 580 bp.通过进一步的序列分析表明,四川黑熊的12S rRNA和16S rRNA基因有较高的进化速率,与美洲黑熊,棕熊,北极熊,眼镜熊及大熊猫的相应基因相比较有较大差异,其中与美洲黑熊的同源性相对较高.

  1. Modifier factors influencing the phenotypic manifestation of the deafness-associated mitochondrial DNA mutations%修饰因子对线粒体DNA突变致聋的影响

    Institute of Scientific and Technical Information of China (English)

    杨爱芬; 郑静; 吕建新; 管敏鑫

    2011-01-01

    Mutations in the mitochondrial DNA have been found to be one of the most important causes of sensorineural hearing loss. In particular, these mutations often occur in the mitochondrial 12S rRNA and tRNA genes. Of these, the homoplasmic A1555G and C1494T mutations in the 12S rRNA have been associated with both aminoglycoside induced and nonsyndromic hearing impairment in many families worldwide. Children carrying the A1555G or C1494T mutation are susceptible to the exposure of ototoxic drugs, thereby inducing or worsening hearing loss. Individuals harboring A1555G or C1494T mutation can also develop hearing loss even in the absence of aminoglycoside exposure. However, matrilineal relatives of intra-families or inter-families carrying the A1555G or C1494T mutation exhibit a wide range of severity,age-at-onset, and audiometric configuration of hearing impairment. These indicate that the A1555G or C1494T mutation is a primary factor underlying the development of deafness but insufficient to produce the clinical phenotype. Thus, other modifier factors, such as aminoglycoside (s), mitochondrial DNA haplotype(s) or nuclear modifier gene(s), play a role in the phenotypic expression of the deafness-associated mitochondrial 12S rRNA A1555G or C1494T mutation. In this review, we summarize the modifier factors for the phenotypic expression of deafness-associated 12S rRNA A1555G and C1494T mutations and propose the molecular pathogenetic mechanism of maternally inherited deafness.%线粒体DNA突变是引起感音神经性耳聋的重要原因之一,这些突变主要位于线粒体12SrRNA和tRNA基因上.其中12S rRNA基因上的同质性A1555G和C1494T突变与氨基糖甙类抗生素造成的耳聋相关.携带这两个突变的个体对耳毒性药物高度敏感,导致临床上常见的"一针致聋"现象.但携带A1555G或C1494T突变的个体在没用药的情况下也能产生非综合征型耳聋,而且同一家系内和不同家系间的母系成员在听力损失

  2. ALLELE DISTRIBUTION OF D12S304, D12S313, D12S1583, D12S1640 AND D12S1708 IN CHINESE HAN POPULATION

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To analyze the genetic polymorphism of 5 STR loci (D12S304, D12S313, D12S1583, D12S1640 and D12S1708) on chromosome 12 in Chinese Han population. Methods EDTA-anticoagulated blood specimens were collected from unrelated individuals of Chinese Han population in Shaanxi province. DNA samples were extracted with the Wizard Genomic DNA purification Kit and were amplified by polymerase chain reaction (PCR) technique. The PCR products were analyzed by ABI 3100 Genetic Analyzer. Results All 5 loci were in Hardy-Weinberg equilibrium. Allele and genotype frequencies, heterozygosity, power of discrimination, polymorphism information content, probability of paternity exclusion and matching probability of each locus were calculated in Excel 2002. Conclusion They are complex loci with lots of evenly distributed alleles and high heterozygosity in Chinese Han population. Thus they are informative polymorphic loci and valuable DNA marker which represents a superior alternative to many established STRs.

  3. An MRPS12 mutation modifies aminoglycoside sensitivity caused by 12S rRNA mutations

    Directory of Open Access Journals (Sweden)

    Sonia eEmperador

    2015-01-01

    Full Text Available Several homoplasmic pathologic mutations in mitochondrial DNA, such as those causing Leber hereditary optic neuropathy or non-syndromic hearing loss, show incomplete penetrance. Therefore, other elements must modify their pathogenicity. Discovery of these modifying factors is not an easy task because in multifactorial diseases conventional genetic approaches may not always be informative.Here, we have taken an evolutionary approach to unmask putative modifying factors for a particular homoplasmic pathologic mutation causing aminoglycoside-induced and non-syndromic hearing loss, the m.1494C>T transition in the mitochondrial DNA. The mutation is located in the decoding site of the mitochondrial ribosomal RNA. We first looked at mammalian species that had fixed the human pathologic mutation. These mutations are called compensated pathogenic deviations because an organism carrying one must also have another that suppresses the deleterious effect of the first. We found that species from the primate family Cercopithecidae (old world monkeys harbor the m.1494T allele even if their auditory function is normal.In humans the m.1494T allele increases the susceptibility to aminoglycosides. However, in primary fibroblasts from a Cercopithecidae species, aminoglycosides do not impair cell growth, respiratory complex IV activity and quantity or the mitochondrial protein synthesis. Interestingly, these species also carry a fixed mutation in the mitochondrial ribosomal protein S12. We show that the expression of this variant in a human m.1494T cell line reduces its susceptibility to aminoglycosides. Because several mutations in this human protein have been described, they may possibly explain the absence of pathologic phenotype in some pedigree members with the most frequent pathologic mutations in mitochondrial ribosomal RNA.

  4. Asymmetric Synthesis of (+)-(11 R,12S)-Mefloquine Hydrochloride

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The asymmetric synthesis of (+)-(11R,12S)-mefloquine hydrochloride, an antimalarial drug, was accomplished from commercially available 2-trifluoromethylaniline, ethyl 4,4,4-trifluoroacetoacetate and cyclopentanone in 7 steps with a 14% overall yield. The key steps were proline-catalyzed asymmetric direct aldol reaction and Beck-mann rearrangement. The absolute configuration was assigned by a Mosher's method.

  5. Molecular phylogenetic inference of the woolly mammoth Mammuthus primigenius, based on complete sequences of mitochondrial cytochrome b and 12S ribosomal RNA genes.

    Science.gov (United States)

    Noro, M; Masuda, R; Dubrovo, I A; Yoshida, M C; Kato, M

    1998-03-01

    Complete sequences of cytochrome b (1,137 bases) and 12S ribosomal RNA (961 bases) genes in mitochondrial DNA were successfully determined from the woolly mammoth (Mammuthus primigenius), African elephant (Loxodonta africana), and Asian elephant (Elephas maximus). From these sequence data, phylogenetic relationships among three genera were examined. Molecular phylogenetic trees reconstructed by the neighbor-joining and the maximum parsimony methods provided an identical topology both for cytochrome b and 12S rRNA genes. These results support the "Mammuthus-Loxodonta" clade, which is contrary to some previous morphological reports that Mammuthus is more closely related to Elephas than to Loxodonta.

  6. PCR primers for metazoan mitochondrial 12S ribosomal DNA sequences.

    Directory of Open Access Journals (Sweden)

    Ryuji J Machida

    Full Text Available BACKGROUND: Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy. METHODOLOGY/PRINCIPAL FINDINGS: A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI's Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans. CONCLUSIONS/SIGNIFICANCE: Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans.

  7. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

    OpenAIRE

    Rozier, Claude; Mache, Régis

    1984-01-01

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloropl...

  8. APPLICATION OF GENETIC DEAFNESS GENE CHIP FOR DETECTION OF GENE MUTATION OF DEAFNESS IN PREGNANT WOMEN

    Institute of Scientific and Technical Information of China (English)

    CHANG Liang; ZHONG Su; ZHAO Nan; LIU Ping; ZHAO Yangyu; QIAO Jie

    2014-01-01

    Objective The study is to identify the carrier rate of common deafness mutation in Chinese pregnant women via detecting deafness gene mutations with gene chip. Methods The pregnant women in obstetric clinic without hearing impairment and hearing disorders family history were selected. The informed consent was signed. Peripheral blood was taken to extract genom-ic DNA. Application of genetic deafness gene chip for detecting 9 mutational hot spot of the most common 4 Chinese deafness genes, namely GJB2 (35delG,176del16bp, 235delC, 299delAT), GJB3 (C538T) ,SLC26A4 ( IVS72A>G, A2168G) and mito-chondrial DNA 12S rRNA (A1555G, C1494T) . Further genetic testing were provided to the spouses and newborns of the screened carriers. Results Peripheral blood of 430 pregnant women were detected,detection of deafness gene mutation carri-ers in 24 cases(4.2%), including 13 cases of the GJB2 heterozygous mutation, 3 cases of SLC26A4 heterozygous mutation, 1 cases of GJB3 heterozygous mutation, and 1 case of mitochondrial 12S rRNA mutation. 18 spouses and 17 newborns took fur-ther genetic tests, and 6 newborns inherited the mutation from their mother. Conclusion The common deafness genes muta-tion has a high carrier rate in pregnant women group,235delC and IVS7-2A>G heterozygous mutations are common.

  9. Chicken rRNA Gene Cluster Structure.

    Directory of Open Access Journals (Sweden)

    Alexander G Dyomin

    Full Text Available Ribosomal RNA (rRNA genes, whose activity results in nucleolus formation, constitute an extremely important part of genome. Despite the extensive exploration into avian genomes, no complete description of avian rRNA gene primary structure has been offered so far. We publish a complete chicken rRNA gene cluster sequence here, including 5'ETS (1836 bp, 18S rRNA gene (1823 bp, ITS1 (2530 bp, 5.8S rRNA gene (157 bp, ITS2 (733 bp, 28S rRNA gene (4441 bp and 3'ETS (343 bp. The rRNA gene cluster sequence of 11863 bp was assembled from raw reads and deposited to GenBank under KT445934 accession number. The assembly was validated through in situ fluorescent hybridization analysis on chicken metaphase chromosomes using computed and synthesized specific probes, as well as through the reference assembly against de novo assembled rRNA gene cluster sequence using sequenced fragments of BAC-clone containing chicken NOR (nucleolus organizer region. The results have confirmed the chicken rRNA gene cluster validity.

  10. 邯郸地区新生儿听力与药物性耳聋易感基因突变联合筛查研究%Neonatal hearing screening combined with drug-induced deafness susceptibility genes mutation screening in Handan area

    Institute of Scientific and Technical Information of China (English)

    陈丁莉; 李守霞; 要跟东; 冯海芹; 郭丽丽; 孙彩霞; 杨志明; 张晓芳

    2013-01-01

    Objectives To evaluate the feasibility and significance of universal neonatal hearing screening combined with mitochondria deafness genes screening using newborn cord blood. Methods One thousand newborns were enrolled in the study. Routine hearing screening was carried out and meanwhile the cord bloods were collected for mitochondria DNA screening. Mutations of A1555G and C1494T on mitochondria gene 12S rRNA were screened using Sanger sequencer. Results In hearing screening, 141 (14.1%) of the 1000 newborns were identified to be at risk, of whom 11 (1.1%) were confirmed to have hearing loss. In gene screening. AI555G mutation was found in 4 (0.4%) of the 1000 newborns and C1494T mutation was found in 2 (0.2%) infants, though they were all identified as normal in the first-step hearing screening. Conclusions Mitochondria deafness genes screening can complement hearing screening, and may effectively help to avoid later healing loss in children sensitive to aminoglycoside antibiotics.%目的 探讨在新生儿听力筛查同时,采用新生儿脐带血进行线粒体耳聋基因筛查的可行性及其意义.方法 收集邯郸地区新生儿1 000例为研究对象,进行常规听力筛查,同时采集脐带血,用于线粒体DNA的筛查.采用Sanger测序法检测线粒体12S rRNA A1555G和C1494T两个突变位点.结果 在1000名新生儿中,初次听力筛查有141例(14.1%) 未通过,经过复筛后仍有11例 (1.1%) 未通过新生儿听力筛查.线粒体12S rRNA检测发现携带A1555G突变4例(0.4%),C1494T突变2例 (0.2%),而该6例新生儿均通过了初次听力筛查.结论 线粒体耳聋基因筛查可以有效补充听力筛查的不足,早期发现对氨基糖苷类抗生素敏感的个体,避免药物性耳聋的发生.

  11. Variations of mitochondrial D-loop region plus downstream gene 12S rRNA-tRNAphe and gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Cheng-Bo Han; Fan Li; Yu-Jie Zhao; Jia-Ming Ma; Dong-Ying Wu; Yu-Kui Zhang; Yan Xin

    2003-01-01

    AIM: To explore the instabilities, polymorphisms and other variations of mitochondrial D-loop region and downstream gene 12S rRNA-tRNAPhe in gastric cancers, and to study their relationship with gastric cancer.METHODS: Three adjacent regions (D-loop, tRNAphe and 12S rRNA) were detected for instabilities, polymorphisms and other variations via PCR amplification followed by direct DNA sequencing in 22 matched gastric cancerous tissues and para-cancerous normal tissues.RESULTS: PolyC or (CA)n instabilities were detected in 13/22(59.1%) gastric cancers and 9/22(40.9 %) in the control (P>0.05). There existed 2/12(16.7%) and 6/10(60%)alterations of 12S rR NA-tRNAphe in well differentiated gastric cancers and poorly differentiated ones, respectively(P0.05).Some new variations were found, among which np 318 and np 321 C-T transitions in D-loop region were two of the five bases for H-strand replication primer. Np 523 AC-deletion and np 527 C-T transition occurred at mtTF1 binding site (mtTFBS), which were associated with the transcription of downstream mitochondrial genome. Seven samples showed the np 16 182 polyC instabilities, five of which simultaneously showed np 16 189 T-C transitions.CONCLUSION: There is no statistic significance of instabilities and polymorphisms in mitochondrial D-loop region between gastric cancerous and para-cancerous normal tissues, which suggests that the instability might relate to heredity or be dependent on aging. There is asignificant correlation between differentiation degree of gastric cancer and variant frequencies of 12S rRNA-tRNAphe. The poorly differentiated gastric cancers are more prone to 12S rRNAtRNAphe variations, or gastric cancers with 12S rRNA-tRNAphe variations are more likely to be poorly differentiated, np 16189 T-C transition may be one of the important reasons for polyC instability in gastric cancer.

  12. 5S rRNA and ribosome.

    Science.gov (United States)

    Gongadze, G M

    2011-12-01

    5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

  13. Evaluation of mitochondrial tRNASer(UCN) G7444A mutation associated with non-syndromic hearing loss in eleven Han Chinese pedigrees%11个携带线粒体tRNASer(UCN)G7444A突变的中国汉族非综合征型耳聋家系分析评估

    Institute of Scientific and Technical Information of China (English)

    唐霄雯; 管敏鑫; 阳娅玲; 高应龙; 肖红利; 何哲耘; 郑静; 郑斌娇; 吕建新; 金龙金

    2014-01-01

    Chinese Han pedigrees with mitochondrial tRNASer(UCN) G7444A mutation underwent audiological testing, deafness associated mutational hot spots screen-ing and pedigree assessment.Results: Among the 2 650 Han Chinese non-syndromic deafness subjects, 22 pa-tients belonging to 11 pedigrees carried mitochondrial tRNASer(UCN) G7444A mutation, account for 0.7%. Num-ber of pedigree carry both mitochondrial tRNASer(UCN) G7444A mutation and mitochondrial 12S rRNA A1555G, mitochondrial 12S rRNA C1494T orGJB2 c.235delC mutation were 3, 1, 2 respectively. Clinical data showed that there were huge difference in the severity of hearing loss, age of onset and penetrance among these 11 Chi-nese Han non-syndromic deafness pedigrees. The average penetrance of these pedigrees carrying mitochondrial tRNASer(UCN) G7444A mutation and mitochondrial 12S rRNA A1555G, C1494T orGJB2 c.235delC mutation were 29.4%, 42.9% and 19.0% respectively. The average penetrance of 4 pedigrees carrying the mitochondrial tRNASer(UCN) G7444A mutation and mitochondrial 12S rRNA A1555G or C1494T mutation is signiifcantly higher than that of carry mitochondrial tRNASer(UCN) G7444A mutation, it was 14.0%.Conclusion: Mitochondrial tRNASer(UCN) G7444A mutation and mitochondrial 12S rRNA A1555G or C1494T mutations may co-modulate the variable penetrance and expressivity of deafness among these Han Chinese non-syndromic pedigrees.

  14. Mapping contacts of the S12-S7 intercistronic region of str operon mRNA with ribosomal protein S7 of E. coli.

    Science.gov (United States)

    Golovin, Andrey; Spiridonova, Vera; Kopylov, Alexei

    2006-10-30

    In E. coli, S7 initiates 30S ribosome assembly by binding to 16S rRNA. It also regulates translation of the S12 and S7 cistrons of the 'streptomycin' operon transcript by binding to the S12-S7 intercistronic region. Here, we describe the contacts of N-terminally His(6)-tagged S7 with this region as mapped by UV-induced cross-linking. The cross-links are located at U(-34), U(-35), quite distant from the start codons of the two cistrons. In order to explain the mechanism of translational repression of S12-S7, we consider a possible conformational rearrangement of the intercistronic RNA structure induced by S7 binding.

  15. Characterization of 12S rRNA gene for meat identification of common wild and domestic small herbivores as an aid to wildlife forensic

    OpenAIRE

    E. Joseph; Sanjeev Singh; Rakesh Ranjan; Parmar, S. N. S.; Nidhi Rajput; Shrivastav, A. B.

    2013-01-01

    Aim: Chital and sambar are the common wild small herbivores, which are vulnerable to poaching for their meat. Many times poachers claim the wild meat to be that of goat or sheep. Hence, authentic evidences are required to stop such wildlife crime. The present investigation was carried out to study the species specific PCR-RFLP patterns for meat identification of chital and sambar and then differentiation from the meat of goat and sheep. Materials and Methods: Extracted DNA from meat samples w...

  16. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose.

    Science.gov (United States)

    Rozier, C; Mache, R

    1984-10-11

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20 degrees C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.

  17. Physical mapping of the human ELA1 gene between D12S361 and D12S347 on chromosome 12q13

    Energy Technology Data Exchange (ETDEWEB)

    Davies, R.L. [Sweet Briar College, VA (United States); Yoon, Sung Joo; Kucherlapati, R. [Albert Einstein College of Medicine, New York, NY (United States)] [and others

    1995-10-10

    ELA1, the pancreatic elastase 1 gene, is conserved in mammalian genomes. ELA1 was previously mapped to chromosome 12 using a panel of mouse-human somatic cell hybrids. We now report the physical and cytogenetic localization of the ELA1 gene. On the physical map, ELA1 is adjacent to the polymorphic marker AFMa283yg1 and between D12S361 and D12S347. Using fluorescence in situ hybridization, we determined that ELA1 maps to 12q13. 13 refs., 2 figs.

  18. Phylogenetic analysis of the order Pleuronectiformes (Teleostei based on sequences of 12S and 16S mitochondrial genes

    Directory of Open Access Journals (Sweden)

    Marisa F.C. Azevedo

    2008-01-01

    Full Text Available The fish order Pleuronectiformes, composed of 14 families, has two suborders: Psettodoidei (with one family and Pleuronectoidei (with thirteen families. The relationships among families of Pleuronectoidei and among the genera of their families have extensively been debated and a consensus has not yet been reached. In the present study, partial sequences of the 12S and 16S mitochondrial rRNA genes were obtained from 19 species belonging to the families Achiridae, Bothidae, Cynoglossidae, Paralichthyidae, Pleuronectidae, Scophthalmidae, and Soleidae. Additional sequences of 42 pleuronectiform species were obtained from GenBank. Phylogenetic analyses were conducted by the methods of maximum-parsimony, maximum-likelihood and Bayesian inference. Our results corroborate the monophyletic status of all families, excluding Paralichthyidae. In the family Achiridae, the genus Catathyridium (freshwater was the sister group of Trinectes (saltwater, and Hypoclinemus (freshwater was the sister group of Achirus (saltwater. Assuming that the putative ancestor of achirids lived in saltwater, it is suggested that the freshwater habitats in South America were colonized independently by different achirid lineages.

  19. Eukaryotic 5S rRNA biogenesis

    Science.gov (United States)

    Ciganda, Martin; Williams, Noreen

    2012-01-01

    The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function. PMID:21957041

  20. Eukaryotic 5S rRNA biogenesis.

    Science.gov (United States)

    Ciganda, Martin; Williams, Noreen

    2011-01-01

    The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function. Copyright © 2011 John Wiley & Sons, Ltd.

  1. ADAM 12-S cleaves IGFBP-3 and IGFBP-5 and is inhibited by TIMP-3

    DEFF Research Database (Denmark)

    Loechel, F; Fox, J W; Murphy, G;

    2000-01-01

    that it cleaves insulin-like growth factor binding protein-3 (IGFBP-3). This result supports a role for ADAM 12-S in the degradation of IGFBP-3 in the blood of pregnant women. Furthermore, we tested for proteolysis of other members of the IGF binding protein family and found that ADAM 12-S cleaves IGFBP-5......ADAMs are a family of multidomain proteins having proteolytic and cell adhesion activities. We have previously shown that ADAM 12-S, the secreted soluble form of human ADAM 12, is a catalytically active protease. We now describe the purification of full-length recombinant ADAM 12-S and demonstrate...... in addition to IGFBP-3, but does not cleave IGFBP-1, -2, -4, or -6. ADAM 12-S may therefore be the IGFBP-5 protease that is secreted by osteoblasts and other cells. Cleavage of both IGFBP-3 and -5 by ADAM 12-S was inhibited by TIMP-3, raising the possibility that TIMP-3 is a physiological inhibitor of ADAM 12...

  2. Teenage-onset non-syndromic deafness associated with a mutation and a polymorphism in the mitochondrial 12S ribsomal RNA gene in a large Zairese pedigree

    Energy Technology Data Exchange (ETDEWEB)

    Matthijs, G.; Claes, S.; Cassiman, J.J. [Univ. of Leuven (Belgium)] [and others

    1994-09-01

    Non-syndromic deafness has been described as both an autosomal dominant and a recessive trait. Recently, Prezant et al. have identified a 1555 A to G substitution in the mitcohondrial 12S rRNA gene associated with deafness, either in an early-onset form with a postulated recessive nuclear defect for phenotypic expression, or after treatment with aminoglycosides. We have analyzed samples from a large pedigree originating from the village KAI SINGINI in Bas-Zaire. Patients have a maternally inherited, sudden-onset and bilateral sensineuronal deafness before the age of 20. To the best of our knowledge, no aminoglycosides have been given to these individuals. Sequencing of the mitochondrial 12S rRNA gene revealed the presence of a homoplasmic 1555 A-G mutation in 8 patients tested. This mutation is invariably associated with a newly described T-C transition at 1420 in the same gene. The 1420 mutation was also found in 1 of 30 unrelated black individuals. It is phylogenetically less conserved than its neighboring bases and than the 1555 mutation. It has been speculated that the 1555 mutation results in greater susceptibility to the effects of aminoglycosides on translational fidelity in the mitochondrial ribosome. It remains to be investigated whether the substitution at 1420 contributes to the ribosomal malfunction and the disease phenotype. Our results add to previous evidence for the 1555 mutation as a pathogenic mutation in non-syndromic deafness. We are currently collecting further family data and samples. The analysis of the entire pedigree will shed light on the possible contribution of nuclear defects in the cochlear-specific damage by impairment of mitochondrial translation.

  3. Increased 5S rRNA oxidation in Alzheimer's disease.

    Science.gov (United States)

    Ding, Qunxing; Zhu, Haiyan; Zhang, Bing; Soriano, Augusto; Burns, Roxanne; Markesbery, William R

    2012-01-01

    It is widely accepted that oxidative stress is involved in neurodegenerative disorders such as Alzheimer's disease (AD). Ribosomal RNA (rRNA) is one of the most abundant molecules in most cells and is affected by oxidative stress in the human brain. Previous data have indicated that total rRNA levels were decreased in the brains of subjects with AD and mild cognitive impairment concomitant with an increase in rRNA oxidation. In addition, level of 5S rRNA, one of the essential components of the ribosome complex, was significantly lower in the inferior parietal lobule (IP) brain area of subjects with AD compared with control subjects. To further evaluate the alteration of 5S rRNA in neurodegenerative human brains, multiple brain regions from both AD and age-matched control subjects were used in this study, including IP, superior and middle temporal gyro, temporal pole, and cerebellum. Different molecular pools including 5S rRNA integrated into ribosome complexes, free 5S rRNA, cytoplasmic 5S rRNA, and nuclear 5S rRNA were studied. Free 5S rRNA levels were significantly decreased in the temporal pole region of AD subjects and the oxidation of ribosome-integrated and free 5S rRNA was significantly increased in multiple brain regions in AD subjects compared with controls. Moreover, a greater amount of oxidized 5S rRNA was detected in the cytoplasm and nucleus of AD subjects compared with controls. These results suggest that the increased oxidation of 5S rRNA, especially the oxidation of free 5S rRNA, may be involved in the neurodegeneration observed in AD.

  4. ADAM12-S stimulates bone growth in transgenic mice by modulating chondrocyte proliferation and maturation

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Albrechtsen, Reidar; Rudkjaer, Lise

    2006-01-01

    ADAM12-S transgenic mice exhibit a pronounced increase in the length of bones, such as femur, tibia, and vertebrae. The effect of ADAM12-S on longitudinal bone growth involves the modulation of chondrocyte proliferation and maturation, likely through proteolytic activities and altered cell......-extracellular matrix interactions in the growth plate. INTRODUCTION: The disintegrin and metalloprotease ADAM12 is expressed in both osteoblasts and osteoclasts, suggesting a regulatory role of ADAM12 in bone. However, thus far, no in vivo function of ADAM12 in the skeleton has been reported. MATERIALS AND METHODS......-extracellular matrix interactions. RESULTS: ADAM12-S transgenic mice exhibit increased longitudinal bone growth. The increased bone length is progressive and age dependent, with a maximum increase of 17% seen in the femur from 6-month-old transgenic mice. The effect is gene dose dependent, being more pronounced...

  5. [Second trimester screening for trisomy 21 using ADAM12-S as a maternal serum marker].

    Science.gov (United States)

    Jiang, Tao; Lv, Ling; Yang, Bing; Sun, Yi-jun; Zhang, Xiao-juan; Sun, Yun; Xu, Qian-jun; Xu, Zheng-feng

    2012-06-01

    To investigate the value of a disintegrin and metalloproteinase 12 secreting form (ADAM12-S) as a maternal serum marker in second trimester screening for trisomy 21 (Down syndrome, DS), and to develop an appropriate prenatal DS screening protocol. Serum samples were collected from 53 pregnant women carrying a trisomy 21 fetus and 621 pregnant women with matched gestational age and weight carrying a healthy fetus. ADAM12-S concentrations were determined with a time-resolved fluorescence immunoassay (TRFIA). Curve fitting by weighted regression and other statistical methods were conducted, and the model was optimized for prenatal trisomy 21 screening program in second trimester. ADAM12-S alone or in combination with other two- or three-combination test was selected as a serum marker for prenatal second-trimester screening of trisomy 21 by calculation of detection rate (DR) and false positive rate (FPR). By comparison, the median multiple of the median (MoM) value of ADAM12-S in DS pregnancy group was higher than that of the control group (PHCG) has increased to 52.80% from 39.62% of the conventional two-combination test (AFP and free β-HCG). For women with a risk between 1/300 and 1/1000 by two-combination test for DS, the DR has increased from 39.62% to 47.12%, but FPR only increased by 0.8% after adding ADAM12-S as a maternal serum marker. Considering the increased DR of pregnancies with a risk between 1/300 and 1/1000 in second trimester, ADAM12-S may provide a feasible maternal serum maker when combined with AFP and free β-HCG. The cost-effectiveness ratio is reasonable.

  6. Non-uniform phenotyping of D12S391 resolved by second generation sequencing

    DEFF Research Database (Denmark)

    Dalsgaard, S; Rockenbauer, E; Buchard, A;

    2014-01-01

    of the electropherograms suggested that the individuals were heterozygous with two alleles that differed in size by one nucleotide. This was confirmed by amplifying the samples with the PowerPlex(®) ESX 17 system. D12S391 is a complex STR with variable numbers of AGAT and AGAC repeats. Second generation sequencing...... revealed that separation of two alleles differing by one nucleotide in length was poor if the number of AGAT repeats in the short allele was higher than in the long allele. A total of 45 individuals with microvariants or off-ladder alleles in D12S391 were sequenced. Thirty different alleles were detected...

  7. Non-uniform phenotyping of D12S391 resolved by second generation sequencing

    DEFF Research Database (Denmark)

    Dalsgaard, S; Rockenbauer, E; Buchard, A

    2014-01-01

    Non-uniform phenotyping of five case work samples were observed in the D12S391 locus. The samples were typed at least twice with the AmpFℓSTR(®) NGM SElect™ PCR Amplification Kit and different alleles were called with GeneMapper(®) ID-X in the different experiments. Detailed analyses of the elect......Non-uniform phenotyping of five case work samples were observed in the D12S391 locus. The samples were typed at least twice with the AmpFℓSTR(®) NGM SElect™ PCR Amplification Kit and different alleles were called with GeneMapper(®) ID-X in the different experiments. Detailed analyses...

  8. Characterization of a Novel 12(S)-HETE Receptor and its Role in Prostate Cancer Progression

    Science.gov (United States)

    2008-01-01

    progression (5). This eicosanoid stimulates several steps of tumor invasion and motility by inducing alterations in the cancer cell cytoskeleton (6...which fall into group A (rhodopsin-like) sub-family. Based on this information, we hypothesize that eicosanoids (such as leukotrienes, prostaglandins...inhibition abilities that various eicosanoids of [3H]-12(S)- HETE binding to the membrane fraction of CHO cells transfected with pcDNA3.1/GPR31. The

  9. Phylogenetic relationships of Salmonella based on rRNA sequences

    DEFF Research Database (Denmark)

    Christensen, H.; Nordentoft, Steen; Olsen, J.E.

    1998-01-01

    To establish the phylogenetic relationships between the subspecies of Salmonella enterica (official name Salmonella choleraesuis), Salmonella bongori and related members of Enterobacteriaceae, sequence comparison of rRNA was performed by maximum-likelihood analysis. The two Salmonella species wer...

  10. Phylogeny of higher taxa of hexapoda according to 12sRNA sequences

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The phylogenetic relationship of Hexapoda has been debated for a long time, which will be resolved mainly depending on the settlement of monophyly, affinities and interrelationships among Protura, Collembola and Diplura. Mitochondrial 12sRNA gene about 355 bp fragments from one proturan species, two collembolan species, two dipluran species and one oribatid species were sequenced. The Kimura 2-parameter distances were calculated and a series of molecular phylogenetic trees were reconstructed by using the N-J method, from which the following points were drawn: (ⅰ) Protura and Collembola compose a monophyletic group representing absent-cerci; (ⅱ) Diplura is not a monophyletic group, in which Campodeoid with filiform cerci belongs to a clade and Japygoid with pincer cerci and Ectognatha com-pose another clade, that is, Insecta s. str. stemmed from Japygoid. So it would be suggested that the phylogenetic relationship of Hexapoda is [Parainsecta (Collembola + Pro-tura) +Campodeoid +Insecta (Japygoid + Ectognatha)].

  11. Routine forensic use of the mitochondrial 12S ribosomal RNA gene for species identification.

    Science.gov (United States)

    Melton, Terry; Holland, Charity

    2007-11-01

    Since July 2004, Mitotyping Technologies has been amplifying and sequencing a approximately 150 base pair fragment of mitochondrial DNA (mtDNA) that codes for 12S ribosomal RNA, to identify the species origin of nonhuman casework samples. The approximately 100 base pair sequence product is searched at http://www.ncbi.nlm.nih.gov/BLAST and the species match is reported. The use of this assay has halved the number of samples for which no mtDNA results are obtained and is especially useful on hairs and degraded samples. The availability of species determination may aid forensic investigators in opening or closing off lines of inquiry where a highly probative but challenging sample has been collected.

  12. The level of ADAM12-S in maternal serum is an early first-trimester marker of fetal trisomy 18

    DEFF Research Database (Denmark)

    Laigaard, Jennie; Christiansen, Michael; Frohlich, Camilla

    2005-01-01

    (DS) fetus. On the basis of this finding, it was suggested that ADAM12-S might be a useful maternal serum marker of fetal chromosomal disease. OBJECTIVE: Retrospective examination of the use of ADAM12-S as a marker for fetal trisomy 18. METHOD: Serum samples were obtained from ten women during...... the first semester of their pregnancies with fetuses that had trisomy 18. An ELISA was used to determine the levels of ADAM12 in maternal serum. Results were compared to ADAM12-S levels, previously measured in the serum of 170 women carrying normal pregnancies during the first trimester. RESULTS: In all...... cases, the ADAM12-S concentration in maternal serum samples was lower in trisomy 18 pregnancies than in normal pregnancies, with a median multiple of the median (MoM) of 0.28 (p trisomy 18...

  13. A phylogeny of the Passerida (Aves:Passeriformes) based on mitochondrial 12S ribosomal RNA gene

    Institute of Scientific and Technical Information of China (English)

    Lina Wu; Yanfeng Sun; Juyong Li; Yaqing Li; Yuefeng Wu; and Dongming Li

    2015-01-01

    Background:Passerida is the largest avian radiation within the order Passeriformes. Current understanding of the high-level relationships within Passerida is based on DNA–DNA hybridizations;however, the phylogenetic relationships within this assemblage have been the subject of many debates. Methods:We analyzed the 12S ribosomal RNA gene from 49 species of Passerida, representing 14 currently recognized families, to outline the phylogenetic relationships within this group. Results:Our results identified the monophyly of the three superfamilies in Passerida:Sylvioidea, Muscicapoidea and Passeroidea. However, current delimitation of some species is at variance with our phylogeny estimate. First, the Parus major, which had been placed as a distinct clade sister to Sylvioidea was identified as a member of the super family;second, the genus Regulus was united with the Sturnidae and nested in the Muscicapoidea clade instead of being a clade of Passerida. Conclusion:Our results were consistent with Johansson’s study of the three superfamilies except for the al ocation of two families, Paridae and Regulidae.

  14. CONTRIBUTION TO THE PHYLOGENY OF THE PANGASIIDAE BASED ON MITOCHONDRIAL 12S RDNA

    Directory of Open Access Journals (Sweden)

    L. Pouyaud

    2016-10-01

    Full Text Available Catfishes are generally one of the economically important groups of fresh and brackish water fishes in the world. In many countries, they form a significant part of inland fisheries, and several species have been  introduced in fish culture. Judging from literature, the main constraint to cultivate wild species and to optimise the production of pangasiid catfishes is due to the poorly documented systematics of this family. In the present contribution, the phylogenetic relationships within Pangasiidae are studied to contribute to a better insight in their taxonomy and evolution. The genetic relatedness is inferred using mitochondrial 12S rDNA gene sequences. To resolve the phylogenetic position of Laides in this group of catfish, five genera of Asian and African Schilbeidae are also considered. The results showed that a species group (complex could be clearly seen in the genetic tree. Pangasius is more derive than the other genera. By using approximate molecular clock/evolutionary calibration from  mitochondrial gene, a new episode of  speciation for the family marked explosive radiation about 5- 8 million years ago (mya. This adaptive radiation extended until the Late Pleistocene. Regarding the relationships between the Pangasiidae and Schilbeidae, two families show an allopatric distribution with slight overlap. The Pangasiidae occur mainly in Southeast Asia, while the Schilbeidae are seen mainly on the Indian subcontinent (including Myanmar and Africa. It confirms the separation between  Schilbeidae and Pangasiidae occurred in the Early Miocene.

  15. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective

    Science.gov (United States)

    Gutell, R. R.; Larsen, N.; Woese, C. R.

    1994-01-01

    The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical

  16. Regulation of Arabidopsis thaliana 5S rRNA Genes.

    Science.gov (United States)

    Vaillant, Isabelle; Tutois, Sylvie; Cuvillier, Claudine; Schubert, Ingo; Tourmente, Sylvette

    2007-05-01

    The Arabidopsis thaliana genome comprises around 1,000 copies of 5S rRNA genes encoding both major and minor 5S rRNAs. In mature wild-type leaves, the minor 5S rRNA genes are silent. Using different mutants of DNA methyltransferases (met1, cmt3 and met1 cmt3), components of the RNAi pathway (ago4) or post-translational histone modifier (hda6/sil1), we show that the corresponding proteins are needed to maintain proper methylation patterns at heterochromatic 5S rDNA repeats. Using reverse transcription-PCR and cytological analyses, we report that a decrease of 5S rDNA methylation at CG or CNG sites in these mutants leads to the release of 5S rRNA gene silencing which occurred without detectable changes of the 5S rDNA chromatin structure. In spite of severely reduced DNA methylation, the met1 cmt3 double mutant revealed no increase in minor 5S rRNA transcripts. Furthermore, the release of silencing of minor 5S rDNAs can be achieved without increased formation of euchromatic loops by 5S rDNA, and is independent from the global heterochromatin content. Additionally, fluorescence in situ hybridization with centromeric 180 bp repeats confirmed that these highly repetitive sequences, in spite of their elevated transcriptional activity in the DNA methyltransferase mutants (met1, cmt3 and met1 cmt3), remain within chromocenters of the mutant nuclei.

  17. SAXS and other spectroscopic analysis of 12S cruciferin isolated from the seeds of Brassica nigra

    Science.gov (United States)

    Khaliq, Binish; Falke, Sven; Negm, Amr; Buck, Friedrich; Munawar, Aisha; Saqib, Maria; Mahmood, Seema; Ahmad, Malik Shoaib; Betzel, Christian; Akrem, Ahmed

    2017-06-01

    Oilseeds of the plant family Brassicaceae are important for providing both lipid and protein contents to human nutrition. Cruciferins (12S globulins) are seed storage proteins, which are getting attention due to their allergenic and pathogenicity related nature. This study describes the purification and characterization of a trimeric (∼190 kDa) cruciferin protein from the seeds of Brassica nigra (L.). Cruciferin was first partially purified by ammonium sulfate precipitation (30% saturation constant) and further purified by size exclusion chromatography. The N-terminal amino-acid sequence analysis showed 82% sequence homology with cruciferin from Arabidopsis thaliana. The 50-55 kDa monomeric cruciferin produced multiple bands of two major molecular weight ranges (α-polypeptides of 28-32 kDa and β-polypeptides of 17-20 kDa) under reduced conditions of SDS-PAGE. The 2D gel electrophoretic analysis showed the further separation of the bands into their isoforms with major pI ranges between 5.7 and 8.0 (α-polypeptides) and 5.5-8.5 (β-polypeptides). The Dynamic Light Scattering (DLS) showed the monodisperse nature of the cruciferin with hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. The Circular Dichroism (CD) spectra showed both α-helices and β-sheets in the native conformation of the trimeric protein. The pure cruciferin protein (40 mg/ml) was successfully crystallized; however, the crystals diffracted only to low resolution data (8 Å). Small-angle x-ray scattering (SAXS) was applied to gain insights into the three-dimensional structure in solution. SAXS showed that the radius of gyration is 4.24 ± 0.25 nm and confirmed the nearly globular shape. The SAXS based ab initio dummy model of B. nigra cruciferin was compared with 11S globulins (PDB ID: pdb:3KGL)

  18. Attempts on producing lymphoid cell line from Penaeus monodon by induction with SV40-T and 12S EIA oncogenes.

    Science.gov (United States)

    Puthumana, Jayesh; Prabhakaran, Priyaja; Philip, Rosamma; Singh, I S Bright

    2015-12-01

    In an attempt of in vitro transformation, transfection mediated expression of Simian virus-40 (T) antigen (SV40-T) and transduction mediated expression of Adenovirus type 12 early region 1A (12S E1A) oncogene were performed in Penaeus monodon lymphoid cells. pSV3-neo vector encoding SV40-T oncogene and a recombinant baculovirus BacP2-12S E1A-GFP encoding 12S E1A oncogene under the control of hybrid promoters were used. Electroporation and lipofection mediated transformation of SV40-T in lymphoid cells confirmed the transgene expression by phenotypic variation and the expression of GFP in co-transfection experiment. The cells transfected by lipofection (≥ 5%) survived for 14 days with lower toxicity (30%), whilst on electroporation, most of the cells succumbed to death (60%) and survived cells lived up to 7 days. Transduction efficiency in primary lymphoid cells was more than 80% within 14 days of post-transduction, however, an incubation period of 7 days post-transduction was observed without detectable expression of 12S E1A. High level of oncogenic 12S E1A expression were observed after 14 day post-transduction and the proliferating cells survived for more than 90 days with GFP expression, however, without in vitro transformation and immortalization. The study put forth the requirement of transduction mediated 'specific' oncogene expression along with telomerase activation and epigenetic induction for the immortalization and establishment of shrimp cell line.

  19. Maternal serum ADAM12s as a potential marker of trisomy 21 prior to 10 weeks of gestation.

    Science.gov (United States)

    Spencer, Kevin; Vereecken, Annie; Cowans, Nicholas J

    2008-03-01

    ADAM12s (a disintegrin and metalloprotease) is a placenta-derived glycoprotein that is involved in growth and differentiation and has been shown to be a potential first-trimester and second-trimester marker of trisomy 21 and other aneuploides. Maternal ADAM12s concentrations show a considerable temporal variation with gestational age and in the initial study levels were found to be significantly reduced in the early first trimester. Here we study the levels prior to 10 weeks of gestation to establish further the effectiveness or otherwise of ADAM12s as an early screening marker. Samples collected as part of routine first-trimester screening were retrieved from storage. In total, ten samples from singleton pregnancies with trisomy 21 were identified and were collected between the 8th and 9th weeks of gestation-of these 80% had been identified by combined first-trimester screening. A series of 62 gestational age-matched samples from singleton pregnancies collected during the same period formed the control group. ADAM12s was measured by a new DELFIA assay incorporating two monoclonals (6E6 and 8F8). Results were expressed as multiples of the median (MoM). The median MoM ADAM12s at a median gestation of 9.3 weeks was 0.61 which was significantly lower than in the controls (p = 0.011) when compared by the Mann-Whitney test. The corresponding median pregnancy associated plasma protein (PAPP-A) was 0.30 and free beta-human chorionic gonadotropin (beta-hCG) 2.02. Combining the data from this study and from the only other published study with data prior to 10 weeks suggests that ADAM12s may have the potential as an early screening marker for trisomy 21, but may not be as reduced as first thought. Copyright (c) 2008 John Wiley & Sons, Ltd.

  20. Study of gene mutation in 21 deaf patients with DNA microarray%基因芯片法检测21例耳聋患者基因突变的研究

    Institute of Scientific and Technical Information of China (English)

    韩崇旭; 任传利; 李贵玲; 汪骅; 张素华; 孙艳; 关兵

    2012-01-01

    Objective To investigate the application of DNA microarray to the screening of deafness gene mutations. Methods Twenty one deaf patients aged from 8 to 18 years were extracted for peripheral blood. DNA microarray was applied to detecting mutations of 9 hot-spots in four most common pathologic genes, namely GJB2 (35delG, 176dell6, 235delC, 299delAT), GJB3 (C538T), SLC26A4 (IVS7-2A>G, A2168G) and mitochondrial 12S rRNA (A1SS5G, C1494T). Results SLC26A4 gene mutations were detected in 8 cases (38% ), including IVS7-2A > G heterozygous mutation in 7 and IVS7-2A >G and 2168A>G heterozygous mutation in one. Four cases (19% ) carried GJB2 gene mutations, including 176 del 16 heterozygous mutation (n = 1 ) , 299 delAT heterozygous mutation ( n = 1 ) , 176 del 16 and 235 del C heterozygous mutation ( n = 1 ) , and 235 del C homozygous mutation ( n = 1 ). Conclusion DNA microarray is a sensitive and specific method for screening sequence variation in deafness gene.%目的 探讨基因芯片在耳聋基因筛查中的应用价值.方法 对扬州市聋哑学校21例8~18岁的耳聋患者中4个耳聋相关基因上的9个热点突变进行检测,包括GJB2( 35 delG、176de116、235 delC及299 delAT)、GJB3 (C538T)、SLC26A4( IVS7-2A>G、A2168G)以及线粒体12S rRNA(A1555G、C1494T).结果 SLC26 A4突变阳性率为38% (8/21),其中IVS7-2A>G杂合突变7例,IVS7-2 A>G与A2168G杂合突变1例;GJB2突变阳性率为19% (4/21),其中176de116杂合突变1例,299 delAT杂合突变1例,176 del 16与235 del C杂合突变1例,235 del C纯合突变1例.结论 基因芯片是一种筛查耳聋基因的高效、经济、简便、灵敏及特异性方法.

  1. The nucleotide sequence of 5S rRNA from a red alga, Porphyra yezoensis.

    OpenAIRE

    Takaiwa, F; Kusuda, M; Saga, N; Sugiura, M

    1982-01-01

    The nucleotide sequence of 5S rRNA from Porphyra yezoensis has been determined to be: pACGUACGGCCAUAUCCGAGACACGCGUACCGGAACCCAUUCCGAAUUCCGAAGUCAAGCGUCCGCGAGUUGGGUUAGU - AAUCUGGUGAAAGAUCACAGGCGAACCCCCAAUGCUGUACGUC. This 5S rRNA sequence is most similar to that of Euglena gracilis (63% homology).

  2. Partial analysis of the central domain of the Bacillus sphaericus JG-A12 S-layer protein

    Energy Technology Data Exchange (ETDEWEB)

    Schnorpfeil, M.; Raff, J.; Flemming, K.; Selenska-Pobell, S.

    2002-05-01

    310 amino acids from the central domain of the B. sphaericus JG-A12 S-layer were analyzed. In contrast to the N-terminal domain of this protein, which possesses a unique structure, the part studied in this work shares a significant identity with the corresponding parts of several other B. sphaericus S-layers. (orig.)

  3. An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Yih-Horng Shiao

    Full Text Available BACKGROUND: Ribosomal RNA (rRNA is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. METHODOLOGY/PRINCIPAL FINDINGS: Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately -1000 nucleotides upstream of the rRNA transcription start site (+1 and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014. During sequence analysis from -388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C in the 5' leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014. Modelling of the secondary structure of the rRNA 5'-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. CONCLUSIONS/SIGNIFICANCE: The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5' leader sequence of the primary rRNA transcript.

  4. Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli

    DEFF Research Database (Denmark)

    Triman, K; Becker, E; Dammel, C;

    1989-01-01

    Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alle...

  5. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Jørgensen, Mads Koustrup

    S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r...... to the presence of filamentous microorganisms was monitored weekly over 4 months. Microthrix was identified as a causative filament and suitable control measures were introduced. The level of Microthrix was reduced after 1-2 months but a number of other filamentous species were still present, with most of them...

  6. The rRNA evolution and procaryotic phylogeny

    Science.gov (United States)

    Fox, G. E.

    1986-01-01

    Studies of ribosomal RNA primary structure allow reconstruction of phylogenetic trees for prokaryotic organisms. Such studies reveal major dichotomy among the bacteria that separates them into eubacteria and archaebacteria. Both groupings are further segmented into several major divisions. The results obtained from 5S rRNA sequences are essentially the same as those obtained with the 16S rRNA data. In the case of Gram negative bacteria the ribosomal RNA sequencing results can also be directly compared with hybridization studies and cytochrome c sequencing studies. There is again excellent agreement among the several methods. It seems likely then that the overall picture of microbial phylogeny that is emerging from the RNA sequence studies is a good approximation of the true history of these organisms. The RNA data allow examination of the evolutionary process in a semi-quantitative way. The secondary structures of these RNAs are largely established. As a result it is possible to recognize examples of local structural evolution. Evolutionary pathways accounting for these events can be proposed and their probability can be assessed.

  7. Higher-order structure of rRNA

    Science.gov (United States)

    Gutell, R. R.; Woese, C. R.

    1986-01-01

    A comparative search for phylogenetically covarying basepair replacements within potential helices has been the only reliable method to determine the correct secondary structure of the 3 rRNAs, 5S, 16S, and 23S. The analysis of 16S from a wide phylogenetic spectrum, that includes various branches of the eubacteria, archaebacteria, eucaryotes, in addition to the mitochondria and chloroplast, is beginning to reveal the constraints on the secondary structures of these rRNAs. Based on the success of this analysis, and the assumption that higher order structure will also be phylogenetically conserved, a comparative search was initiated for positions that show co-variation not involved in secondary structure helices. From a list of potential higher order interactions within 16S rRNA, two higher-order interactions are presented. The first of these interactions involves positions 570 and 866. Based on the extent of phylogenetic covariation between these positions while maintaining Watson-Crick pairing, this higher-order interaction is considered proven. The other interaction involves a minimum of six positions between the 1400 and 1500 regions of the 16S rRNA. Although these patterns of covariation are not as striking as the 570/866 interaction, the fact that they all exist in an anti-parallel fashion and that experimental methods previously implicated these two regions of the molecule in tRNA function suggests that these interactions be given serious consideration.

  8. The rRNA evolution and procaryotic phylogeny

    Science.gov (United States)

    Fox, G. E.

    1986-01-01

    Studies of ribosomal RNA primary structure allow reconstruction of phylogenetic trees for prokaryotic organisms. Such studies reveal major dichotomy among the bacteria that separates them into eubacteria and archaebacteria. Both groupings are further segmented into several major divisions. The results obtained from 5S rRNA sequences are essentially the same as those obtained with the 16S rRNA data. In the case of Gram negative bacteria the ribosomal RNA sequencing results can also be directly compared with hybridization studies and cytochrome c sequencing studies. There is again excellent agreement among the several methods. It seems likely then that the overall picture of microbial phylogeny that is emerging from the RNA sequence studies is a good approximation of the true history of these organisms. The RNA data allow examination of the evolutionary process in a semi-quantitative way. The secondary structures of these RNAs are largely established. As a result it is possible to recognize examples of local structural evolution. Evolutionary pathways accounting for these events can be proposed and their probability can be assessed.

  9. First trimester screening for trisomy 21 in gestational week 8-10 by ADAM12-S as a maternal serum marker

    Directory of Open Access Journals (Sweden)

    Guitton Marie

    2010-10-01

    Full Text Available Abstract Background A disintegrin and metalloprotease 12 (ADAM12-S has previously been reported to be significantly reduced in maternal serum from women with fetal aneuploidy early in the first trimester and to significantly improve the quality of risk assessment for fetal trisomy 21 in prenatal screening. The aim of this study was to determine whether ADAM12-S is a useful serum marker for fetal trisomy 21 using the mixture model. Method In this case control study ADAM12-S was measured by KRYPTOR ADAM12-S immunoassay in maternal serum from gestational weeks 8 to 11 in 46 samples of fetal trisomy 21 and in 645 controls. Comparison of sensitivity and specificity of first trimester screening for fetal trisomy 21 with or without ADAM12-S included in the risk assessment using the mixture model. Results The concentration of ADAM12-S increased from week 8 to 11 and was negatively correlated with maternal weight. Log MoM ADAM12-S was positively correlated with log MoM PAPP-A (r = 0.39, P Conclusion The data show moderately decreased levels of ADAM12-S in cases of fetal aneuploidy in gestational weeks 8-11. However, including ADAM12-S in the routine risk does not improve the performance of first trimester screening for fetal trisomy 21.

  10. Phylogenetic analysis based on 28S rRNA of Babesia spp. in ruminants in China.

    Science.gov (United States)

    Gou, Huitian; Guan, Guiquan; Ma, Miling; Liu, Aihong; Liu, Zhijie; Ren, Qiaoyun; Li, Youquan; Yang, Jifei; Chen, Ze; Yin, Hong; Luo, Jianxun

    2013-04-01

    Molecular phylogenetic analyses are mainly based on the small ribosomal RNA subunit (18S rRNA), internal transcribed spacer regions, and other molecular markers. We compared the phylogenetic relationships of Babesia spp. using large subunit ribosomal RNA, i.e., 28S rRNA, and the united 28S + 18S rRNA sequence fragments from 11 isolates of Babesia spp. collected in China. Due to sequence length and variability, the 28S rRNA gene contained more information than the 18S rRNA gene and could be used to elucidate the phlyogenetic relationships of B. motasi, B. major, and B. bovis. Thus, 28S rRNA is another candidate marker that can be used for the phylogenetic analysis of Babesia spp. However, the united fragment (28S + 18S) analysis provided better supported phylogenetic relationships than single genes for Babesia spp. in China.

  11. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Science.gov (United States)

    Lucero, David E; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W; Peña, Reynaldo; Morrissey, Leslie A; Rizzo, Donna M; Stevens, Lori

    2014-12-01

    In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  12. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Directory of Open Access Journals (Sweden)

    David E Lucero

    2014-12-01

    Full Text Available In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps. Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens, five for chicken (Gallus gallus and unicolored blackbird (Agelasticus cyanopus, and one for opossum (Monodelphis domestica. Using the qPCR assay we detected chicken (13 vectors, and human (14 vectors blood meals as well as an additional blood meal source, Canis sp. (4 vectors.We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  13. First trimester screening for trisomy 21 in gestational week 8-10 by ADAM12-S as a maternal serum marker.

    Science.gov (United States)

    Tørring, Niels; Ball, Susan; Wright, Dave; Sarkissian, Gaïané; Guitton, Marie; Darbouret, Bruno

    2010-10-29

    A disintegrin and metalloprotease 12 (ADAM12-S) has previously been reported to be significantly reduced in maternal serum from women with fetal aneuploidy early in the first trimester and to significantly improve the quality of risk assessment for fetal trisomy 21 in prenatal screening. The aim of this study was to determine whether ADAM12-S is a useful serum marker for fetal trisomy 21 using the mixture model. In this case control study ADAM12-S was measured by KRYPTOR ADAM12-S immunoassay in maternal serum from gestational weeks 8 to 11 in 46 samples of fetal trisomy 21 and in 645 controls. Comparison of sensitivity and specificity of first trimester screening for fetal trisomy 21 with or without ADAM12-S included in the risk assessment using the mixture model. The concentration of ADAM12-S increased from week 8 to 11 and was negatively correlated with maternal weight. Log MoM ADAM12-S was positively correlated with log MoM PAPP-A (r = 0.39, P hCG (r = 0.21, P trisomy 21 in gestational week 8 was 0.66 increasing to approx. 0.9 MoM in week 9 and 10. The use of ADAM12-S along with biochemical markers from the combined test (PAPP-A, free beta hCG) with or without nuchal translucency measurement did not affect the detection rate or false positive rate of fetal aneuploidy as compared to routine screening using PAPP-A and free β-hCG with or without nuchal translucency. The data show moderately decreased levels of ADAM12-S in cases of fetal aneuploidy in gestational weeks 8-11. However, including ADAM12-S in the routine risk does not improve the performance of first trimester screening for fetal trisomy 21.

  14. Visual analysis of the yeast 5S rRNA gene transcriptome: regulation and role of La protein.

    Science.gov (United States)

    French, Sarah L; Osheim, Yvonne N; Schneider, David A; Sikes, Martha L; Fernandez, Cesar F; Copela, Laura A; Misra, Vikram A; Nomura, Masayasu; Wolin, Sandra L; Beyer, Ann L

    2008-07-01

    5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, approximately 132 nucleotides (nt), were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a fourfold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (presence of rapamycin or postexponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. The polymerase III elongation rate was estimated to be in the range of 60 to 75 nt/s, with a reinitiation interval of approximately 1.2 s. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on the amount or size of 5S RNA produced yet resulted in more polymerases per gene on average, consistent with a non-rate-limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.

  15. Insights into the phylogenetic positions of photosynthetic bacteria obtained from 5S rRNA and 16S rRNA sequence data

    Science.gov (United States)

    Fox, G. E.

    1985-01-01

    Comparisons of complete 16S ribosomal ribonucleic acid (rRNA) sequences established that the secondary structure of these molecules is highly conserved. Earlier work with 5S rRNA secondary structure revealed that when structural conservation exists the alignment of sequences is straightforward. The constancy of structure implies minimal functional change. Under these conditions a uniform evolutionary rate can be expected so that conditions are favorable for phylogenetic tree construction.

  16. Diversity of 23S rRNA genes within individual prokaryotic genomes.

    Directory of Open Access Journals (Sweden)

    Anna Pei

    Full Text Available BACKGROUND: The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons. METHODOLOGY/PRINCIPAL FINDINGS: Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4% genomes (mean 0.40%, range 0.01%-4.04%. Significant (1.17%-4.04% intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition. In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS, ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes. CONCLUSIONS/SIGNIFICANCE: These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy.

  17. Mechanistic study on the nuclear modifier gene MSS1 mutation suppressing neomycin sensitivity of the mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhou, Qiyin; Wang, Wei; He, Xiangyu; Zhu, Xiaoyu; Shen, Yaoyao; Yu, Zhe; Wang, Xuexiang; Qi, Xuchen; Zhang, Xuan; Fan, Mingjie; Dai, Yu; Yang, Shuxu; Yan, Qingfeng

    2014-01-01

    The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations.

  18. Characteristic archaebacterial 16S rRNA oligonucleotides

    Science.gov (United States)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  19. Defects in 18 S or 28 S rRNA processing activate the p53 pathway.

    Science.gov (United States)

    Hölzel, Michael; Orban, Mathias; Hochstatter, Julia; Rohrmoser, Michaela; Harasim, Thomas; Malamoussi, Anastassia; Kremmer, Elisabeth; Längst, Gernot; Eick, Dirk

    2010-02-26

    The p53 tumor suppressor pathway is activated by defective ribosome synthesis. Ribosomal proteins are released from the nucleolus and block human double minute-2 (Hdm2) that targets p53 for degradation. However, it remained elusive how abrogation of individual rRNA processing pathways contributes to p53 stabilization. Here, we show that selective inhibition of 18 S rRNA processing provokes accumulation of p53 as efficiently as abrogated 28 S rRNA maturation. We describe hUTP18 as a novel mammalian rRNA processing factor that is specifically involved in 18 S rRNA production. hUTP18 was essential for the cleavage of the 5'-external transcribed spacer leader sequence from the primary polymerase I transcript, but was dispensable for rRNA transcription. Because maturation of the 28 S rRNA was unaffected in hUTP18-depleted cells, our results suggest that the integrity of both the 18 S and 28 S rRNA synthesis pathways can be monitored independently by the p53 pathway. Interestingly, accumulation of p53 after hUTP18 knock down required the ribosomal protein L11. Therefore, cells survey the maturation of the small and large ribosomal subunits by separate molecular routes, which may merge in an L11-dependent signaling pathway for p53 stabilization.

  20. Fragmentary 5S rRNA gene in the human mitochondrial genome

    Energy Technology Data Exchange (ETDEWEB)

    Nierlich, D.P.

    1982-02-01

    The human mitochondrial genoma contains a 23-nucleodtide sequence that is homologous to a part of the 5S rRNA's of bacteria. This homology, the structure of the likely transcript, and the location of the sequence relative to the mitochondrial rRNA genes suggest that the sequence represents a fragmentary 5S rRNA gene.

  1. Magnetic Signatures of Quantum Critical Points of the Ferrimagnetic Mixed Spin-(1/2, S) Heisenberg Chains at Finite Temperatures

    Science.gov (United States)

    Strečka, Jozef; Verkholyak, Taras

    2016-10-01

    Magnetic properties of the ferrimagnetic mixed spin-(1/2,S) Heisenberg chains are examined using quantum Monte Carlo simulations for two different quantum spin numbers S=1 and 3/2. The calculated magnetization curves at finite temperatures are confronted with zero-temperature magnetization data obtained within the density matrix renormalization group method, which imply an existence of two quantum critical points determining a breakdown of the gapped Lieb-Mattis ferrimagnetic phase and Tomonaga-Luttinger spin-liquid phase, respectively. While a square root behavior of the magnetization accompanying each quantum critical point is gradually smoothed upon rising temperature, the susceptibility and isothermal entropy change data at low temperatures provide a stronger evidence of the zero-temperature quantum critical points through marked local maxima and minima, respectively.

  2. Binding of 12-s-12 dimeric surfactants to calf thymus DNA: Evaluation of the spacer length influence.

    Science.gov (United States)

    Sarrión, Beatriz; Bernal, Eva; Martín, Victoria Isabel; López-López, Manuel; López-Cornejo, Pilar; García-Calderón, Margarita; Moyá, María Luisa

    2016-08-01

    Several cationic dimeric surfactants have shown high affinity towards DNA. Bis-quaternary ammonium salts (m-s-m) have been the most common type of dimeric surfactants investigated and it is generally admitted that those that posses a short spacer (s≤3) show better efficiency to bind or compact DNA. However, experimental results in this work show that 12-s-12 surfactants with long spacers make the surfactant/ctDNA complexation more favorable than those with short spacers. A larger contribution of the hydrophobic interactions, which control the binding Gibbs energy, as well as a higher average charge of the surfactant molecules bound to the nucleic acid, which favors the electrostatic attractions, could explain the experimental observations. Dimeric surfactants with intermediate spacer length seem to be the less efficient for DNA binding.

  3. Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kiparisov, Sergey; Petrov, Alexey; Meskauskas, Arturas; Sergiev, Petr V; Dontsova, Olga A; Dinman, Jonathan D

    2005-10-01

    5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semi-dominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression.

  4. rRNA maturation as a "quality" control step in ribosomal subunit assembly in Dictyostelium discoideum.

    Science.gov (United States)

    Mangiarotti, G; Chiaberge, S; Bulfone, S

    1997-10-31

    In Dictyostelium discoideum, newly assembled ribosomal subunits enter polyribosomes while they still contain immature rRNA. rRNA maturation requires the engagement of the subunits in protein synthesis and leads to stabilization of their structure. Maturation of pre-17 S rRNA occurs only after the newly formed 40 S ribosomal particle has entered an 80 S ribosome and participated at least in the formation of one peptide bond or in one translocation event; maturation of pre-26 S rRNA requires the presence on the 80 S particle of a peptidyl-tRNA containing at least 6 amino acids. Newly assembled particles that cannot fulfill these requirements for structural reasons are disassembled into free immature rRNA and ribosomal proteins.

  5. Resurrection of an ancestral 5S rRNA

    Directory of Open Access Journals (Sweden)

    Fox George E

    2011-07-01

    to resurrect additional meaningful 5S rRNA ancestors as well as ancestral sequences of many different types of RNA.

  6. Resurrection of an ancestral 5S rRNA.

    Science.gov (United States)

    Lu, Qing; Fox, George E

    2011-07-22

    In addition to providing phylogenetic relationships, tree making procedures such as parsimony and maximum likelihood can make specific predictions of actual historical sequences. Resurrection of such sequences can be used to understand early events in evolution. In the case of RNA, the nature of parsimony is such that when applied to multiple RNA sequences it typically predicts ancestral sequences that satisfy the base pairing constraints associated with secondary structure. The case for such sequences being actual ancestors is greatly improved, if they can be shown to be biologically functional. A unique common ancestral sequence of 28 Vibrio 5S ribosomal RNA sequences predicted by parsimony was resurrected and found to be functional in the context of the E. coli cellular environment. The functionality of various point variants and intermediates that were constructed as part of the resurrection were examined in detail. When separately introduced the changes at single stranded positions and individual double variants at base-paired positions were also viable. An additional double variant was examined at a different base-paired position and it was also valid. The results show that at least in the case of the 5S rRNAs considered here, ancestors predicted by parsimony are likely to be realistic when the prediction is not overly influenced by single outliers. It is especially noteworthy that the phenotype of the predicted ancestors could be anticipated as a cumulative consequence of the phenotypes of the individual variants that comprised them. Thus, point mutation data is potentially useful in evaluating the reasonableness of ancestral sequences predicted by parsimony or other methods. The results also suggest that in the absence of significant tertiary structure constraints double variants that preserve pairing in stem regions will typically be accepted. Overall, the results suggest that it will be feasible to resurrect additional meaningful 5S rRNA ancestors as well

  7. Synthesis, Structure, and Electrical Properties of the Mo12 Cluster Sulfide Hg∼2.8KMo12S14 Containing Mercury Chains.

    Science.gov (United States)

    Gougeon, Patrick; Gall, Philippe

    2017-03-20

    The new compound Hg∼2.8KMo12S14 was synthesized by diffusing mercury into the metastable KMo12S14 compound at 350 °C. Its crystallographic structure, solved from single-crystal X-ray diffraction, shows that the Mo-S framework is maintained during the synthesis. It is based on Mo12S14S6 units interlinked via Mo-S bonds as in the parent compound. The mercury forms linear chains with Hg-Hg distances of about 2.72 Å in the tunnels delimited by the Mo12S14S6 units. Superconductivity was observed below 2.5 K by electrical resistivity and magnetic susceptibility measurements.

  8. Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification.

    Directory of Open Access Journals (Sweden)

    Markus Buchhaupt

    Full Text Available Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2'-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in the 18S rRNA of S.cerevisiae are methylated at 2'-O ribose sugar. Polysomes also contain a similar heterogeneity for methylated Am100, which shows that 40S ribosome subunits with and without Am100 participate in translation. Introduction of a multicopy plasmid containing the corresponding methylation guide snoRNA gene SNR51 led to an increased A100 methylation, suggesting the cellular snR51 level to limit the extent of this modification. Partial rRNA modification demonstrates a new level of ribosome heterogeneity in eukaryotic cells that might have substantial impact on regulation and fine-tuning of the translation process.

  9. Dicistronic tRNA-5S rRNA genes in Yarrowia lipolytica: an alternative TFIIIA-independent way for expression of 5S rRNA genes.

    Science.gov (United States)

    Acker, Joël; Ozanne, Christophe; Kachouri-Lafond, Rym; Gaillardin, Claude; Neuvéglise, Cécile; Marck, Christian

    2008-10-01

    In eukaryotes, genes transcribed by RNA polymerase III (Pol III) carry their own internal promoters and as such, are transcribed as individual units. Indeed, a very few cases of dicistronic Pol III genes are yet known. In contrast to other hemiascomycetes, 5S rRNA genes of Yarrowia lipolytica are not embedded into the tandemly repeated rDNA units, but appear scattered throughout the genome. We report here an unprecedented genomic organization: 48 over the 108 copies of the 5S rRNA genes are located 3' of tRNA genes. We show that these peculiar tRNA-5S rRNA dicistronic genes are expressed in vitro and in vivo as Pol III transcriptional fusions without the need of the 5S rRNA gene-specific factor TFIIIA, the deletion of which displays a viable phenotype. We also report the existence of a novel putative non-coding Pol III RNA of unknown function about 70 nucleotide-long (RUF70), the 13 genes of which are devoid of internal Pol III promoters and located 3' of the 13 copies of the tDNA-Trp (CCA). All genes embedded in the various dicistronic genes, fused 5S rRNA genes, RUF70 genes and their leader tRNA genes appear to be efficiently transcribed and their products correctly processed in vivo.

  10. Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification.

    Science.gov (United States)

    Buchhaupt, Markus; Sharma, Sunny; Kellner, Stefanie; Oswald, Stefanie; Paetzold, Melanie; Peifer, Christian; Watzinger, Peter; Schrader, Jens; Helm, Mark; Entian, Karl-Dieter

    2014-01-01

    Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2'-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in the 18S rRNA of S.cerevisiae are methylated at 2'-O ribose sugar. Polysomes also contain a similar heterogeneity for methylated Am100, which shows that 40S ribosome subunits with and without Am100 participate in translation. Introduction of a multicopy plasmid containing the corresponding methylation guide snoRNA gene SNR51 led to an increased A100 methylation, suggesting the cellular snR51 level to limit the extent of this modification. Partial rRNA modification demonstrates a new level of ribosome heterogeneity in eukaryotic cells that might have substantial impact on regulation and fine-tuning of the translation process.

  11. 18S rRNA degradation is not accompanied by altered rRNA transport at early times following irradiation of HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuchs, P.; Krolak, J.M.; McClain, D.; Minton, K.W.

    1990-01-01

    In recent investigations on the effects of radiation on rRNA processing in HeLa S3 cells, the authors pulse-labeled the cells with uridine immediately prior to irradiation. The 45 S rRNA precursor, which undergoes nuclear processing to form one each of its major daughter species, 28S and 18S rRNA, was separated from the daughter species by gel electrophoresis and the radiolabel in each species determined at various times after irradiation. By pulse-labeling the cells prior to irradiation, superimposed effects caused by radiation-induced alterations of rRNA transcription and Refs. therein were minimized, permitting selective analysis of the processing of that fraction of 45S precursor that had been synthesized (radiolabeled) predominantly prior to irradiation. They now report more detailed studies on 45S rRNA processing within the first 2 h following irradiation in which they have found a maximum 28 S:18 S ratio of 2:1 that is observed about 1 h following irradiation of 5 or 10 Gy.

  12. Epigenetic Programming of the rRNA Promoter by MBD3

    OpenAIRE

    2008-01-01

    Within the human genome there are hundreds of copies of the rRNA gene, but only a fraction of these genes are active. Silencing through epigenetics has been extensively studied; however, it is essential to understand how active rRNA genes are maintained. Here, we propose a role for the methyl-CpG binding domain protein MBD3 in epigenetically maintaining active rRNA promoters. We show that MBD3 is localized to the nucleolus, colocalizes with upstream binding factor, and binds to unmethylated r...

  13. Evolutionary dynamics of rRNA gene clusters in cichlid fish

    Directory of Open Access Journals (Sweden)

    Nakajima Rafael T

    2012-10-01

    Full Text Available Abstract Background Among multigene families, ribosomal RNA (rRNA genes are the most frequently studied and have been explored as cytogenetic markers to study the evolutionary history of karyotypes among animals and plants. In this report, we applied cytogenetic and genomic methods to investigate the organization of rRNA genes among cichlid fishes. Cichlids are a group of fishes that are of increasing scientific interest due to their rapid and convergent adaptive radiation, which has led to extensive ecological diversity. Results The present paper reports the cytogenetic mapping of the 5S rRNA genes from 18 South American, 22 African and one Asian species and the 18S rRNA genes from 3 African species. The data obtained were comparatively analyzed with previously published information related to the mapping of rRNA genes in cichlids. The number of 5S rRNA clusters per diploid genome ranged from 2 to 15, with the most common pattern being the presence of 2 chromosomes bearing a 5S rDNA cluster. Regarding 18S rDNA mapping, the number of sites ranged from 2 to 6, with the most common pattern being the presence of 2 sites per diploid genome. Furthermore, searching the Oreochromis niloticus genome database led to the identification of a total of 59 copies of 5S rRNA and 38 copies of 18S rRNA genes that were distributed in several genomic scaffolds. The rRNA genes were frequently flanked by transposable elements (TEs and spread throughout the genome, complementing the FISH analysis that detect only clustered copies of rRNA genes. Conclusions The organization of rRNA gene clusters seems to reflect their intense and particular evolutionary pathway and not the evolutionary history of the associated taxa. The possible role of TEs as one source of rRNA gene movement, that could generates the spreading of ribosomal clusters/copies, is discussed. The present paper reinforces the notion that the integration of cytogenetic data and genomic analysis provides a

  14. Comparative structural analysis of cytoplasmic and chloroplastic 5S rRNA from spinach.

    OpenAIRE

    Pieler, T; Digweed, M; Bartsch, M; Erdmann, V A

    1983-01-01

    5S rRNAs from Spinacea oleracea cytoplasmic and chloroplastic ribosomes have been subjected to digestion with the single strand specific nuclease S1 and to chemical modification of cytidines by sodium bisulphite in order to probe the RNA structure. According to these data, cytoplasmic 5S rRNA can be folded as proposed in the general eukaryotic 5S rRNA structure (1) and 5S rRNA from chloroplastides is shown to be more related to the general eubacterial structure (2).

  15. Evolutionary dynamics of rRNA gene clusters in cichlid fish

    Science.gov (United States)

    2012-01-01

    Background Among multigene families, ribosomal RNA (rRNA) genes are the most frequently studied and have been explored as cytogenetic markers to study the evolutionary history of karyotypes among animals and plants. In this report, we applied cytogenetic and genomic methods to investigate the organization of rRNA genes among cichlid fishes. Cichlids are a group of fishes that are of increasing scientific interest due to their rapid and convergent adaptive radiation, which has led to extensive ecological diversity. Results The present paper reports the cytogenetic mapping of the 5S rRNA genes from 18 South American, 22 African and one Asian species and the 18S rRNA genes from 3 African species. The data obtained were comparatively analyzed with previously published information related to the mapping of rRNA genes in cichlids. The number of 5S rRNA clusters per diploid genome ranged from 2 to 15, with the most common pattern being the presence of 2 chromosomes bearing a 5S rDNA cluster. Regarding 18S rDNA mapping, the number of sites ranged from 2 to 6, with the most common pattern being the presence of 2 sites per diploid genome. Furthermore, searching the Oreochromis niloticus genome database led to the identification of a total of 59 copies of 5S rRNA and 38 copies of 18S rRNA genes that were distributed in several genomic scaffolds. The rRNA genes were frequently flanked by transposable elements (TEs) and spread throughout the genome, complementing the FISH analysis that detect only clustered copies of rRNA genes. Conclusions The organization of rRNA gene clusters seems to reflect their intense and particular evolutionary pathway and not the evolutionary history of the associated taxa. The possible role of TEs as one source of rRNA gene movement, that could generates the spreading of ribosomal clusters/copies, is discussed. The present paper reinforces the notion that the integration of cytogenetic data and genomic analysis provides a more complete picture for

  16. Strain identification and 5S rRNA gene characterization of the hyperthermophilic archaebacterium Sulfolobus acidocaldarius.

    OpenAIRE

    Durovic, P; Kutay, U.; Schleper, C.; Dennis, P. P.

    1994-01-01

    A commonly used laboratory Sulfolobus strain has been unambiguously identified as Sulfolobus acidocaldarius DSM639. The 5S rRNA gene from this strain was cloned and sequenced. It differs at 17 of 124 positions from the identical 5S rRNA sequences from Sulfolobus solfataricus and a strain apparently misidentified as S. acidocaldarius. Analysis of the transcripts from the 5S rRNA gene failed to identify any precursor extending a significant distance beyond the 5' or 3' boundary of the 5S rRNA-c...

  17. Identification and chromosomal distribution of 5S rRNA genes in Neurospora crassa.

    OpenAIRE

    Metzenberg, R L; Stevens, J N; Selker, E U; Morzycka-Wroblewska, E

    1985-01-01

    The 5S rRNA genes of Neurospora crassa, unlike those of most organisms, are not tandemly arranged, and they are found imbedded in a variety of unique sequences. The 5S rRNA regions of most of the genes are of one type, alpha; however, several other "isotypes" (beta, gamma, delta, zeta, and eta) are also found. We asked whether Neurospora 5S rRNA genes are dispersed on a chromosomal scale and whether genes of different isotypes are spatially segregated. We identified, by DNA sequencing, 5S rRN...

  18. Strain identification and 5S rRNA gene characterization of the hyperthermophilic archaebacterium Sulfolobus acidocaldarius.

    OpenAIRE

    Durovic, P; Kutay, U.; Schleper, C; Dennis, P P

    1994-01-01

    A commonly used laboratory Sulfolobus strain has been unambiguously identified as Sulfolobus acidocaldarius DSM639. The 5S rRNA gene from this strain was cloned and sequenced. It differs at 17 of 124 positions from the identical 5S rRNA sequences from Sulfolobus solfataricus and a strain apparently misidentified as S. acidocaldarius. Analysis of the transcripts from the 5S rRNA gene failed to identify any precursor extending a significant distance beyond the 5' or 3' boundary of the 5S rRNA-c...

  19. A phylogeny of the Passerida(Aves: Passeriformes)based on mitochondrial 12S ribosomal RNA gene

    Institute of Scientific and Technical Information of China (English)

    Lina; Wu; Yanfeng; Sun; Juyong; Li; Yaqing; Li; Yuefeng; Wu; Dongming; Li

    2015-01-01

    Background: Passerida is the largest avian radiation within the order Passeriformes. Current understanding of the high-level relationships within Passerida is based on DNA–DNA hybridizations; however, the phylogenetic relationships within this assemblage have been the subject of many debates.Methods: We analyzed the 12 S ribosomal RNA gene from 49 species of Passerida, representing 14 currently recognized families, to outline the phylogenetic relationships within this group.Results: Our results identified the monophyly of the three superfamilies in Passerida: Sylvioidea, Muscicapoidea and Passeroidea. However, current delimitation of some species is at variance with our phylogeny estimate. First, the Parus major, which had been placed as a distinct clade sister to Sylvioidea was identified as a member of the super family;second, the genus Regulus was united with the Sturnidae and nested in the Muscicapoidea clade instead of being a clade of Passerida.Conclusion: Our results were consistent with Johansson’s study of the three superfamilies except for the al ocation of two families, Paridae and Regulidae.

  20. EXTraS discovery of an 1.2-s X-ray pulsar in M 31

    CERN Document Server

    Esposito, P; Belfiore, A; Novara, G; Sidoli, L; Castillo, G A Rodríguez; De Luca, A; Tiengo, A; Haberl, F; Salvaterra, R; Read, A M; Salvetti, D; Sandrelli, S; Marelli, M; Wilms, J; D'Agostino, D

    2015-01-01

    During a search for coherent signals in the X-ray archival data of XMM-Newton, we discovered a modulation at 1.2 s in 3XMM J004301.4+413017 (3X J0043), a source lying in the direction of an external arm of M 31. This short period indicates a neutron star (NS). Between 2000 and 2013, the position of 3X J0043 was imaged by public XMM-Newton observations 35 times. The analysis of these data allowed us to detect an orbital modulation at 1.27 d and study the long-term properties of the source. The emission of the pulsar was rather hard (most spectra are described by a power law with $\\Gamma < 1$) and, assuming the distance to M 31, the 0.3-10 keV luminosity was variable, from $\\sim$$3\\times10^{37}$ to $2\\times10^{38}$ erg s$^{-1}$. The analysis of optical data shows that, while 3X J0043 is likely associated to a globular cluster in M 31, a counterpart with $V\\gtrsim22$ outside the cluster cannot be excluded. Considering our findings, there are two main viable scenarios for 3X J0043: a peculiar low-mass X-ray bi...

  1. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group.

    Energy Technology Data Exchange (ETDEWEB)

    Bayvkin, S. G.; Lysov, Y. P.; Zakhariev, V.; Kelly, J. J.; Jackman, J.; Stahl, D. A.; Cherni, A.; Engelhardt Inst. of Molecular Biology; Loyola Univ.; Johns Hopkins Univ.; Univ. of Washington

    2004-08-01

    In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.

  2. YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

    DEFF Research Database (Denmark)

    Andersen, Niels Møller; Douthwaite, Stephen

    2006-01-01

    The rRNAs in Escherichia coli contain methylations at 24 nucleotides, which collectively are important for ribosome function. Three of these methylations are m5C modifications located at nucleotides C967 and C1407 in 16S rRNA and at nucleotide C1962 in 23S rRNA. Bacterial rRNA modifications gener...... methyltransferase gene rsmF, and that the nomenclature system be extended to include the rRNA methyltransferases that still await identification....

  3. 12S rRNA基因序列探讨中国10种壁虎的系统关系%PHYLOGENY OF TEN SPECIES OF CHINESE GEKKONID LIZARDS (GEKKONIDAE: LACERTILIA) INFERRED FROM 12 S rDAN DNA SEQUENCES

    Institute of Scientific and Technical Information of China (English)

    韩德民; 周开亚; 王义权

    2001-01-01

    对肌肉样品采用SDS/蛋白酶K裂解、酚-氯仿抽提和乙醇沉淀提取总DNA,用线粒体12S rDNA通用引物扩增,用银染测序和ABI Prism 310型遗传分析仪测定了Gekko japonicus, G. swinhonis, G. hokouensis, G. gecko, Cyrtopodion elongatus, C. russowi, Teratoscincus roborowskii, Hemidactylus bowringii, H. frenatus和Gehyra mutilata 10种壁虎的线粒体12S rRNA基因片段的序列。对位排列后的序列长421 bp,含201个变异位点。属间核苷酸变异范围为0.228~0.282,属内是0.005~0.263。重建的分子系统树将10种壁虎聚为三支:第1支是大壁虎和截趾虎;第2支由壁虎属其余的3个种构成一个单系;第三支由其余3个属的5个种组成。研究结果表明:截趾虎与壁虎属4物种亲缘关系较近;吐鲁番沙虎和弯脚虎属2物种、蜥虎属2物种之间的亲缘关系较近,支持Russell(1976)关于弯脚虎属与蜥虎属间进化关系的研究结果而不支持Kluge(1987)将沙虎属另立亚科的系统发生假说;铅山壁虎与无蹼壁虎最先聚为姐妹群,再与多疣壁虎相聚组成一个单系。%The studies on phylogenetic relationships of gekkonid lizards have been largely based on morphological characters before, including the structure of digits and internal anatomy. In China, the phylogeny among some gekkonid lizards based on karyotype, nucleolar organizer and LDH (lactic dehydrogenase) isozyme was r e ported. However, nucleotide sequence data have not yet been employed to study th e phylogeny of gekkonid lizards. In the present study, phylogenetic relationship among 10 Chinese gekkonid lizard species was inferred based on sequences of 1 2S rRNA gene fragment.  1. Material and method   Total DNA was extracted from about 50 mg of the muscle tissue from 10 gekkonid lizards (Gekko japonicus, G. swinhonis, G. hokouensis, G. gecko, Cyrtopodion elongatus, C. russowi, Teratoscincus roborowskii, Hemidactylus bowringii, H. frenatus and

  4. Development of a dual-internal-reference technique to improve accuracy when determining bacterial 16S rRNA:16S rRNA gene ratio with application to Escherichia coli liquid and aerosol samples.

    Science.gov (United States)

    Zhen, Huajun; Krumins, Valdis; Fennell, Donna E; Mainelis, Gediminas

    2015-10-01

    Accurate enumeration of rRNA content in microbial cells, e.g. by using the 16S rRNA:16S rRNA gene ratio, is critical to properly understand its relationship to microbial activities. However, few studies have considered possible methodological artifacts that may contribute to the variability of rRNA analysis results. In this study, a technique utilizing genomic DNA and 16S rRNA from an exogenous species (Pseudomonas fluorescens) as dual internal references was developed to improve accuracy when determining the 16S rRNA:16S rRNA gene ratio of a target organism, Escherichia coli. This technique was able to adequately control the variability in sample processing and analysis procedures due to nucleic acid (DNA and RNA) losses, inefficient reverse transcription of RNA, and inefficient PCR amplification. The measured 16S rRNA:16S rRNA gene ratio of E. coli increased by 2-3 fold when E. coli 16S rRNA gene and 16S rRNA quantities were normalized to the sample-specific fractional recoveries of reference (P. fluorescens) 16S rRNA gene and 16S rRNA, respectively. In addition, the intra-sample variation of this ratio, represented by coefficients of variation from replicate samples, decreased significantly after normalization. This technique was applied to investigate the temporal variation of 16S rRNA:16S rRNA gene ratio of E. coli during its non-steady-state growth in a complex liquid medium, and to E. coli aerosols when exposed to particle-free air after their collection on a filter. The 16S rRNA:16S rRNA gene ratio of E. coli increased significantly during its early exponential phase of growth; when E. coli aerosols were exposed to extended filtration stress after sample collection, the ratio also increased. In contrast, no significant temporal trend in E. coli 16S rRNA:16S rRNA gene ratio was observed when the determined ratios were not normalized based on the recoveries of dual references. The developed technique could be widely applied in studies of relationship between

  5. Diversity of 5S rRNA genes within individual prokaryotic genomes.

    Science.gov (United States)

    Pei, Anna; Li, Hongru; Oberdorf, William E; Alekseyenko, Alexander V; Parsons, Tamasha; Yang, Liying; Gerz, Erika A; Lee, Peng; Xiang, Charlie; Nossa, Carlos W; Pei, Zhiheng

    2012-10-01

    We examined intragenomic variation of paralogous 5S rRNA genes to evaluate the concept of ribosomal constraints. In a dataset containing 1161 genomes from 779 unique species, 96 species exhibited > 3% diversity. Twenty-seven species with > 10% diversity contained a total of 421 mismatches between all pairs of the most dissimilar copies of 5S rRNA genes. The large majority (401 of 421) of the diversified positions were conserved at the secondary structure level. The high diversity was associated with partial rRNA operon, split operon, or spacer length-related divergence. In total, these findings indicated that there are tight ribosomal constraints on paralogous 5S rRNA genes in a genome despite of the high degree of diversity at the primary structure level. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. An Archaea 5S rRNA analog is stably expressed in Escherichia coli

    Science.gov (United States)

    Yang, Y.; Fox, G. E.

    1996-01-01

    Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

  7. Dinoflagellate 17S rRNA sequence inferred from the gene sequence: Evolutionary implications

    Science.gov (United States)

    Herzog, Michel; Maroteaux, Luc

    1986-01-01

    We present the complete sequence of the nuclear-encoded small-ribosomal-subunit RNA inferred from the cloned gene sequence of the dinoflagellate Prorocentrum micans. The dinoflagellate 17S rRNA sequence of 1798 nucleotides is contained in a family of 200 tandemly repeated genes per haploid genome. A tentative model of the secondary structure of P. micans 17S rRNA is presented. This sequence is compared with the small-ribosomal-subunit rRNA of Xenopus laevis (Animalia), Saccharomyces cerevisiae (Fungi), Zea mays (Planta), Dictyostelium discoideum (Protoctista), and Halobacterium volcanii (Monera). Although the secondary structure of the dinoflagellate 17S rRNA presents most of the eukaryotic characteristics, it contains sufficient archaeobacterial-like structural features to reinforce the view that dinoflagellates branch off very early from the eukaryotic lineage. PMID:16578795

  8. Inhibition of Escherichia coli precursor-16S rRNA processing by mouse intestinal contents

    DEFF Research Database (Denmark)

    Licht, Tine Rask; Tolker-Nielsen, Tim; Holmstrøm, Kim;

    1999-01-01

    . We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coli cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial......The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems...... growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coli growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher...

  9. Detection of Babesia microti parasites by highly sensitive 18S rRNA reverse transcription PCR.

    Science.gov (United States)

    Hanron, Amelia E; Billman, Zachary P; Seilie, Annette M; Chang, Ming; Murphy, Sean C

    2017-03-01

    Babesia are increasingly appreciated as a cause of transfusion-transmitted infection. Sensitive methods are needed to screen blood products. We report herein that B. microti 18S rRNA is over 1,000-fold more abundant than its coding genes, making reverse transcription PCR (RT-PCR) much more sensitive than PCR. Babesia 18S rRNA may be useful for screening the blood supply.

  10. Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

    Science.gov (United States)

    Hotto, Amber M.; Castandet, Benoît; Gilet, Laetitia; Higdon, Andrea; Condon, Ciarán; Stern, David B.

    2015-01-01

    RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3′ end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3′ extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation. PMID:25724636

  11. Low abundant spacer 5S rRNA transcripts are frequently polyadenylated in Nicotiana.

    Science.gov (United States)

    Fulnecek, Jaroslav; Kovarik, Ales

    2007-11-01

    In plants, 5S rRNA genes (5S rDNA) encoding 120-nt structural RNA molecules of ribosomes are organized in tandem arrays comprising thousands of units. Failure to correctly terminate transcription would generate longer inaccurately processed transcripts interfering with ribosome biogenesis. Hence multiple termination signals occur immediately after the 5S rRNA coding sequence. To obtain information about the efficiency of termination of 5S rDNA transcription in plants we analyzed 5S rRNA pools in three Nicotiana species, N. sylvestris, N. tomentosiformis and N. tabacum. In addition to highly abundant 120-nt 5S rRNA transcripts, we also detected RNA species composed of a genic region and variable lengths of intergenic sequences. These genic-intergenic RNA molecules occur at a frequency severalfold lower than the mature 120-nt transcripts, and are posttranscriptionally modified by polyadenylation at their 3' end in contrast to 120-nt transcripts. An absence of 5S small RNAs (smRNA) argue against a dominant role for the smRNA biosynthesis pathway in the degradation of aberrant 5S rRNA in Nicotiana. This work is the first description of polyadenylated 5S rRNA species in higher eukaryotes originating from a read-through transcription into the intergenic spacer. We propose that polyadenylation may function in a "quality control" pathway ensuring that only correctly processed molecules enter the ribosome biogenesis.

  12. rRNA Pseudogenes in Filamentous Ascomycetes as Revealed by Genome Data

    Directory of Open Access Journals (Sweden)

    Yi Li

    2017-08-01

    Full Text Available The nuclear ribosomal DNA (rDNA is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP mutation was probably responsible for the formation of those rRNA pseudogenes.

  13. Phylogenetic analysis of Pomacea canaliculata isolates collected from rice fields in different origins of China by combined mitochondrial 12S and 16S genes.

    Science.gov (United States)

    Li, Xiao-Yan; Bian, Qing-Qing; Zhao, Guang-Hui

    2015-02-01

    To study the genetic relationships of Pomacea canaliculata collected from rice fields in China, the mitochondrial (mt) 12S and 16S of 9 P. canaliculata isolates from 5 southern provinces in China were sequenced and analyzed. The intra-specific sequence variations of P. canaliculata were 0-1.1% for 12S and 0--0.6% for 16S, while the inter-specific variations among common Pomacea species in mt 12S and 16S were 3.0-11.7% and 2.3-10.1%, respectively. Phylogenetic analysis based on combined sequences of mt 12S and 16S revealed complex genetic structure of P. canaliculata in China. Two phylogenetic groups of P. canaliculata were indicated in China with one group sistered to P. canaliculata isolates from USA, and two groups were even found in the same province. The phylogenetic relationships of Pomacea spp. also could be effectively inferred by combined sequences of mt 12S and 16S. These findings provided basic information for further study of population genetics and diffusion pattern of P. canaliculata in China as well as in the world.

  14. Na2.9KMo12S14: a novel quaternary reduced molybdenum sulfide containing Mo12 clusters with a channel structure

    Directory of Open Access Journals (Sweden)

    Patrick Gougeon

    2013-06-01

    Full Text Available The crystal structure of trisodium potassium dodecamolybdenum tetradecasulfide, Na2.9 (2KMo12S14, consists of Mo12S14S6 cluster units interconnected through interunit Mo—S bonds and delimiting channels in which the Na+ cations are disordered. The cluster units are centered at Wyckoff positions 2d and have point-group symmetry 3.2. The K atom lies on sites with 3.2 symmetry (Wyckoff site 2c between two consecutive Mo12S14S6 units. One of the three independent S atoms and one Na atom lie on sites with 3.. symmetry (Wyckoff sites 4e and 4f. The other Na atom occupies a 2b position with -3.. symmetry. The crystal studied was a merohedral twin with refined components of 0.4951 (13 and 0.5049 (13.

  15. Uncultivated microbial eukaryotic diversity: a method to link ssu rRNA gene sequences with morphology.

    Directory of Open Access Journals (Sweden)

    Marissa B Hirst

    Full Text Available Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA "phylotypes" from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of

  16. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    Science.gov (United States)

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.

  17. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    Directory of Open Access Journals (Sweden)

    Huijing Hao

    Full Text Available API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.

  18. Rapid in vivo exploration of a 5S rRNA neutral network.

    Science.gov (United States)

    Zhang, Zhengdong D; Nayar, Madhavi; Ammons, David; Rampersad, Joanne; Fox, George E

    2009-02-01

    A partial knockout compensation method to screen 5S ribosomal RNA sequence variants in vivo is described. The system utilizes an Escherichia coli strain in which five of eight genomic 5S rRNA genes were deleted in conjunction with a plasmid which is compensatory when carrying a functionally active 5S rRNA. The partial knockout strain is transformed with a population of potentially compensatory plasmids each carrying a randomly generated 5S rRNA gene variant. a The ability to compensate the slow growth rate of the knockout strain is used in conjunction with sequencing to rapidly identify variant 5S rRNAs that are functional as well as those that likely are not. The assay is validated by showing that the growth rate of 15 variants separately expressed in the partial knockout strain can be accurately correlated with in vivo assessments of the potential validity of the same variants. A region of 5S rRNA was mutagenized with this approach and nine novel variants were recovered and characterized. Unlike a complete knockout system, the method allows recovery of both deleterious and functional variants.. The method can be used to study variants of any 5S rRNA in the E. coli context including those of E. coli.

  19. A critical role for noncoding 5S rRNA in regulating Mdmx stability.

    Science.gov (United States)

    Li, Muyang; Gu, Wei

    2011-09-16

    Both p53 and Mdmx are ubiquitinated and degraded by the same E3 ligase Mdm2; interestingly, however, while p53 is rapidly degraded by Mdm2, Mdmx is a stable protein in most cancer cells. Thus, the mechanism by which Mdmx is degraded by Mdm2 needs further elucidation. Here, we identified the noncoding 5S rRNA as a major component of Mdmx-associated complexes from human cells. We show that 5S rRNA acts as a natural inhibitor of Mdmx degradation by Mdm2. RNAi-mediated knockdown of endogenous 5S rRNA, while not affecting p53 levels, significantly induces Mdmx degradation and, subsequently, activates p53-dependent growth arrest. Notably, 5S rRNA binds the RING domain of Mdmx and blocks its ubiquitination by Mdm2, whereas Mdm2-mediated p53 ubiquitination remains intact. These results provide insights into the differential effects on p53 and Mdmx by Mdm2 in vivo and reveal a critical role for noncoding 5S rRNA in modulating the p53-Mdmx axis. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA.

    Science.gov (United States)

    Fu, Yan; Bannach, Oliver; Chen, Hao; Teune, Jan-Hendrik; Schmitz, Axel; Steger, Gerhard; Xiong, Liming; Barbazuk, W Brad

    2009-05-01

    Identifying conserved alternative splicing (AS) events among evolutionarily distant species can prioritize AS events for functional characterization and help uncover relevant cis- and trans-regulatory factors. A genome-wide search for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNA-derived exon in TFIIIA gene exists in all representative land plant species but not in green algae and nonplant species, suggesting it is specific to land plants. TFIIIA is essential for RNA polymerase III-based transcription of 5S rRNA in eukaryotes. Integrating comparative genomics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense-mediated mRNA decay. Utilizing multiple independent Arabidopsis overexpressing TFIIIA transgenic lines under osmotic and salt stress, strong accordance between phenotypic and molecular evidence reveals the biological relevance of AS of the exonized 5S rRNA in quantitative autoregulation of TFIIIA homeostasis. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized noncoding element.

  1. 5S rRNA gene arrangements in protists: a case of nonadaptive evolution.

    Science.gov (United States)

    Drouin, Guy; Tsang, Corey

    2012-06-01

    Given their high copy number and high level of expression, one might expect that both the sequence and organization of eukaryotic ribosomal RNA genes would be conserved during evolution. Although the organization of 18S, 5.8S and 28S ribosomal RNA genes is indeed relatively well conserved, that of 5S rRNA genes is much more variable. Here, we review the different types of 5S rRNA gene arrangements which have been observed in protists. This includes linkages to the other ribosomal RNA genes as well as linkages to ubiquitin, splice-leader, snRNA and tRNA genes. Mapping these linkages to independently derived phylogenies shows that these diverse linkages have repeatedly been gained and lost during evolution. This argues against such linkages being the primitive condition not only in protists but also in other eukaryote species. Because the only characteristic the diverse genes with which 5S rRNA genes are found linked with is that they are tandemly repeated, these arrangements are unlikely to provide any selective advantage. Rather, the observed high variability in 5S rRNA genes arrangements is likely the result of the fact that 5S rRNA genes contain internal promoters, that these genes are often transposed by diverse recombination mechanisms and that these new gene arrangements are rapidly homogenized by unequal crossingovers and/or by gene conversions events in species with short generation times and frequent founder events.

  2. Novel essential gene Involved in 16S rRNA processing in Escherichia coli.

    Science.gov (United States)

    Kurata, Tatsuaki; Nakanishi, Shinobu; Hashimoto, Masayuki; Taoka, Masato; Yamazaki, Yukiko; Isobe, Toshiaki; Kato, Jun-ichi

    2015-02-27

    Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three-dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase-H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgF(ts)) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5' end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgF(ts) mutant cells showed that the expression of genes containing multiple Shine-Dalgarno-like sequences was observed to be lower than in wild type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation.

  3. Deep sequencing of subseafloor eukaryotic rRNA reveals active Fungi across marine subsurface provinces.

    Directory of Open Access Journals (Sweden)

    William Orsi

    Full Text Available The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC, nitrate, sulfide, and dissolved inorganic carbon (DIC. These correlations are supported by terminal restriction length polymorphism (TRFLP analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.

  4. Decreases in average bacterial community rRNA operon copy number during succession.

    Science.gov (United States)

    Nemergut, Diana R; Knelman, Joseph E; Ferrenberg, Scott; Bilinski, Teresa; Melbourne, Brett; Jiang, Lin; Violle, Cyrille; Darcy, John L; Prest, Tiffany; Schmidt, Steven K; Townsend, Alan R

    2016-05-01

    Trait-based studies can help clarify the mechanisms driving patterns of microbial community assembly and coexistence. Here, we use a trait-based approach to explore the importance of rRNA operon copy number in microbial succession, building on prior evidence that organisms with higher copy numbers respond more rapidly to nutrient inputs. We set flasks of heterotrophic media into the environment and examined bacterial community assembly at seven time points. Communities were arrayed along a geographic gradient to introduce stochasticity via dispersal processes and were analyzed using 16 S rRNA gene pyrosequencing, and rRNA operon copy number was modeled using ancestral trait reconstruction. We found that taxonomic composition was similar between communities at the beginning of the experiment and then diverged through time; as well, phylogenetic clustering within communities decreased over time. The average rRNA operon copy number decreased over the experiment, and variance in rRNA operon copy number was lowest both early and late in succession. We then analyzed bacterial community data from other soil and sediment primary and secondary successional sequences from three markedly different ecosystem types. Our results demonstrate that decreases in average copy number are a consistent feature of communities across various drivers of ecological succession. Importantly, our work supports the scaling of the copy number trait over multiple levels of biological organization, ranging from cells to populations and communities, with implications for both microbial ecology and evolution.

  5. Mitochondrial 12S Ribosomal RNA A1555G Mutation Associated with Cardiomyopathy and Hearing Loss following High-Dose Chemotherapy and Repeated Aminoglycoside Exposure

    DEFF Research Database (Denmark)

    Skou, Anne-Sofie; Tranebjærg, Lisbeth; Jensen, Tim

    2014-01-01

    A 19-month-old girl with the A1555G mitochondrial mutation in the 12S ribosomal RNA gene and acute myelogenous leukemia developed dilated cardiomyopathy and bilateral sensorineural hearing loss before undergoing allogeneic stem cell transplantation. She had received gentamicin during episodes...

  6. Phylogenetic relationships of the Gomphales based on nuc-25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined sequences

    Science.gov (United States)

    Admir J. Giachini; Kentaro Hosaka; Eduardo Nouhra; Joseph Spatafora; James M. Trappe

    2010-01-01

    Phylogenetic relationships among Geastrales, Gomphales, Hysterangiales, and Phallales were estimated via combined sequences: nuclear large subunit ribosomal DNA (nuc-25S-rDNA), mitochondrial small subunit ribosomal DNA (mit-12S-rDNA), and mitochondrial atp6 DNA (mit-atp6-DNA). Eighty-one taxa comprising 19 genera and 58 species...

  7. Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.

  8. Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA

    DEFF Research Database (Denmark)

    Ostergaard, P; Phan, H; Johansen, L B

    1998-01-01

    The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one prim...... approach was used to map the putative binding regions on domain V of protein L9 and the 5 S RNA-L5-L18 complex.......The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one......+VI. This indicates that there are two major protein assembly centres located at the ends of the 23 S rRNA, which is consistent with an earlier view that in vitro protein assembly nucleates around proteins L24 and L3. Although similar protein assembly patterns were observed over a range of temperature and magnesium...

  9. Specific features of 5S rRNA structure - its interactions with macromolecules and possible functions.

    Science.gov (United States)

    Smirnov, A V; Entelis, N S; Krasheninnikov, I A; Martin, R; Tarassov, I A

    2008-12-01

    Small non-coding RNAs are today a topic of great interest for molecular biologists because they can be regarded as relicts of a hypothetical "RNA world" which, apparently, preceded the modern stage of organic evolution on Earth. The small molecule of 5S rRNA (approximately 120 nucleotides) is a component of large ribosomal subunits of all living beings (5S rRNAs are not found only in mitoribosomes of fungi and metazoans). This molecule interacts with various protein factors and 23S (28S) rRNA. This review contains the accumulated data to date concerning 5S rRNA structure, interactions with other biological macromolecules, intracellular traffic, and functions in the cell.

  10. Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S rRNA Gene.

    Science.gov (United States)

    Tian, Ren-Mao; Cai, Lin; Zhang, Wei-Peng; Cao, Hui-Luo; Qian, Pei-Yuan

    2015-07-27

    Horizontal gene transfer (HGT) of operational genes has been widely reported in prokaryotic organisms. However, informational genes such as those involved in transcription and translation processes are very difficult to be horizontally transferred, as described by Woese's complexity hypothesis. Here, we analyzed all of the completed prokaryotic genome sequences (2,143 genomes) in the NCBI (National Center for Biotechnology Information) database, scanned for genomes with high intragenomic heterogeneity of 16S rRNA gene copies, and explored potential HGT events of ribosomal RNA genes based on the phylogeny, genomic organization, and secondary structures of the ribosomal RNA genes. Our results revealed 28 genomes with relatively high intragenomic heterogeneity of multiple 16S rRNA gene copies (lowest pairwise identity 16S rRNA gene only occurred at intragenus or intraspecies levels, which is quite different from the HGT of operational genes. Our results improve our understanding regarding the exchange of informational genes.

  11. Recognition determinants for proteins and antibiotics within 23S rRNA

    DEFF Research Database (Denmark)

    Douthwaite, Stephen Roger; Voldborg, Bjørn Gunnar Rude; Hansen, Lykke Haastrup

    1995-01-01

    Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination of molecu......Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination......-proteins L10.(L12)4 and L11 and is inhibited by interaction with the antibiotic thiostrepton. The peptidyltransferase center within domain V is inhibited by macrolide, lincosamide, and streptogramin B antibiotics, which interact with the rRNA around nucleotide A2058. Drug resistance is conferred by mutations...

  12. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18.

    Science.gov (United States)

    Smirnov, Alexandre; Entelis, Nina; Martin, Robert P; Tarassov, Ivan

    2011-06-15

    5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes.

  13. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda.

    Directory of Open Access Journals (Sweden)

    Shu Wu

    Full Text Available Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9 within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%; and could aid in species-level analyses, but with some limitations; 2 nearly-whole-length sequences and some partial regions (around V2, V4, and V9 of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%; 3 compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%; and 4 V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

  14. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda.

    Science.gov (United States)

    Wu, Shu; Xiong, Jie; Yu, Yuhe

    2015-01-01

    Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

  15. rRNA sequence comparison of Beauveria bassiana, Tolypocladium cylindrosporum, and Tolypocladium extinguens.

    Science.gov (United States)

    Rakotonirainy, M S; Dutertre, M; Brygoo, Y; Riba, G

    1991-01-01

    Five strains of Tolypocladium cylindrosporum, one strain of Tolypocladium extinguens, and nine strains of Beauveria bassiana were analyzed using a rapid rRNA sequencing technique. The sequences of two highly variable domains (D1 and D2) located at the 5' end of the 28S-like rRNA molecule were determined. The phylogenetic tree computed from the absolute number of nucleotide differences shows the separation between the genus Beauveria and the genus Tolypocladium and points out that T. cylindrosporum and T. extinguens probably do not belong to the same genus.

  16. Strength and Regulation of Seven rRNA Promoters in Escherichia coli.

    Science.gov (United States)

    Maeda, Michihisa; Shimada, Tomohiro; Ishihama, Akira

    2015-01-01

    The model prokaryote Escherichia coli contains seven copies of the rRNA operon in the genome. The presence of multiple rRNA operons is an advantage for increasing the level of ribosome, the key apparatus of translation, in response to environmental conditions. The complete sequence of E. coli genome, however, indicated the micro heterogeneity between seven rRNA operons, raising the possibility in functional heterogeneity and/or differential mode of expression. The aim of this research is to determine the strength and regulation of the promoter of each rRNA operon in E. coli. For this purpose, we used the double-fluorescent protein reporter pBRP system that was developed for accurate and precise determination of the promoter strength of protein-coding genes. For application of this promoter assay vector for measurement of the rRNA operon promoters devoid of the signal for translation, a synthetic SD sequence was added at the initiation codon of the reporter GFP gene, and then approximately 500 bp-sequence upstream each 16S rRNA was inserted in front of this SD sequence. Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector. Results indicated that: the promoter activity was the highest for the rrnE promoter under all growth conditions analyzed, including different growth phases of wild-type E. coli grown in various media; but the promoter strength of other six rrn promoters was various depending on the culture conditions. These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations. Possible difference in the functional role of each rRNA operon is also discussed.

  17. Phylogeny of protostome worms derived from 18S rRNA sequences.

    Science.gov (United States)

    Winnepenninckx, B; Backeljau, T; De Wachter, R

    1995-07-01

    The phylogenetic relationships of protostome worms were studied by comparing new complete 18S rRNA sequences of Vestimentifera, Pogonophora, Sipuncula, Echiura, Nemertea, and Annelida with existing 18S rRNA sequences of Mollusca, Arthropoda, Chordata, and Platyhelminthes. Phylogenetic trees were inferred via neighbor-joining and maximum parsimony analyses. These suggest that (1) Sipuncula and Echiura are not sister groups; (2) Nemertea are protostomes; (3) Vestimentifera and Pogonophora are protostomes that have a common ancestor with Echiura; and (4) Vestimentifera and Pogonophora are a monophyletic clade.

  18. Multiple independent insertions of 5S rRNA genes in the spliced-leader gene family of trypanosome species.

    Science.gov (United States)

    Beauparlant, Marc A; Drouin, Guy

    2014-02-01

    Analyses of the 5S rRNA genes found in the spliced-leader (SL) gene repeat units of numerous trypanosome species suggest that such linkages were not inherited from a common ancestor, but were the result of independent 5S rRNA gene insertions. In trypanosomes, 5S rRNA genes are found either in the tandemly repeated units coding for SL genes or in independent tandemly repeated units. Given that trypanosome species where 5S rRNA genes are within the tandemly repeated units coding for SL genes are phylogenetically related, one might hypothesize that this arrangement is the result of an ancestral insertion of 5S rRNA genes into the tandemly repeated SL gene family of trypanosomes. Here, we use the types of 5S rRNA genes found associated with SL genes, the flanking regions of the inserted 5S rRNA genes and the position of these insertions to show that most of the 5S rRNA genes found within SL gene repeat units of trypanosome species were not acquired from a common ancestor but are the results of independent insertions. These multiple 5S rRNA genes insertion events in trypanosomes are likely the result of frequent founder events in different hosts and/or geographical locations in species having short generation times.

  19. Cloning and Sequence Analysis on COX1 and 1 2 S rRNA Genes of Two Seed Beetle Species%2种豆象线粒体COX1和12SrRNA基因序列的克隆分析

    Institute of Scientific and Technical Information of China (English)

    吴培福; 佟友贵; 王琳; 潘涌智; 熊忠平; 窦报坤

    2014-01-01

    分别提取银合欢豆象和合欢豆象的基因组,对其COX1和12 S rRNA基因进行测序分析。结果表明:2种豆象的COX1和12 S rRNA基因A、T含量较丰富;碱基C和T间有最高的转换率;偏向使用以A或T结尾的密码子;银合欢豆象与合欢豆象进化距离较远,与A.oblongoguttatus进化距离最近,合欢豆象与多型豆象属聚在一个大的分支上。%The genome DNA was extracted respectively from Acanthoscelides macrophthalmus and Bruchidius ter-renus,and the COX1 and 12S rRNA genes were sequenced and analyzed to establish the basis for the further molecu-lar study.The results showed that COX1 and 12S rRNA of the two beetle species presented higher content of A+T, nucleotide C and T showed higher conversion rate.The two genes had codon usage bias as using codons with the third base A or T.It was showed that Acanthoscelides macrophthalmus shared a longer distance with Bruchidius terrenus, but a shorter distance with Acanthoscelides oblongoguttatus.While,Bruchidius terrenus would be grouped into the same branch of the genus Bruchidius.However,the more accurate phylogenetic roles of Acanthoscelides macrophthal-mus and Bruchidius terrenus need to be further studied due to limited nucleotide data in GenBank.

  20. Phylogenetic placement of the spider genus Nephila (Araneae: Araneoidea) inferred from rRNA and MaSp1 gene sequences.

    Science.gov (United States)

    Pan, Hong-Chun; Zhou, Kai-Ya; Song, Da-Xiang; Qiu, Yang

    2004-03-01

    The family status of the genus Nephila, which belongs to Tetragnathidae currently but Araneidae formerly, was reexamined based on molecular phylogenetic analyses. In the present study, 12S and 18S rRNA gene fragments of eight species of spiders were amplified and sequenced. In addition, 3'-end partial cDNA of major ampullate spidroin-1 (MaSp1) gene of Argiope amoena was cloned and sequenced, and the 3'-end non-repetitive region's cDNA sequence of MaSp1 gene and the predicted amino acid sequence of C-terminal non-repetitive region of MaSp1 were aligned with some previously known sequences. The resulting phylogeny showed that Araneidae and Tetragnathidae are not a sister group in the superfamily Araneoidea, and the genus Nephila is closer to the genera of the family Araneidae rather than to those of Tetragnathidae. We suggest that the genus Nephila should be transferred back to Araneidae. Or the subfamily Nephilinae might be elevated to family level after it was redefined and redelimited. Furthermore, the study showed that 3'-end non-repetitive region's cDNA sequence of MaSp1 gene and C-terminal non-repetitive region's amino acid sequence of MaSp1 are useful molecular markers for phylogenetic analysis of spiders.

  1. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    NARCIS (Netherlands)

    Ziesemer, K.A.; Mann, A.E.; Sankaranarayanan, K.; Schroeder, H.; Ozga, A.T.; Brandt, B.W.; Zaura, E.; Waters-Rist, A.; Hoogland, M.; Salazar-García, D.C.; Aldenderfer, M.; Speller, C.; Hendy, J.; Weston, D.A.; MacDonald, S.J.; Thomas, G.H.; Collins, M.J.; Lewis, C.M.; Hofman, C.; Warinner, C.

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gen

  2. Rapid identification of rumen protozoa by restriction analysis of amplified 18S rRNA gene

    NARCIS (Netherlands)

    Regensbogenova, M.; Kisidayova, S.; Michalowski, T.; Javorsky, P.; Moon-van der Staay, S.Y.; Hackstein, J.H.P.; McEwan, N.R.; Jouany, J.P.; Newbold, J.C.; Pristas, P.

    2004-01-01

    A rapid method has been developed for molecular identification of rumen ciliates without the need for cultivation. Total DNA was isolated from single protozoal cells by the Chelex method and nearly complete protozoal 18S rRNA genes were amplified and subjected to restriction fragment length polymorp

  3. Prosthetic joint infection due to Lysobacter thermophilus diagnosed by 16S rRNA gene sequencing.

    Science.gov (United States)

    Dhawan, B; Sebastian, S; Malhotra, R; Kapil, A; Gautam, D

    2016-01-01

    We report the first case of prosthetic joint infection caused by Lysobacter thermophilus which was identified by 16S rRNA gene sequencing. Removal of prosthesis followed by antibiotic treatment resulted in good clinical outcome. This case illustrates the use of molecular diagnostics to detect uncommon organisms in suspected prosthetic infections.

  4. NOF1 encodes an Arabidopsis protein involved in the control of rRNA expression.

    Directory of Open Access Journals (Sweden)

    Erwana Harscoët

    Full Text Available The control of ribosomal RNA biogenesis is essential for the regulation of protein synthesis in eukaryotic cells. Here, we report the characterization of NOF1 that encodes a putative nucleolar protein involved in the control of rRNA expression in Arabidopsis. The gene has been isolated by T-DNA tagging and its function verified by the characterization of a second allele and genetic complementation of the mutants. The nof1 mutants are affected in female gametogenesis and embryo development. This result is consistent with the detection of NOF1 mRNA in all tissues throughout plant life's cycle, and preferentially in differentiating cells. Interestingly, the closely related proteins from zebra fish and yeast are also necessary for cell division and differentiation. We showed that the nof1-1 mutant displays higher rRNA expression and hypomethylation of rRNA promoter. Taken together, the results presented here demonstrated that NOF1 is an Arabidopsis gene involved in the control of rRNA expression, and suggested that it encodes a putative nucleolar protein, the function of which may be conserved in eukaryotes.

  5. Direct regulation of rRNA transcription by fibroblast growth factor 2.

    Science.gov (United States)

    Sheng, Zhi; Liang, Yanping; Lin, Chih-Yin; Comai, Lucio; Chirico, William J

    2005-11-01

    Fibroblast growth factor 2 (FGF-2), which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Five isoforms (18 to approximately 34 kDa) of FGF-2 are derived from alternative initiation codons of a single mRNA. The 18-kDa FGF-2 isoform is released from cells by a nonclassical secretory pathway and regulates gene expression by binding to cell surface receptors. This isoform also localizes to the nucleolus, raising the possibility that it may directly regulate ribosome biogenesis, a rate-limiting process in cell growth. Although several growth factors have been shown to accumulate in the nucleolus, their function and mechanism of action remain unclear. Here we show that 18-kDa FGF-2 interacts with upstream binding factor (UBF), an architectural transcription factor essential for rRNA transcription. The maximal activation of rRNA transcription in vitro by 18-kDa FGF-2 requires UBF. The 18-kDa FGF-2 localizes to rRNA genes and is necessary for the full activation of pre-rRNA synthesis in vivo. Our results demonstrate that 18-kDa FGF-2 directly regulates rRNA transcription.

  6. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

    Science.gov (United States)

    Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2015-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

  7. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten;

    1994-01-01

    Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows...

  8. Transcription analysis of the Streptomyces coelicolor A3(2) rrnA operon

    DEFF Research Database (Denmark)

    van Wezel, G P; Krab, I M; Douthwaite, S

    1994-01-01

    Transcription start sites and processing sites of the Streptomyces coelicolor A3(2) rrnA operon have been investigated by a combination of in vivo and in vitro transcription analyses. The data from these approaches are consistent with the existence of four in vivo transcription sites, corresponding...

  9. Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase

    DEFF Research Database (Denmark)

    Nielsen, A K; Douthwaite, S; Vester, B

    1999-01-01

    Erm methyltransferases modify bacterial 23S ribosomal RNA at adenosine 2058 (A2058, Escherichia coli numbering) conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics. The motif that is recognized by Erm methyltransferases is contained within helix 73 of 23S rRNA a...

  10. A comparison of the proinflammatory effects of 12(R)- and 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid in human skin.

    Science.gov (United States)

    Wollard, P M; Cunnigham, F M; Murphy, G M; Camp, R D; Derm, F F; Greaves, M W

    1989-10-01

    Topical application of racemic 12-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R,S)-HETE] produces erythema and leucocyte accumulation in human skin. Since 12(R)-HETE is more potent than its epimer 12(S)-HETE as a neutrophil chemoattractant in vitro, their proinflammatory effects have now been compared in vivo. 12(R)- and 12(S)-HETE (0.5 - 20 ug/site) were applied topically to the forearm skin of 5 healthy volunteers and the sites occluded for 6 h. Five ug each of the two enantiomers were also applied to the opposite forearm. At 6 and 24 h blood flow and the areas of erythematous responses were measured. The 5 ug application sites were biopsied at 24 h. Both enantiomers caused dose related erythema and increased blood flow at 6 and 24 h, which were not significantly different at either of the time points tested. In contrast, pronounced neutrophil infiltrates were seen in the epidermis (25.2 +/- 13 cells/hpf) and dermis (13.2 +/- 5.1 cells/hpf) 24 h after application of 12(R)-, but not 12(S)-HETE (0.02 +/- 0.02 and 1.02 +/- 0.7 cells/hpf in epidermis and dermis respectively). However, the numbers of dermal mononuclear cells accumulating in response to the two enantiomers were similar. 12(R)-HETE thus appears to be a more potent neutrophil chemoattractant than 12(S)-HETE in human skin in vivo and may be of potential importance as a mediator of inflammation in man.

  11. Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine.

    Science.gov (United States)

    Kouvela, Ekaterini C; Gerbanas, George V; Xaplanteri, Maria A; Petropoulos, Alexandros D; Dinos, George P; Kalpaxis, Dimitrios L

    2007-01-01

    5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N1-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA(Phe) at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent 'loosening' of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.

  12. Common 5S rRNA variants are likely to be accepted in many sequence contexts

    Science.gov (United States)

    Zhang, Zhengdong; D'Souza, Lisa M.; Lee, Youn-Hyung; Fox, George E.

    2003-01-01

    Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The

  13. Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca(2+) signalling.

    Science.gov (United States)

    Stadler, Serena; Nguyen, Chi Huu; Schachner, Helga; Milovanovic, Daniela; Holzner, Silvio; Brenner, Stefan; Eichsteininger, Julia; Stadler, Mira; Senfter, Daniel; Krenn, Liselotte; Schmidt, Wolfgang M; Huttary, Nicole; Krieger, Sigurd; Koperek, Oskar; Bago-Horvath, Zsuzsanna; Brendel, Konstantin Alexander; Marian, Brigitte; de Wever, Oliver; Mader, Robert M; Giessrigl, Benedikt; Jäger, Walter; Dolznig, Helmut; Krupitza, Georg

    2016-12-24

    Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca(2+) levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca(2+), Ca(2+)-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca(2+) release. Thus, Ca(2+) signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.

  14. Organism-specific rRNA capture system for application in next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Sai-Kam Li

    Full Text Available RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA and transfer RNA (tRNA have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by in vitro transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.

  15. Growth increase of Arabidopsis by forced expression of rice 45S rRNA gene.

    Science.gov (United States)

    Makabe, So; Motohashi, Reiko; Nakamura, Ikuo

    2017-02-01

    Forced expression of rice 45S rRNA gene conferred ca. 2-fold increase of above-ground growth in transgenic Arabidopsis . This growth increase was probably brought by cell proliferation, not by cell enlargement. Recent increase in carbon dioxide emissions is causing global climate change. The use of plant biomass as alternative energy source is one way to reduce these emissions. Therefore, reinforcement of plant biomass production is an urgent key issue to overcome both depletion of fossil energies and emission of carbon dioxide. Here, we created transgenic Arabidopsis with a 2-fold increase in above-ground growth by forced expression of the rice 45S rRNA gene using the maize ubiquitin promoter. Although the size of guard cells and ploidy of leaf-cells were similar between transgenic and control plants, numbers of stomata and pavement cells were much increased in the transgenic leaf. This data suggested that cell number, not cell expansion, was responsible for the growth increase, which might be brought by the forced expression of exogenous and full-length 45S rRNA gene. The expression level of rice 45S rRNA transcripts was very low, possibly triggering unknown machinery to enhance cell proliferation. Although microarray analysis showed enhanced expression of ethylene-responsive transcription factors, these factors might respond to ethylene induced by abiotic/biotic stresses or genomic incompatibility, which might be involved in the expression of species-specific internal transcribed spacer (ITS) sequences within rice 45S rRNA transcripts. Further analysis of the mechanism underlying the growth increase will contribute to understanding the regulation of the cell proliferation and the mechanism of hybrid vigor.

  16. Oral microbiome profiles: 16S rRNA pyrosequencing and microarray assay comparison.

    Directory of Open Access Journals (Sweden)

    Jiyoung Ahn

    Full Text Available OBJECTIVES: The human oral microbiome is potentially related to diverse health conditions and high-throughput technology provides the possibility of surveying microbial community structure at high resolution. We compared two oral microbiome survey methods: broad-based microbiome identification by 16S rRNA gene sequencing and targeted characterization of microbes by custom DNA microarray. METHODS: Oral wash samples were collected from 20 individuals at Memorial Sloan-Kettering Cancer Center. 16S rRNA gene survey was performed by 454 pyrosequencing of the V3-V5 region (450 bp. Targeted identification by DNA microarray was carried out with the Human Oral Microbe Identification Microarray (HOMIM. Correlations and relative abundance were compared at phylum and genus level, between 16S rRNA sequence read ratio and HOMIM hybridization intensity. RESULTS: The major phyla, Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were identified with high correlation by the two methods (r = 0.70∼0.86. 16S rRNA gene pyrosequencing identified 77 genera and HOMIM identified 49, with 37 genera detected by both methods; more than 98% of classified bacteria were assigned in these 37 genera. Concordance by the two assays (presence/absence and correlations were high for common genera (Streptococcus, Veillonella, Leptotrichia, Prevotella, and Haemophilus; Correlation = 0.70-0.84. CONCLUSION: Microbiome community profiles assessed by 16S rRNA pyrosequencing and HOMIM were highly correlated at the phylum level and, when comparing the more commonly detected taxa, also at the genus level. Both methods are currently suitable for high-throughput epidemiologic investigations relating identified and more common oral microbial taxa to disease risk; yet, pyrosequencing may provide a broader spectrum of taxa identification, a distinct sequence-read record, and greater detection sensitivity.

  17. Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis.

    Science.gov (United States)

    Floyd, Brice E; Morriss, Stephanie C; MacIntosh, Gustavo C; Bassham, Diane C

    2015-01-01

    Ribosomes account for a majority of the cell's RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2-2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2-2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2-2, atg9-4, atg5-1, rns2-2 atg9-4, and rns2-2 atg5-1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2-2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2-2 atg5-1 double mutant but not by an rns2-2 atg9-4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole.

  18. Linking Maternal and Somatic 5S rRNA types with Different Sequence-Specific Non-LTR Retrotransposons

    NARCIS (Netherlands)

    Locati, M.D.; Pagano, J.F.B.; Ensink, W.A.; van Olst, M.; van Leeuwen, S.; Nehrdich, U.; Zhu, K.; Spaink, H.P.; Girard, G.; Rauwerda, H.; Jonker, M.J.; Dekker, R.J.; Breit, T.M.

    5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo and adult tissue,

  19. The nucleotide sequence of chloroplast 4.5S rRNA from a fern, Dryopteris acuminata.

    OpenAIRE

    Takaiwa, F.; Kusuda, M; SUGIURA, M.

    1982-01-01

    The 4.5S rRNA was isolated from the chloroplast ribosomes from Dryopteris acuminata. The complete nucleotide sequence was determined to be: OHUAAGGUCACGGCAAGACGAGCCGUUUAUCACCACGAUAGGUGCUAAGUGGAGGUGCAGUAAUGUAUGCAGCUGAGGC AUCCUAAUAGACCGAGAGGUUUGAACOH. The 4.5S rRNA is composed of 103 nucleotides and shows strong homology with those from flowering plants.

  20. Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons.

    Science.gov (United States)

    Kamke, Janine; Taylor, Michael W; Schmitt, Susanne

    2010-04-01

    The phylogenetic diversity of microorganisms in marine sponges is becoming increasingly well described, yet relatively little is known about the activities of these symbionts. Given the seemingly favourable environment provided to microbes by their sponge hosts, as indicated by the extraordinarily high abundance of sponge symbionts, we hypothesized that the majority of sponge-associated bacteria are active in situ. To test this hypothesis we compared, for the first time in sponges, 16S rRNA gene- vs 16S rRNA-derived bacterial community profiles to gain insights into symbiont composition and activity, respectively. Clone libraries revealed a highly diverse bacterial community in Ancorina alata, and a much lower diversity in Polymastia sp., which were identified by electron microscopy as a high- and a low-microbial abundance sponge, respectively. Substantial overlap between DNA and RNA libraries was evident at both phylum and phylotype levels, indicating in situ activity for a large fraction of sponge-associated bacteria. This active fraction included uncultivated, sponge-specific lineages within, for example, Actinobacteria, Chloroflexi and Gemmatimonadetes. This study shows the potential of RNA vs DNA comparisons based on the 16S rRNA gene to provide insights into the activity of sponge-associated microorganisms.

  1. Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro.

    Science.gov (United States)

    Sharwood, Robert E; Hotto, Amber M; Bollenbach, Thomas J; Stern, David B

    2011-02-01

    Post-transcriptional regulation in the chloroplast is exerted by nucleus-encoded ribonucleases and RNA-binding proteins. One of these ribonucleases is RNR1, a 3'-to-5' exoribonuclease of the RNase II family. We have previously shown that Arabidopsis rnr1-null mutants exhibit specific abnormalities in the expression of the rRNA operon, including the accumulation of precursor 23S, 16S, and 4.5S species and a concomitant decrease in the mature species. 5S rRNA transcripts, however, accumulate to a very low level in both precursor and mature forms, suggesting that they are unstable in the rnr1 background. Here we demonstrate that rnr1 plants overaccumulate an antisense RNA, AS5, that is complementary to the 5S rRNA, its intergenic spacer, and the downstream trnR gene, which encodes tRNA(Arg), raising the possibility that AS5 destabilizes 5S rRNA or its precursor and/or blocks rRNA maturation. To investigate this, we used an in vitro system that supports 5S rRNA and trnR processing. We show that AS5 inhibits 5S rRNA maturation from a 5S-trnR precursor, and shorter versions of AS5 demonstrate that inhibition requires intergenic sequences. To test whether the sense and antisense RNAs form double-stranded regions in vitro, treatment with the single-strand-specific mung bean nuclease was used. These results suggest that 5S-AS5 duplexes interfere with a sense-strand secondary structure near the endonucleolytic cleavage site downstream from the 5S rRNA coding region. We hypothesize that these duplexes are degraded by a dsRNA-specific ribonuclease in vivo, contributing to the 5S rRNA deficiency observed in rnr1.

  2. 16S rRNA beacons for bacterial monitoring during human space missions.

    Science.gov (United States)

    Larios-Sanz, Maia; Kourentzi, Katerina D; Warmflash, David; Jones, Jeffrey; Pierson, Duane L; Willson, Richard C; Fox, George E

    2007-04-01

    Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.

  3. Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment.

    Directory of Open Access Journals (Sweden)

    Jolanta Kwasniewska

    Full Text Available In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley by maleic hydrazide (MH cells was performed. Simultaneously fluorescence in situ hybridization (FISH with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment.

  4. A renaissance for the pioneering 16S rRNA gene

    Energy Technology Data Exchange (ETDEWEB)

    Tringe, Susannah; Hugenholtz, Philip

    2008-09-07

    Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the last quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

  5. A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples.

    Science.gov (United States)

    Dollive, Serena; Peterfreund, Gregory L; Sherrill-Mix, Scott; Bittinger, Kyle; Sinha, Rohini; Hoffmann, Christian; Nabel, Christopher S; Hill, David A; Artis, David; Bachman, Michael A; Custers-Allen, Rebecca; Grunberg, Stephanie; Wu, Gary D; Lewis, James D; Bushman, Frederic D

    2012-07-03

    Eukaryotic microorganisms are important but understudied components of the human microbiome. Here we present a pipeline for analysis of deep sequencing data on single cell eukaryotes. We designed a new 18S rRNA gene-specific PCR primer set and compared a published rRNA gene internal transcribed spacer (ITS) gene primer set. Amplicons were tested against 24 specimens from defined eukaryotes and eight well-characterized human stool samples. A software pipeline https://sourceforge.net/projects/brocc/ was developed for taxonomic attribution, validated against simulated data, and tested on pyrosequence data. This study provides a well-characterized tool kit for sequence-based enumeration of eukaryotic organisms in human microbiome samples.

  6. Improving oligonucleotide fingerprinting of rRNA genes by implementation of polony microarray technology

    Science.gov (United States)

    Ruegger, Paul M.; Bent, Elizabeth; Li, Wei; Jeske, Daniel R.; Cui, Xinping; Braun, Jonathan; Jiang, Tao; Borneman, James

    2012-01-01

    Improvements to oligonucleotide fingerprinting of rRNA genes (OFRG) were obtained by implementing polony microarray technology. OFRG is an array-based method for analyzing microbial community composition. Polonies are discrete clusters of DNA, produced by solid-phase PCR in hydrogels, and derived from individual, spatially isolated DNA molecules. The advantages of a polony-based OFRG method include higher throughput and reductions in the PCR-induced errors and compositional skew inherent in all other PCR-based community composition methods, including high throughput sequencing of rRNA genes. Given the similarities between polony microarrays and certain aspects of sequencing methods such as the Illumina platform, we suggest that if concepts presented in this study were implemented in high throughput sequencing protocols, a reduction of PCR-induced errors and compositional skew may be realized. PMID:22640891

  7. Phylogenetic analysis of freshwater mussel corbicula regularis by 18s rRNA gene sequencing

    Directory of Open Access Journals (Sweden)

    Magare V N

    2015-04-01

    Full Text Available Corbicula regularis is a freshwater mussel found in the Indian sub-continent. In the present study, phylogenetic characterization of this important bivalve was attempted using 18S ribosomal RNA gene markers. Genomic DNA was extracted and 18S rRNA gene was amplified by universal primers. The amplification product was sequenced and compared with the nucleotide databases available online to evaluate phylogenetic relationship of the animal under study. Results indicated that 18S rRNA gene sequences of C. regularis showed high degree of similarity to another freshwater mussel, C. fluminea. This work constitutes the first ever sequence deposition of the C. regularis in the nucleotide databases highlighting the usefulness of 18S ribosomal gene markers for phylogenetic analysis.

  8. 5755例新生儿GJB2、12S rRNA耳聋基因检测结果分析

    Institute of Scientific and Technical Information of China (English)

    杨晶群; 余红

    2016-01-01

    目的 探讨在新生儿中开展耳聋基因GJB2及线粒体12S rRNA筛查的意义.方法 对5755例新生儿出生后2-3天采集足跟血,利用飞行时间质谱技术对GJB2耳聋基因的5个突变位点235delC、299-300delAT、35delG、176-191de116、167delT突变,及线粒体12S rRNA耳聋基因的2个突变位点1494C-T、1555A-G检测.结果 5755例新生儿检出GJB2基因183例,阳性率3.18%.1例GJB2 235del纯合突变及1例GJB2 235del&299-30 delAT复合杂合突变经ABR检查确诊为中重度、重度听力损失,2例GJB2 235del杂合突变分别为轻度,重度听力损失,1例176-191de116杂合突变为中度听力损失.检出9例线粒体12S rRNA基因阳性,阳性率0.16%,其新生儿听力初筛均通过.结论 在新生儿中开展GJB2、线粒体12S rRNA耳聋基因筛查,可以早期发现先天遗传性聋,避免药物相关性耳聋,以及对遗传咨询,婚育指导具有重要意义.

  9. Impaired rRNA synthesis triggers homeostatic responses in hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Anna eKiryk

    2013-11-01

    Full Text Available Decreased rRNA synthesis and nucleolar disruption, known as nucleolar stress, are primary signs of cellular stress associated with aging and neurodegenerative disorders. Silencing of rDNA occurs during early stages of Alzheimer´s disease (AD and may play a role in dementia. Moreover aberrant regulation of the protein synthesis machinery is present in the brain of suicide victims and implicates the epigenetic modulation of rRNA. Recently, we developed unique mouse models characterized by nucleolar stress in neurons. We inhibited RNA polymerase I by genetic ablation of the basal transcription factor TIF-IA in adult hippocampal neurons. Nucleolar stress resulted in progressive neurodegeneration, although with a differential vulnerability within the CA1, CA3 and dentate gyrus. Here, we investigate the consequences of nucleolar stress on learning and memory. The mutant mice show normal performance in the Morris water maze and in other behavioral tests, suggesting the activation of adaptive mechanisms. In fact, we observe a significantly enhanced learning and re-learning corresponding to the initial inhibition of rRNA transcription. This phenomenon is accompanied by aberrant synaptic plasticity. By the analysis of nucleolar function and integrity, we find that the synthesis of rRNA is later restored. Gene expression profiling shows that thirty-six transcripts are differentially expressed in comparison to the control group in absence of neurodegeneration. Additionally, we observe a significant enrichment of the putative serum response factor (SRF binding sites in the promoters of the genes with changed expression, indicating potential adaptive mechanisms mediated by the mitogen-activated protein kinase pathway. In the dentate gyrus a neurogenetic response might compensate the initial molecular deficits. These results underscore the role of nucleolar stress in neuronal homeostasis and open a new ground for therapeutic strategies aiming at preserving

  10. Impaired rRNA synthesis triggers homeostatic responses in hippocampal neurons.

    Science.gov (United States)

    Kiryk, Anna; Sowodniok, Katharina; Kreiner, Grzegorz; Rodriguez-Parkitna, Jan; Sönmez, Aynur; Górkiewicz, Tomasz; Bierhoff, Holger; Wawrzyniak, Marcin; Janusz, Artur K; Liss, Birgit; Konopka, Witold; Schütz, Günther; Kaczmarek, Leszek; Parlato, Rosanna

    2013-01-01

    Decreased rRNA synthesis and nucleolar disruption, known as nucleolar stress, are primary signs of cellular stress associated with aging and neurodegenerative disorders. Silencing of rDNA occurs during early stages of Alzheimer's disease (AD) and may play a role in dementia. Moreover, aberrant regulation of the protein synthesis machinery is present in the brain of suicide victims and implicates the epigenetic modulation of rRNA. Recently, we developed unique mouse models characterized by nucleolar stress in neurons. We inhibited RNA polymerase I by genetic ablation of the basal transcription factor TIF-IA in adult hippocampal neurons. Nucleolar stress resulted in progressive neurodegeneration, although with a differential vulnerability within the CA1, CA3, and dentate gyrus (DG). Here, we investigate the consequences of nucleolar stress on learning and memory. The mutant mice show normal performance in the Morris water maze and in other behavioral tests, suggesting the activation of adaptive mechanisms. In fact, we observe a significantly enhanced learning and re-learning corresponding to the initial inhibition of rRNA transcription. This phenomenon is accompanied by aberrant synaptic plasticity. By the analysis of nucleolar function and integrity, we find that the synthesis of rRNA is later restored. Gene expression profiling shows that 36 transcripts are differentially expressed in comparison to the control group in absence of neurodegeneration. Additionally, we observe a significant enrichment of the putative serum response factor (SRF) binding sites in the promoters of the genes with changed expression, indicating potential adaptive mechanisms mediated by the mitogen-activated protein kinase pathway. In the DG a neurogenetic response might compensate the initial molecular deficits. These results underscore the role of nucleolar stress in neuronal homeostasis and open a new ground for therapeutic strategies aiming at preserving neuronal function.

  11. Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

    Energy Technology Data Exchange (ETDEWEB)

    DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.

    2006-02-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  12. PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

    Science.gov (United States)

    Hafez, Mohamed; Guha, Tuhin Kumar; Shen, Chen; Sethuraman, Jyothi; Hausner, Georg

    2014-01-01

    Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

  13. Stimulation of the mouse rRNA gene promoter by a distal spacer promoter.

    OpenAIRE

    Paalman, M H; Henderson, S L; Sollner-Webb, B

    1995-01-01

    We show that the mouse ribosomal DNA (rDNA) spacer promoter acts in vivo to stimulate transcription from a downstream rRNA gene promoter. This augmentation of mammalian RNA polymerase I transcription is observed in transient-transfection experiments with three different rodent cell lines, under noncompetitive as well as competitive transcription conditions, over a wide range of template concentrations, whether or not the enhancer repeats alone stimulate or repress expression from the downstre...

  14. Novel variants of the 5S rRNA genes in Eruca sativa.

    Science.gov (United States)

    Singh, K; Bhatia, S; Lakshmikumaran, M

    1994-02-01

    The 5S ribosomal RNA (rRNA) genes of Eruca sativa were cloned and characterized. They are organized into clusters of tandemly repeated units. Each repeat unit consists of a 119-bp coding region followed by a noncoding spacer region that separates it from the coding region of the next repeat unit. Our study reports novel gene variants of the 5S rRNA genes in plants. Two families of the 5S rDNA, the 0.5-kb size family and the 1-kb size family, coexist in the E. sativa genome. The 0.5-kb size family consists of the 5S rRNA genes (S4) that have coding regions similar to those of other reported plant 5S rDNA sequences, whereas the 1-kb size family consists of the 5S rRNA gene variants (S1) that exist as 1-kb BamHI tandem repeats. S1 is made up of two variant units (V1 and V2) of 5S rDNA where the BamHI site between the two units is mutated. Sequence heterogeneity among S4, V1, and V2 units exists throughout the sequence and is not limited to the noncoding spacer region only. The coding regions of V1 and V2 show approximately 20% dissimilarity to the coding regions of S4 and other reported plant 5S rDNA sequences. Such a large variation in the coding regions of the 5S rDNA units within the same plant species has been observed for the first time. Restriction site variation is observed between the two size classes of 5S rDNA in E. sativa.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Environmental rRNA inventories miss over half of protistan diversity

    Directory of Open Access Journals (Sweden)

    Hong Sunhee

    2008-12-01

    Full Text Available Abstract Background The main tool to discover novel microbial eukaryotes is the rRNA approach. This approach has important biases, including PCR discrimination against certain rRNA gene species, which makes molecular inventories skewed relative to the source communities. The degree of this bias has not been quantified, and it remains unclear whether species missed from clone libraries could be recovered by increasing sequencing efforts, or whether they cannot be detected in principle. Here we attempt to discriminate between these possibilities by statistically analysing four protistan inventories obtained using different general eukaryotic PCR primers. Results We show that each PCR primer set-specific clone library is not a sample from the community diversity but rather from a fraction of this diversity. Therefore, even sequencing such clone libraries to saturation would only recover that fraction, which, according to the parametric models, varies between 17 ± 4% to 49 ± 10%, depending on the set of primers. The pooled data is thus qualitatively richer than individual libraries, even if normalized to the same sequencing effort. Conclusion The use of a single pair of primers leads to significant underestimation of the true community richness at all levels of taxonomic hierarchy. The majority of available protistan rRNA gene surveys likely sampled less than half of the target diversity, and might have completely missed the rest. The use of multiple PCR primers reduces this bias but does not necessarily eliminate it.

  16. 4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response.

    Science.gov (United States)

    Burger, Kaspar; Mühl, Bastian; Kellner, Markus; Rohrmoser, Michaela; Gruber-Eber, Anita; Windhager, Lukas; Friedel, Caroline C; Dölken, Lars; Eick, Dirk

    2013-10-01

    High concentrations (> 100 µM) of the ribonucleoside analog 4-thiouridine (4sU) is widely used in methods for RNA analysis like photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and nascent messenger (m)RNA labeling (4sU-tagging). Here, we show that 4sU-tagging at low concentrations ≤ 10 µM can be used to measure production and processing of ribosomal (r)RNA. However, elevated concentrations of 4sU (> 50 µM), which are usually used for mRNA labeling experiments, inhibit production and processing of 47S rRNA. The inhibition of rRNA synthesis is accompanied by nucleoplasmic translocation of nucleolar nucleophosmin (NPM1), induction of the tumor suppressor p53, and inhibition of proliferation. We conclude that metabolic labeling of RNA by 4sU triggers a nucleolar stress response, which might influence the interpretation of results. Therefore, functional ribosome biogenesis, nucleolar integrity, and cell cycle should be addressed in 4sU labeling experiments.

  17. Proteins associated with rRNA in the Escherichia coli ribosome.

    Science.gov (United States)

    Bernabeu, C; Vazquez, D; Ballesta, J P

    1978-04-27

    Ribosomal proteins located near the rRNA have been identified by cross linking to [14C]spermine with 1,5-difluoro-2,4-dinitrobenzene. The polyamine binds to double-stranded rRNA; those proteins showing radioactivity covalently bound after treatment with the bifunctional reagent should therefore be located in the vicinity of these regions of rRNA. Six proteins from the small subunit, S4, S5, S9, S18, S19 and S20 and ten proteins from the large subunit L2, L6, L13, L14, L16, L17, L18, L19, L22 and L27 preferentially take up the label. The results obtained with three proteins from the large subunit, L6, L16 and L27, show a high degree of variability that could reflect differences of conformation in the subunit population. Several proteins were drastically modified by the cross-linking agent but were not detected in the two-dimensional gel electrophoresis (e.g., S1, S11, S21, L7, L8 and L12) and therefore could not be studied.

  18. Purine and pyrimidine composition in 5S rRNA and its mutational significance

    Directory of Open Access Journals (Sweden)

    Subacius Sandra Maria Rodrigues

    1998-01-01

    Full Text Available Variations observed in 5S rRNA base compositions are almost entirely due to fixation of point mutations. As a consequence, 5S rRNA size has remained relatively constant during evolution and, therefore, dependencies among the four bases can be predicted. In order to characterize the nature and to determine the degree of such dependencies, correlation analysis followed by principal component factorial analysis was conducted on a large sample of 5S rRNA sequences. The results show that the purine and pyrimidine contents tend to remain constant, so that A + G = Kpur and C + U = Kpyr. The composition of the four bases expressed now by Kpur/Kpyr relationships is also constant (Ks. These relationships imply that the behavior of the mutations in the variable sites of the molecule follows rules imposed by the chemical nature of the bases involved. Consequently, transition mutations would be more favored than substitutions in transversion sites and also than insertion-deletion (rare in 5S rRNAs, since transitions would not significantly alter the values of the Ks-index.

  19. Crystallization and X-ray diffraction data of Thermus flavus 5S rRNA helices

    Science.gov (United States)

    Vallazza, Marco; Senge, Andrea; Lippmann, Corinna; Perbandt, Markus; Betzel, Christian; Bald, Rolf; Erdmann, Volker A.

    2001-11-01

    5S rRNA is an essential component of the large ribosomal subunit in prokaryotes and eukaryotes. Its unknown function in the ribosome will eventually be revealed in part by structural studies. To promote crystallization and enhance resolution in X-ray diffraction the molecule was subdivided into five domains A-E. Several RNA oligonucleotides were chemically produced by solid-phase phosphoramidite synthesis in order to construct the domains of the 5S rRNA. An improved RNA-MPD-screen was applied in crystallization which covers a complete 2D matrix for the components used. Crystallization analysis resulted in preferred combinations of pH, polyamine, monovalent and divalent cations for short RNA molecules. Six types of crystals corresponding to the domains B, C and E of Thermus flavus 5S rRNA could be obtained which were suitable for X-ray diffraction. Four RNA helices consist of seven base pairs and two of eight base pairs. As special features, they contain two adenines in a bulge position or G : U wobble base pairs assumed to be involved in RNA-protein recognition. With an increase in crystal size an increase in resolution by X-ray analysis was observed. X-ray diffraction data were collected to 1.5 Å resolution using synchrotron radiation and cryogenic cooling techniques.

  20. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    Science.gov (United States)

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-11-13

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

  1. Characterization of Hydrocortisone Biometabolites and 18S rRNA Gene in Chlamydomonas reinhardtii Cultures

    Directory of Open Access Journals (Sweden)

    Seyed Bagher Mosavi-Azam

    2008-10-01

    Full Text Available A unicellular microalga, Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1. This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25ºC for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11b,17b-Dihydroxyandrost-4-en-3-one (2, 11b-hydroxyandrost-4-en-3,17-dione (3, 11b,17a,20b,21-tetrahydroxypregn-4-en-3-one (4 and prednisolone (5 were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.

  2. Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

    Science.gov (United States)

    Thota, Swarna Gowri; Unnikannan, C P; Thampatty, Sitalakshmi R; Manorama, R; Bhandari, Rashna

    2015-02-15

    Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

  3. The Role of 16S rRNA Gene Sequencing in Confirmation of Suspected Neonatal Sepsis.

    Science.gov (United States)

    El Gawhary, Somaia; El-Anany, Mervat; Hassan, Reem; Ali, Doaa; El Gameel, El Qassem

    2016-02-01

    Different molecular assays for the detection of bacterial DNA in the peripheral blood represented a diagnostic tool for neonatal sepsis. We targeted to evaluate the role of 16S rRNA gene sequencing to screen for bacteremia to confirm suspected neonatal sepsis (NS) and compare with risk factors and septic screen testing. Sixty-two neonates with suspected NS were enrolled. White blood cells count, I/T ratio, C-reactive protein, blood culture and 16S rRNA sequencing were performed. Blood culture was positive in 26% of cases, and PCR was positive in 26% of cases. Evaluation of PCR for the diagnosis of NS showed sensitivity 62.5%, specificity 86.9%, PPV 62.5%, NPV 86.9% and accuracy of 79.7%. 16S rRNA PCR increased the sensitivity of detecting bacterial DNA in newborns with signs of sepsis from 26 to 35.4%, and its use can be limited to cases with the most significant risk factors and positive septic screen. © The Author [2015]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  4. Pseudoknot in domain II of 23 S rRNA is essential for ribosome function

    DEFF Research Database (Denmark)

    Rosendahl, G; Hansen, L H; Douthwaite, S

    1995-01-01

    The structure of domain II in all 23 S (and 23 S-like) rRNAs is constrained by a pseudoknot formed between nucleotides 1005 and 1138, and between 1006 and 1137 (Escherichia coli numbering). These nucleotides are exclusively conserved as 1005C.1138G and 1006C.1137G pairs in all Bacteria, Archaea...... and chloroplasts, whereas 1005G.1138C and 1006U.1137A pairs occur in Eukarya. We have mutagenized nucleotides 1005C-->G, 1006C-->U, 1137G-->A and 1138G-->C, both individually and in combinations, in a 23 S rRNA gene from the bacterium E. coli. The ability of 23 S rRNA to support cell growth is reduced when either...... "eukaryal" (1005G.1138C or 1006U.1137A) pair and one "bacterial" C.G pair largely restores the structure and function of the rRNA. Bacterial ribosomes containing both these eukaryal pairs also participate in protein synthesis, although at much reduced efficiency, and the structure of their pseudoknot region...

  5. Improved identification of Gordonia, Rhodococcus and Tsukamurella species by 5'-end 16S rRNA gene sequencing.

    Science.gov (United States)

    Wang, Tao; Kong, Fanrong; Chen, Sharon; Xiao, Meng; Sorrell, Tania; Wang, Xiaoyan; Wang, Shuo; Sintchenko, Vitali

    2011-01-01

    The identification of fastidious aerobic Actinomycetes such as Gordonia, Rhodococcus, and Tsukamurella has remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5'-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus, and Tsukamurella, and defined potential reference sequences for species from each of these genera. The 16S rRNA gene sequence based identification algorithm for species identification was used and enhanced by aligning test sequences with reference sequences from the List of Prokaryotic Names with Standing in Nomenclature. Conventional PCR based 16S rRNA gene sequencing and the alignment of the isolate 16S rRNA gene sequence with reference sequences accurately identified 100% of clinical strains of aerobic Actinomycetes. While partial 16S rRNA gene sequences of reference type strains matched with the 16S rRNA gene sequences of 19 isolates in our data set, another 13 strains demonstrated a degree of polymorphism with a 1-4 bp difference in the regions of difference. 5'-end 606 bp 16S rRNA gene sequencing, coupled with the assignment of well defined reference sequences to clinically relevant species of bacteria, can be a useful strategy for improving the identification of clinically relevant aerobic Actinomycetes.

  6. [Phylogenetic comparison between Spirulina and Arthrospira based on 16S rRNA and rpoC1 gene].

    Science.gov (United States)

    Wu, Yuemei; Wang, Suying; Dong, Shirui

    2016-02-04

    Based on 16S rRNA and rpoC1 gene sequences, the phylogenetic relationship between Spirulina and Arthrospira were studied and compared. We amplified, sequenced and analyzed 16S rRNA and rpoC1 of 84 strains. Then the phylogenetic trees were constructed and compared. The conserved sites percentage, average G+C content and sequence identity of rpoC1 were 49.7%, 47.7%, 76%-100% respectively, significantly lower than 79.4%, 55.6% and 91%-100% of 16S rRNA, and the heterogeneity degree was higher. The trees generated with two different genes showed similar topologies and thus inferred consistent phylogenetic relationships. Eighty-four experimental strains were divided into 3 groups belonging to 2 genera: F-35 1, F-904-2, F-1070 and TJBC14 were Spirulina and the rest were Arthrospira. Although morphospecies and geographical species could not be distinguished based on 16S rRNA and rpoC1 gene sequences, the bootstrap value of rpoC1 (100%) was higher than that of 16S rRNA (99%). Moreover, clustering effect of rpoC1 for Spirulina and Arthrospirai was better than 16S rRNA. Spirulina and Arthrospira were different genera, rpoC1 gene has more advantage to distinguish the strains in the same genus than that of 16S rRNA gene.

  7. Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria.

    Science.gov (United States)

    Smirnov, Alexandre; Tarassov, Ivan; Mager-Heckel, Anne-Marie; Letzelter, Michel; Martin, Robert P; Krasheninnikov, Igor A; Entelis, Nina

    2008-04-01

    RNA import into mitochondria is a widespread phenomenon. Studied in details for yeast, protists, and plants, it still awaits thorough investigation for human cells, in which the nuclear DNA-encoded 5S rRNA is imported. Only the general requirements for this pathway have been described, whereas specific protein factors needed for 5S rRNA delivery into mitochondria and its structural determinants of import remain unknown. In this study, a systematic analysis of the possible role of human 5S rRNA structural elements in import was performed. Our experiments in vitro and in vivo show that two distinct regions of the human 5S rRNA molecule are needed for its mitochondrial targeting. One of them is located in the proximal part of the helix I and contains a conserved uncompensated G:U pair. The second and most important one is associated with the loop E-helix IV region with several noncanonical structural features. Destruction or even destabilization of these sites leads to a significant decrease of the 5S rRNA import efficiency. On the contrary, the beta-domain of the 5S rRNA was proven to be dispensable for import, and thus it can be deleted or substituted without affecting the 5S rRNA importability. This finding was used to demonstrate that the 5S rRNA can function as a vector for delivering heterologous RNA sequences into human mitochondria. 5S rRNA-based vectors containing a substitution of a part of the beta-domain by a foreign RNA sequence were shown to be much more efficiently imported in vivo than the wild-type 5S rRNA.

  8. Intragenomic heterogeneity of the 16S rRNA-23S rRNA internal transcribed spacer among Pseudomonas syringae and Pseudomonas fluorescens strains.

    Science.gov (United States)

    Milyutina, Irina A; Bobrova, Vera K; Matveeva, Eugenia V; Schaad, Norman W; Troitsky, Alexey V

    2004-10-01

    The 16S-23S rRNA internal transcribed spacer regions (ITS1) from 14 strains of Pseudomonas syringae and P. fluorescens were sequenced. ITS1 exhibited significant sequence variability among different operons within a single genome. From 1 to 4 types of ITS1 were found in individual genomes of the P. syringae and P. fluorescens strains. A total of eight ITS1 types were identified among strains studied. The ITS1 nucleotide sequences consisted of conserved blocks including, among others, a stem-forming region of box B, tRNAIle and tRNAAla genes and several variable blocks. The differences in the variable regions were mostly due to insertions and/or deletions of nucleotide blocks. The intragenomic heterogeneity of ITS1 was brought about by different combinations of variable blocks, which possibly have resulted from recombination and horizontal transfer.

  9. rRNA gene restriction patterns of Haemophilus influenzae biogroup aegyptius strains associated with Brazilian purpuric fever.

    Science.gov (United States)

    Irino, K; Grimont, F; Casin, I; Grimont, P A

    1988-08-01

    The rRNA gene restriction patterns of 92 isolates of Haemophilus influenzae biogroup aegyptius, associated with conjunctivitis or Brazilian purpuric fever in the State of São Paulo, Brazil, were studied with 16 + 23S rRNA from Escherichia coli as a probe. All strains were classified into 15 patterns. Isolates from Brazilian purpuric fever cases were seen only in patterns 3 (most frequently) and 4 (rarely), whereas isolates from conjunctivitis were found in all 15 patterns. The study demonstrated that rRNA from E. coli can serve as a probe for molecular epidemiology.

  10. The Final Step in 5.8S rRNA Processing Is Cytoplasmic in Saccharomyces cerevisiae▿ †

    OpenAIRE

    Thomson, Emma; TOLLERVEY, DAVID

    2009-01-01

    The 18S rRNA component of yeast (Saccharomyces cerevisiae) 40S ribosomes undergoes cytoplasmic 3' cleavage following nuclear export, whereas exported pre-60S subunits were believed to contain only mature 5.8S and 25S rRNAs. However, in situ hybridization detected 3'-extended forms of 5.8S rRNA in the cytoplasm, which were lost when Crm1-dependent preribosome export was blocked by treatment with leptomycin B (LMB). LMB treatment rapidly blocked processing of 6S pre-rRNA to 5.8S rRNA, leading t...

  11. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    Institute of Scientific and Technical Information of China (English)

    Carlos; W; Nossa; William; E; Oberdorf; Jφrn; A; Aas; Bruce; J; Paster; Todd; Z; DeSantis; Eoin; L; Brodie; Daniel; Malamud; Michael; A; Poles

    2010-01-01

    AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classifica...

  12. Evolution of RNA editing sites in the mitochondrial small subunit rRNA of the Myxomycota.

    Science.gov (United States)

    Krishnan, Uma; Barsamian, Arpi; Miller, Dennis L

    2007-01-01

    Because of their unique and unprecedented character, it is often difficult to imagine how and why the different, diverse types of RNA editing have evolved. Information about the evolution of a particular RNA editing system can be obtained by comparing RNA editing characteristics in contemporary organisms whose phylogenetic relationships are known so that editing patterns in ancestral organisms can be inferred. This information can then be used to build models of the origins, constraints, variability, and mechanisms of RNA editing. As an example of the types of information that can be obtained from these analyses, we describe how we have used cDNA, covariation, and phylogenetic analyses to study the evolution of the variation in RNA editing site location in the core region of the small subunit rRNA gene in the mtDNA of seven myxomycetes, including Physarum polycephalum. We find that the unique type of insertional RNA editing present in mitochondria of P. polycephalum is also present in the mitochondrial small subunit (SSU) rRNA of the other six myxomycetes. As in Physarum, this editing predominantly consists of cytidine insertions, but also includes uridine insertions and certain dinucleotide insertions such that any of the four canonical ribonucleotides can be inserted. Although the characteristics of RNA editing in these organisms are the same as in Physarum, the location of the insertion sites varies among the seven organisms relative to the conserved primary sequence and secondary structure of the rRNA. Nucleotide insertions have been identified at 29 different sites within this core region of the rRNA, but no one organism has more than 10 of these insertion sites, suggesting that editing sites have been created and/or eliminated since the divergence of these organisms. To determine the order in which editing sites have been created or eliminated, the sequences of the mitochondrial SSU rRNA have been aligned and this alignment has been used to produce

  13. DNA sequencing reveals limited heterogeneity in the 16S rRNA gene from the rrnB operon among five Mycoplasma hominis isolates

    DEFF Research Database (Denmark)

    Mygind, T; Birkelund, Svend; Christiansen, Gunna

    1998-01-01

    To investigate the intraspecies heterogeneity within the 16S rRNA gene of Mycoplasma hominis, five isolates with diverse antigenic profiles, variable/identical P120 hypervariable domains, and different 16S rRNA gene RFLP patterns were analysed. The 16S rRNA gene from the rrnB operon was amplified...

  14. Design and experimental application of a novel non-degenerate universal primer set that amplifies prokaryotic 16S rRNA genes with a low possibility to amplify eukaryotic rRNA genes.

    Science.gov (United States)

    Mori, Hiroshi; Maruyama, Fumito; Kato, Hiromi; Toyoda, Atsushi; Dozono, Ayumi; Ohtsubo, Yoshiyuki; Nagata, Yuji; Fujiyama, Asao; Tsuda, Masataka; Kurokawa, Ken

    2014-01-01

    The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.

  15. Transcription of the 5S rRNA heterochromatic genes is epigenetically controlled in Arabidopsis thaliana and Xenopus laevis.

    Science.gov (United States)

    Douet, J; Tourmente, S

    2007-07-01

    5S ribosomal DNA is a highly conserved tandemly repeated multigenic family. As suggested for a long time, we have shown that only a fraction of the 5S rRNA genes are expressed in Arabidopsis thaliana. In Xenopus laevis, there is a developmental control of the expression of the 5S rRNA genes with only one of the two 5S rDNA families expressed during oogenesis. For both Arabidopsis and Xenopus, the strongest transcription of 5S rRNA, respectively in the seed and during oogenesis is correlated with heterogeneity in the transcribed 5S rRNAs. Epigenetic mechanisms such as modification of the chromatin structure are involved in the transcriptional regulation of the 5S rRNA genes in both organisms. In Arabidopsis, two silencing pathways, methylation-dependent (RNAi) and methylation-independent (MOM pathway), are involved in the silencing of a 5S rDNA fraction.

  16. Identification of Swertia mussotii and its adulterant Swertia species by 5S rRNA gene spacer.

    Science.gov (United States)

    Yu, Man-Tang; Wong, Ka-Lok; Zong, Yu-Ying; Shaw, Pang-Chui; Che, Chun-Tao

    2008-03-01

    This research focused on analyzing the differences of 5S rRNA gene spacer sequences on Swertia mussotii and its commonly used adulterants, including S. franchetiana, S. wolfangiana and S. chirayita. DNA was extracted from the collected Swertia samples. 5S rRNA intergenic spacers were amplified by PCR, sequenced and analyzed. 5S rRNA gene spacer sequences were different between S. mussotii and its other three adulterants. Sequence divergence among species ranged from 30.6% to 65.0%. 5S rRNA spacers may be used as molecular authentication markers to differentiate S. mussotii and other commonly used Swertia adulterants. This result provides reliable and simple reference for the authentication of Swertia genus species.

  17. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    National Research Council Canada - National Science Library

    Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jørn A; Paster, Bruce J; Desantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

    2010-01-01

    .... A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species...

  18. Community structure, cellular rRNA content, and activity of sulfate-reducing bacteria in marine Arctic sediments

    DEFF Research Database (Denmark)

    Ravenschlag, K.; Sahm, K.; Knoblauch, C.;

    2000-01-01

    The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburg-fjorden, Svalbard) a-as characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes...... that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5...... mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface, Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell(-1...

  19. The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition.

    Science.gov (United States)

    Onofrillo, Carmine; Galbiati, Alice; Montanaro, Lorenzo; Derenzini, Massimo

    2017-01-17

    Pre-ribosomal complex RPL5/RPL11/5S rRNA (5S RNP) is considered the central MDM2 inhibitory complex that control p53 stabilization during ribosome biogenesis inhibition. Despite its role is well defined, the dynamic of 5S RNP assembly still requires further characterization. In the present work, we report that MDM2 inhibition is dependent by a pre-existing population of 5S rRNA.

  20. Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

    Science.gov (United States)

    Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

    2012-07-01

    Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  1. Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection.

    Directory of Open Access Journals (Sweden)

    Chenyu Zhang

    Full Text Available Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial

  2. YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

    DEFF Research Database (Denmark)

    Purta, Elzbieta; O'Connor, Michelle; Bujnicki, Janusz M

    2008-01-01

    . coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m(5)C methyltransferases RsmB and RsmF and is in fact more similar to known m(5)U (5-methyluridine) RNA methyltransferases. In keeping with the previously...... proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI....

  3. RNase MRP is required for entry of 35S precursor rRNA into the canonical processing pathway.

    Science.gov (United States)

    Lindahl, Lasse; Bommankanti, Ananth; Li, Xing; Hayden, Lauren; Jones, Adrienne; Khan, Miriam; Oni, Tolulope; Zengel, Janice M

    2009-07-01

    RNase MRP is a nucleolar RNA-protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.

  4. Short alleles revealed by PCR demonstrate no heterozygote deficiency at minisatellite loci D1S7, D7S21, and D12S11

    Energy Technology Data Exchange (ETDEWEB)

    Alonso, S.; Castro, A.; Fernandez-Fernandez, I.; Pancorbo, M.M. de [Universidad del Pais Vasco, Vizcaya (Spain)

    1997-02-01

    Short VNTR alleles that go undetected after conventional Southern blot hybridization may constitute an alternative explanation for the heterozygosity deficiency observed at some minisatellite loci. To examine this hypothesis, we have employed a screening procedure based on PCR amplification of those individuals classified as homozygotes in our databases for the loci D1S7, D7S21, and D12S11. The results obtained indicate that the frequency of these short alleles is related to the heterozygosity deficiency observed. For the most polymorphic locus, D1S7, {approximately}60% of those individuals previously classified as homozygotes were in fact heterozygotes for a short allele. After the inclusion of these new alleles, the agreement between observed and expected heterozygosity, along with other statistical tests employed, provide additional evidence for lack of population substructuring. Comparisons of allele frequency distributions reveal greater differences between racial groups than between closely related populations. 45 refs., 3 figs., 6 tabs.

  5. Novel 12S mtDNA findings in sloths (Pilosa, Folivora and anteaters (Pilosa, Vermilingua suggest a true case of long branch attraction

    Directory of Open Access Journals (Sweden)

    Maria Claudene Barros

    2008-01-01

    Full Text Available We sequenced 12S RNA mtDNA for the majority of the extant species of sloths and anteaters and compared our results with previous data obtained by our group using 16S RNA mtDNA in the same specimens and to GenBank sequences of the extinct giant sloth Mylodon. Our results suggest that pigmy-anteaters may be a case of the long-branch attraction phenomenon and also show the large genetic difference between the Amazonian and Atlantic forest three-toed sloths, contrasting with the small differences observed between the two non-Atlantic forest forms of sloths. These results have important implications for the taxonomy of sloths and anteaters and strongly suggest the placement of pigmy anteaters in their own family (Cyclopidae and raising the taxonomic status of Bradypus torquatus to a genus.

  6. Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes.

    Science.gov (United States)

    Soni, Dharmendra Kumar; Dubey, Suresh Kumar

    2014-12-01

    The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity.

  7. Hypomethylation and hypermethylation of the tandem repetitive 5S rRNA genes in Arabidopsis.

    Science.gov (United States)

    Vaillant, Isabelle; Tutois, Sylvie; Jasencakova, Zuzana; Douet, Julien; Schubert, Ingo; Tourmente, Sylvette

    2008-04-01

    5S ribosomal DNA (5S rDNA) is organized in tandem repeats on chromosomes 3, 4 and 5 in Arabidopsis thaliana. One part of the 5S rDNA is located within the heterochromatic chromocenters, and the other fraction forms loops with euchromatic features that emanate from the chromocenters. We investigated whether the A. thaliana heterochromatin, and particularly the 5S rDNA, is modified when changing the culture conditions (cultivation in growth chamber versus greenhouse). Nuclei from challenged tissues displayed larger total, as well as 5S rDNA, heterochromatic fractions, and the DNA methyltransferase mutants met1 and cmt3 had different impacts in Arabidopsis. The enlarged fraction of heterochromatic 5S rDNA was observed, together with the reversal of the silencing of some 5S rRNA genes known as minor genes. We observed hypermethylation at CATG sites, and a concomitant DNA hypomethylation at CG/CXG sites in 5S rDNA. Our results show that the asymmetrical hypermethylation is correlated with the ageing of the plants, whereas hypomethylation results from the growth chamber/culture conditions. In spite of severely reduced DNA methylation, the met1 mutant revealed no increase in minor 5S rRNA transcripts in these conditions. The increasing proportion of cytosines in asymmetrical contexts during transition from the euchromatic to the heterochromatic state in the 5S rDNA array suggests that 5S rDNA units are differently affected by the (hypo and hyper)methylation patterns along the 5S rDNA locus. This might explain the different behaviour of 5S rDNA subpopulations inside a 5S array in terms of chromatin compaction and expression, i.e. some 5S rRNA genes would become derepressed, whereas others would join the heterochromatic fraction.

  8. rRNA maturation in yeast cells depleted of large ribosomal subunit proteins.

    Directory of Open Access Journals (Sweden)

    Gisela Pöll

    Full Text Available The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins. They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein-rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i how individual r-proteins control the productive processing of the major 5' end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

  9. Diversity and evolution of 5S rRNA gene family organization in Pythium.

    Science.gov (United States)

    Bedard, James E J; Schurko, Andrew M; de Cock, Arthur W A M; Klassen, Glen R

    2006-01-01

    The 5S rRNA gene family organization among 87 species and varieties of Pythium was investigated to assess evolutionary stability of the two patterns detected and to determine which pattern is likely the ancestral state in the genus. Species with filamentous sporangia (Groups A-C according to the ITS phylogenetic tree for Pythium) had 5S genes linked to the rDNA repeat that were predominantly coded for on the DNA strand opposite to the one with the other rRNA genes ('inverted' orientation). A small group of species with contiguous sporangia (Group D) is related to Groups A-C but had unlinked 5S genes. The main group of species with spherical zoosporangia (Groups E-J) generally had unlinked 5S genes in tandem arrays. The six species in Group K, although they also have spherical sporangia, had linked genes on the same strand as the other rRNA genes 'non-inverted' and most of them had pairs of tandem 5S genes. The evolutionary stability of 5S sequence organization was compared with the stability of morphological characters as interpreted from a phylogeny based on ITS sequence analysis. Features of 5S sequence organization were found to be just as consistent within groups as were the morphological characters. To determine the ancestral type of 5S family organization, a survey of Phytophthora strains was conducted to supply an outgroup reference. The most parsimonious interpretation of the data in this survey yielded the tentative conclusion that the linked condition of the 5S sequences was ancestral.

  10. Molecular epidemiology of Plasmodium species prevalent in Yemen based on 18 s rRNA

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    A Azazy Ahmed

    2010-11-01

    Full Text Available Abstract Background Malaria is an endemic disease in Yemen and is responsible for 4.9 deaths per 100,000 population per year and 43,000 disability adjusted life years lost. Although malaria in Yemen is caused mainly by Plasmodium falciparum and Plasmodium vivax, there are no sequence data available on the two species. This study was conducted to investigate the distribution of the Plasmodium species based on the molecular detection and to study the molecular phylogeny of these parasites. Methods Blood samples from 511 febrile patients were collected and a partial region of the 18 s ribosomal RNA (18 s rRNA gene was amplified using nested PCR. From the 86 positive blood samples, 13 Plasmodium falciparum and 4 Plasmodium vivax were selected and underwent cloning and, subsequently, sequencing and the sequences were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. Results Malaria was detected by PCR in 86 samples (16.8%. The majority of the single infections were caused by P. falciparum (80.3%, followed by P. vivax (5.8%. Mixed infection rates of P. falciparum + P. vivax and P. falciparum + P. malariae were 11.6% and 2.3%, respectively. All P. falciparum isolates were grouped with the strain 3D7, while P. vivax isolates were grouped with the strain Salvador1. Phylogenetic trees based on 18 s rRNA placed the P. falciparum isolates into three sub-clusters and P. vivax into one cluster. Sequence alignment analysis showed 5-14.8% SNP in the partial sequences of the 18 s rRNA of P. falciparum. Conclusions Although P. falciparum is predominant, P. vivax, P. malariae and mixed infections are more prevalent than has been revealed by microscopy. This overlooked distribution should be considered by malaria control strategy makers. The genetic polymorphisms warrant further investigation.

  11. Global Perspectives on Activated Sludge Community Composition analyzed using 16S rRNA amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Saunders, Aaron Marc; Albertsen, Mads

    communities, and in this study activated sludge sampled from 32 Wastewater Treatment Plants (WWTPs) around the world was described and compared. The top abundant bacteria in the global activated sludge ecosystem were found and the core population shared by multiple samples was investigated. The results......Activated sludge is the most commonly applied bioprocess throughout the world for wastewater treatment. Microorganisms are key to the process, yet our knowledge of their identity and function is still limited. High-througput16S rRNA amplicon sequencing can reliably characterize microbial...

  12. Structures of nucleolus and transcription sites of rRNA genes in rat liver cells

    Institute of Scientific and Technical Information of China (English)

    陶伟; 焦明大; 赫杰; 何孟元; 郝水

    2000-01-01

    We observed the ultrastructure of nucleolus in rat liver cells by conventional electron microscopy, and employed cytochemistry NAMA-Ur DNA specific stain method to analyze the distribution and position of nucleolar DNA in situ. The results showed that nucleolar DNA of rat liver cells comes from nucleolus-associated chromatin, and continuously extends in the dense fibrillar component (DFC) of nucleolus, localizes at the periphery of fibrillar center (FC) and in DFC. Furthermore, by employing anti-DNA/RNA hybrid antibodies, we directly and selectively labeled transcription sites of rRNA genes and testified that localization of transcription sites not only to DFC but also to the periphery of FC.

  13. Greengenes, a Chimera-checked 16S rRNA gene database and workbenchcompatible with ARB

    Energy Technology Data Exchange (ETDEWEB)

    DeSantis, Todd Z.; Hugenholtz, Philip; Larsen, Neils; Rojas,Mark; Brodie, Eoin L.; Keller, Keith; Huber, Thomas; Dalevi, Daniel; Hu,Ping; Andersen, Gary L.

    2006-04-10

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that in congruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3 percent of environmental sequences and 0.2 percent of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  14. Novel Acanthamoeba 18S rRNA gene sequence type from an environmental isolate.

    Science.gov (United States)

    Magnet, A; Henriques-Gil, N; Galván-Diaz, A L; Izquiedo, F; Fenoy, S; del Aguila, C

    2014-08-01

    The free-living amoebae, Acanthamoeba, can act as opportunistic parasites on a wide range of vertebrates and are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices, and their ubiquitous distribution. Subgenus classification based on morphology is being replaced by a classification based on the sequences of the 18S rRNA gene with a total of 18 different genotypes (T1-T18). A new environmental strain of Acanthamoeba isolated from a waste water treatment plant is presented in this study as a candidate for the description of the novel genotype T19 after phylogenetic analysis.

  15. Structures of nucleolus and transcription sites of rRNA genes in rat liver cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    We observed the ultrastructure of nucleolus in rat liver cells by conventional electronmicroscopy, and employed cytochemistry NAMA-Ur DNA specific stain method to analyze the distributionand position of nucleolar DNA in situ. The results showed that nucleolar DNA of rat livercells comes from nucleolus-associated chromatin, and continuously extends in the dense fibrillarcomponent (DFC) of nucleolus, localizes at the periphery of fibrillar center (FC) and in DFC. Furthermore,by employing anti-DNA/RNA hybrid antibodies, we directly and selectively labeled transcriptionsites of rRNA genes and testified that localization of transcription sites not only to DFC butalso to the periphery of FC.

  16. Mutations in 23S rRNA Confer Resistance against Azithromycin in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Marvig, Rasmus Lykke; Søndergaard, Mette S. R.; Pedersen, Søren Damkiær

    2012-01-01

    The emergence of antibiotic-resistant Pseudomonas aeruginosa is an important concern in the treatment of long-term airway infections in cystic fibrosis patients. In this study, we report the occurrence of azithromycin resistance among clinical P. aeruginosa DK2 isolates. We demonstrate that resis...... that resistance is associated with specific mutations (A2058G, A2059G, and C2611T in Escherichia coli numbering) in domain V of 23S rRNA and that introduction of A2058G and C2611T into strain PAO1 results in azithromycin resistance....

  17. Interaction of tRNA with domain II of 23S rRNA.

    Science.gov (United States)

    Hill, W E; Tassanakajohn, A; Tapprich, W E

    1990-08-27

    The interaction of tRNA with domain II of 23S rRNA in E. coli ribosomes has been probed using short, complementary DNA oligodeoxyribonucleotides. Specifically, cDNA oligomers to the region 801-811 of the 23S rRNA were used to ascertain the interaction of this region with tRNA. It was found that when tRNA was bound to the P site, considerable competition occurred between tRNA and the cDNA oligomers which base paired with the nucleotides 807-811. However, A-site bound tRNA neither displaced, nor was displaced, by cDNA oligomers to this region. Additionally, the binding of tRNA lacking the CACCA nucleotides on the 3' terminus was unaffected by the presence a cDNA oligomer complementary to nucleotides 803-811, indicating that the cDNA-tRNA competition was dependent on the 3' terminal nucleotides of tRNA.

  18. Coevolution in RNA molecules driven by selective constraints: evidence from 5S rRNA.

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    Nan Cheng

    Full Text Available Understanding intra-molecular coevolution helps to elucidate various structural and functional constraints acting on molecules and might have practical applications in predicting molecular structure and interactions. In this study, we used 5S rRNA as a template to investigate how selective constraints have shaped the RNA evolution. We have observed the nonrandom occurrence of paired differences along the phylogenetic trees, the high rate of compensatory evolution, and the high TIR scores (the ratio of the numbers of terminal to intermediate states, all of which indicate that significant positive selection has driven the evolution of 5S rRNA. We found three mechanisms of compensatory evolution: Watson-Crick interaction (the primary one, complex interactions between multiple sites within a stem, and interplay of stems and loops. Coevolutionary interactions between sites were observed to be highly dependent on the structural and functional environment in which they occurred. Coevolution occurred mostly in those sites closest to loops or bulges within structurally or functionally important helices, which may be under weaker selective constraints than other stem positions. Breaking these pairs would directly increase the size of the adjoining loop or bulge, causing a partial or total structural rearrangement. In conclusion, our results indicate that sequence coevolution is a direct result of maintaining optimal structural and functional integrity.

  19. Coevolution in RNA molecules driven by selective constraints: evidence from 5S rRNA.

    Science.gov (United States)

    Cheng, Nan; Mao, Yuanhui; Shi, Youyi; Tao, Shiheng

    2012-01-01

    Understanding intra-molecular coevolution helps to elucidate various structural and functional constraints acting on molecules and might have practical applications in predicting molecular structure and interactions. In this study, we used 5S rRNA as a template to investigate how selective constraints have shaped the RNA evolution. We have observed the nonrandom occurrence of paired differences along the phylogenetic trees, the high rate of compensatory evolution, and the high TIR scores (the ratio of the numbers of terminal to intermediate states), all of which indicate that significant positive selection has driven the evolution of 5S rRNA. We found three mechanisms of compensatory evolution: Watson-Crick interaction (the primary one), complex interactions between multiple sites within a stem, and interplay of stems and loops. Coevolutionary interactions between sites were observed to be highly dependent on the structural and functional environment in which they occurred. Coevolution occurred mostly in those sites closest to loops or bulges within structurally or functionally important helices, which may be under weaker selective constraints than other stem positions. Breaking these pairs would directly increase the size of the adjoining loop or bulge, causing a partial or total structural rearrangement. In conclusion, our results indicate that sequence coevolution is a direct result of maintaining optimal structural and functional integrity.

  20. Midkine accumulated in nucleolus of HepG2 cells involved in rRNA transcription

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Jian-Zhong Shao; Li-Shan Min; Yong-Tao Xiao; Li-Xin Xiang; Zhi-Hong Ma

    2008-01-01

    AIM: To invesgate the ultrastructural location of midkine (MK) in nucleolus and function corresponding to its location. METHODS: To investigate the ultrastructural location of MK in nucleolus with immunoelectronic microscopy. To study the role that MK plays in ribosomal biogenesis by real-time PCR. The effect of MK on anti-apoptotic activity of HepG2 cells was studied with FITC-conjugated annexin V and propidium iodide PI double staining through FACS assay. RESULTS: MK mainly localized in the granular component (GC), dense fibrillar component (DFC) and the border between the DF-C and fibrillar center (FC). The production of 45S precursor rRNA level was decreased significantly in the presence of IK antisense oligonucleotide in the HepG2 cells. Furthermore, it was found that exogenous MK could protect HepG2 from apoptosis significantly. CONCLUSION: NK was constitutively translocated to the nucleolus of HepG2 cells, where it accumulated and mostly distributed at DFC, GC components and at the region between FC and DFC, MK played an important role in rRNA transcription, ribosome biogenesis, and cell proliferation in HepG2 cells. MK might serve as a molecular target for therapeutic intervention of human carcinomas.

  1. Comparison of two approaches for the classification of 16S rRNA gene sequences.

    Science.gov (United States)

    Chatellier, Sonia; Mugnier, Nathalie; Allard, Françoise; Bonnaud, Bertrand; Collin, Valérie; van Belkum, Alex; Veyrieras, Jean-Baptiste; Emler, Stefan

    2014-10-01

    The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.

  2. PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries.

    Science.gov (United States)

    Potvin, Marianne; Lovejoy, Connie

    2009-01-01

    Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.

  3. Transcription analysis of the Streptomyces coelicolor A3(2) rrnA operon

    DEFF Research Database (Denmark)

    van Wezel, G P; Krab, I M; Douthwaite, S

    1994-01-01

    to the promoters P1-P4. The transcription start sites are located at -597, -416, -334 and -254 relative to the start of the 16S rRNA gene. Two putative processing sites were identified, one of which is similar to a sequence reported earlier in S. coelicolor and other eubacteria. The P1 promoter is likely...... to be recognized by the RNA polymerase holoenzyme containing sigma hrdB, the principal sigma factor in S. coelicolor. P2 also shares homology with the consensus for vegetative promoters, but has a sequence overlapping the consensus -35 region that is also present in the -35 regions of P3 and P4. The -35 sequence...... common to P2, P3 and P4 is not similar to any other known consensus promoter sequence. In fast-growing mycelium, P2 appears to be the most frequently used promoter. Transcription from all of the rrnA promoters decreased during the transition from exponential to stationary phase, although transcription...

  4. Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians

    Directory of Open Access Journals (Sweden)

    Chiari Ylenia

    2005-03-01

    Full Text Available Abstract Background Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI has been proposed as universal marker for this purpose among animals. Results Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%, with low degrees of pairwise haplotype divergence within populations (0–1%. Conclusion We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.

  5. Control of rRNA Synthesis in Escherichia coli: a Systems Biology Approach†

    Science.gov (United States)

    Dennis, Patrick P.; Ehrenberg, Mans; Bremer, Hans

    2004-01-01

    The first part of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years. The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media. This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp. These absolute promoter activities are evaluated in terms of rrn promoter strength (Vmax/Km) and free RNA polymerase concentrations. Three major conclusions emerge from this evaluation. First, the rrn promoters are not saturated with RNA polymerase. As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities. Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes. Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis. This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation. PMID:15590778

  6. GENE 16S RRNA SEQUENCE PHYLOGENETIC ANALYSIS OF LYSINE PRODUCERS STRAINS

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    G. S. Andriiash

    2014-12-01

    Full Text Available The phylogenetic relationships of strainsproducers of essential amino acids of aspartate family Brevibacterium sp. UCM Ac-674 (Brevibacterium sp. 90, Brevibacterium sp. IMV Ac-5004 (Brevibacterium sp. 90H, Brevibacterium sp. UCM Ac-675 (Brevibacterium sp. E531, mutant strain Brevibacterium sp. IMV B-7447 from the «Collections strains and lines of plants for food and agricultural biotechnology SO “Institute for Food Biotechnology and Genomics” of National Academy of Sciences of Ukraine were investigated. The affiliation strain Brevibacterium sp. IMV B-7447 to the genus Brevibacterium within the sequences of the genes based on 16S rRNA was confirmed. The dendogram of phylogenetic relationships of studied strains and related strains Brevibacterium from database GenBank was constructed. It was shown that by the criterion of homology gene sequences based on 16S rRNA the investigated strains-producers belong to three phylogenetic groups. It was established that the mutant strain Brevibacterium sp. ІMV B-7447 has no analogues in the database GenBank.

  7. [Characterization of 5S rRNA gene sequence and secondary structure in gymnosperms].

    Science.gov (United States)

    Liu, Zhan-Lin; Zhang, Da-Ming; Wang, Xiao-Ru

    2003-01-01

    In higher plants the primary and the secondary structures of 5S ribosomal RNA gene are considered highly conservative. Little is known about the 5S rRNA gene structure, organization and variation in gyimnosperms. In this study we analyzed sequence and structure variation of 5S rRNA gene in Pinus through cloning and sequencing multiple copies of 5S rDNA repeats from individual trees of five pines, P. bungeana, P. tabulaeformis, P. yunnanensis, P. massoniana and P. densata. Pinus bungeana is from the subgenus Strobus while the other four are from the subgenus Pinus (diploxylon pines). Our results revealed variations in both primary and secondary structure among copies of 5S rDNA within individual genomes and between species. 5S rRNA gene in Pinus is 120 bp long in most of the 122 clones we sequenced except for one or two deletions in three clones. Among these clones 50 unique sequences were identified and they were shared by different pine species. Our sequences were compared to 13 sequences each representing a different gymnosperm species, and to six sequences representing both angiosperm monocots and dicots. Average sequence similarity was 97.1% among Pinus species and 94.3% between Pinus and other gymnosperms. Between gymnosperms and angiosperms the sequence similarity decreased to 88.1%. Similar to other molecular data, significant sequence divergence was found between the two Pinus subgenera. The 5S gene tree (neighbor-joining tree) grouped the four diploxylon pines together and separated them distinctly from P. bungeana. Comparison of sequence divergence within individuals and between species suggested that concerted evolution has been very weak especially after the divergence of the four diploxylon pines. The phylogenetic information contained in the 5S rRNA gene is limited due to its shorter length and the difficulties in identifying orthologous and paralogous copies of rDNA multigene family further complicate its phylogenetic application. Pinus densata is a

  8. Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

    DEFF Research Database (Denmark)

    Hansen, M.C.; Nielsen, A.K.; Molin, Søren

    2001-01-01

    experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates...... the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell...... in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining...

  9. A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA.

    Science.gov (United States)

    Nishimura, Kenji; Ashida, Hiroki; Ogawa, Taro; Yokota, Akiho

    2010-09-01

    In plant chloroplasts, the ribosomal RNA (rRNA) of the large subunit of the ribosome undergoes post-maturation fragmentation processing. This processing consists of site-specific cleavage that generates gapped, discontinuous rRNA molecules. However, the molecular mechanism underlying introduction of the gap structure (the 'hidden break') is poorly understood. Here, we found that the DEAD box protein RH39 plays a key role in introduction of the hidden break into the 23S rRNA in Arabidopsis chloroplasts. Genetic screening for an Arabidopsis plant with a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase identified an RH39 mutant. The levels of other chloroplast-encoded photosynthetic proteins were also severely reduced. The reductions were not due to a failure of transcription, but rather inefficiency in translation. RNA gel blotting revealed incomplete fragmentation of 23S rRNA in chloroplasts during maturation. In vitro analysis with recombinant RH39 suggested that the protein binds to the adjacent sequence upstream of the hidden break site to exert its function. We propose a molecular mechanism for the RH39-mediated fragmentation processing of 23S rRNA in chloroplasts.

  10. Binding site for Xenopus ribosomal protein L5 and accompanying structural changes in 5S rRNA.

    Science.gov (United States)

    Scripture, J Benjamin; Huber, Paul W

    2011-05-10

    The structure of the eukaryotic L5-5S rRNA complex was investigated in protection and interference experiments and is compared with the corresponding structure (L18-5S rRNA) in the Haloarcula marismortui 50S subunit. In close correspondence with the archaeal structure, the contact sites for the eukaryotic ribosomal protein are located primarily in helix III and loop C and secondarily in loop A and helix V. While the former is unique to L5, the latter is also a critical contact site for transcription factor IIIA (TFIIIA), accounting for the mutually exclusive binding of these two proteins to 5S RNA. The binding of L5 causes structural changes in loops B and C that expose nucleotides that contact the Xenopus L11 ortholog in H. marismortui. This induced change in the structure of the RNA reveals the origins of the cooperative binding to 5S rRNA that has been observed for the bacterial counterparts of these proteins. The native structure of helix IV and loop D antagonizes binding of L5, indicating that this region of the RNA is dynamic and also influenced by the protein. Examination of the crystal structures of Thermus thermophilus ribosomes in the pre- and post-translocation states identified changes in loop D and in the surrounding region of 23S rRNA that support the proposal that 5S rRNA acts to transmit information between different functional domains of the large subunit.

  11. A critical role for the non-coding 5S rRNA in regulating Mdmx stability

    Science.gov (United States)

    Li, Muyang; Gu, Wei

    2013-01-01

    Summary Both p53 and Mdmx are ubiquitinated and degraded by the same E3 ligase Mdm2; interestingly, however, while p53 is rapidly degraded by Mdm2, Mdmx is a stable protein in most of cancer cells. Thus, the mechanism by which Mdmx is degraded by Mdm2 needs further elucidation. Here, we identified the noncoding 5S rRNA as a major component of Mdmx-associated complexes from human cells. We show that 5S rRNA acts as a natural inhibitor of Mdmx degradation by Mdm2. RNAi-mediated knockdown of endogenous 5S rRNA, while not affecting p53 levels, significantly induces Mdmx degradation and subsequently, activates p53-dependent growth arrest. Notably, 5S rRNA binds the RING domain of Mdmx and blocks its ubiquitination by Mdm2 whereas Mdm2-mediated p53 ubiquitination remains intact. These results provide insights into the differential effects on p53 and Mdmx by Mdm2 in vivo and reveal an critical role of noncoding 5S rRNA in modulating the p53-Mdmx axis. PMID:21925390

  12. [Strategy of selecting 16S rRNA hypervariable regions for metagenome-phylogenetic marker genes based analysis].

    Science.gov (United States)

    Zhang, Jun-yi; Zhu, Bing-chuan; Xu, Chao; Ding, Xiao; Li, Jun-feng; Zhang, Xue-gong; Lu, Zu-hong

    2015-11-01

    The advent of next generation sequencing technology enables parallel analysis of the whole microbial community from multiple samples. Particularly, sequencing 16S rRNA hypervariable tags has become the most efficient and cost-effective method for assessing microbial diversity. Due to its short read length of the 2nd-generation sequencing methods that cannot cover the full 16S rRNA genomic region, specific hypervariable regions or V-regions must be selected to act as the proxy. Over the past decade, selection of V-regions has not been consistent in assessing microbial diversity. Here we evaluated the current strategies of selecting 16S rRNA hypervariable regions for surveying microbial diversity. The environmental condition was considered as one of the important factors for selection of 16S rRNA hypervariable regions. We suggested that a pilot study to test different V-regions is required in bacterial diversity studies based on 16S rRNA genes.

  13. Modification of universal 12S rDNA primers for specific amplification of contaminated Taenia spp. (Cestoda) gDNA enabling phylogenetic studies.

    Science.gov (United States)

    von Nickisch-Rosenegk, M; Silva-Gonzalez, R; Lucius, R

    1999-10-01

    The construction of new specific tapeworm primers allowed synthesis of a 311-bp fragment of the mitochondrial 12S rDNA of 11 Taenia species and two Echinococcus species by PCR. After direct sequencing and construction of an alignment, the DNA sequences were calculated by three different phylogenetic algorithms. The phylogenetic trees were tested by 1000 bootstrap replications. Reliability of the nodes was tested by splits testing. All three algorithms revealed a clear monophyletic phylum Taenia, suggesting it may be paraphyletic with respect to the genus Echinococcus. Within the genus Taenia, the first secure group was composed by Taenia saginata, T. solium, T. serialis, T. ovis and T. hydatigena. A delimited second group was formed by T. martis, T. taeniaeformis, T. mustelae and T. parva. All of them were opposed to the genus Echinococcus using other cyclophyllideans as an outgroup. In this study Echinococcus was used as an outgroup, being the closest species against which the ingroup could be routed. The findings of this publication reflect Verster's basic morphologically based grouping of the Taeniidae.

  14. Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

    Science.gov (United States)

    Dorsch, M; Lane, D; Stackebrandt, E

    1992-01-01

    The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.

  15. Trends in evolution of 5S rRNA of deuterostomes: bases and homogeneous clusters

    Directory of Open Access Journals (Sweden)

    Sandra Maria Rodrigues Subacius

    2002-01-01

    Full Text Available Evolution of metazoan 5S rRNA sequences was analyzed through base composition and types, location and frequency of clustered bases. Characters from sequences of protostomes did not show regular trends as compared with paleontology dating or organism complexity. Trends of increasing G and C, stronger in G clusters, and decreasing A and U, were detected in deuterostomes, in parallel with evolution of complexity. The multifunctional domain 71-104 was highlighted among conserved stretches. Clusters of C were typical of helices. Those of G were longer, extending from helices into loops or related to bulges, which is suggestive of functional significance. Deuterostomian trends were installed early in the lineage and reached full development in aquatic organisms, not increasing further after reptiles. It can be suggested that ribosomal RNA structures participated in deuterostomian high regulatory complexity, either specifically or as part of the widespread processes of chromosomal regionalization.

  16. Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of common Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to h1 and the h3 haplotype is found only in the coastal waters of China. AG transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

  17. Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on genetic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the relationship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K.bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

  18. Functional interactions within 23S rRNA involving the peptidyltransferase center

    DEFF Research Database (Denmark)

    Douthwaite, S

    1992-01-01

    A molecular genetic approach has been employed to investigate functional interactions within 23S rRNA. Each of the three base substitutions at guanine 2032 has been made. The 2032A mutation confers resistance to the antibiotics chloramphenicol and clindamycin, which interact with the 23S r...... that also confer antibiotic resistance. Both the domain II deletion and the 2057A mutation relieve the hypersensitive effect of the 2032A mutation, producing an erythromycin-resistant phenotype; in addition, the combination of the 2032A and 2057A mutations confers a higher level of chloramphenicol...... and chloramphenicol. Introduction of the domain II deletion into these double-mutation constructs gives rise to erythromycin resistance. The results are interpreted as indicating that position 2032 interacts with the peptidyltransferase loop and that there is a functional connection between domains II and V....

  19. Fluoroscence in situ hybridization of chicken intestinal samples with bacterial rRNA targeted oligonucleotide probes

    DEFF Research Database (Denmark)

    Olsen, Katja Nyholm; Francesch, M.; Christensen, Henrik

    2006-01-01

    The objective was to develop a fast and accurate molecular method for the quantification of the intestinal flora in chickens by rRNA fluorescence in situ hybridization (FISH). Seven weeks old conventionally reared Lohmann hens were used to set up the method. To sample ileal intestinal content......, the distal part from Meckels diverticulum to the ileo-caecal junction was removed. Fixation was performed in ethanol and phosphate buffered saline. After washing by centrifugation, the sample was resuspended in pre-heated hybridization buffer with oligonucleotide probe labelled with Cy3 (10ng/µl). The cells...... were hybridized for 24-72h, centrifuged, washed with pre-heated hybridization buffer, centrifuged and resuspended in Millipore quality water before filtration onto a 0.22 µm black polycarbonate filter. The probes used in this study were, LGC354A, LGC354B, LGC354C, Strc493, Bacto1080, Sal3, Chis150, EUB...

  20. PHYLOGENETIC STATUS OF BABYLONIA ZEYLANICA (FAMILY BABYLONIIDAE BASED ON 18S rRNA GENE FRAGMENT

    Directory of Open Access Journals (Sweden)

    Vaithilingam RAVITCHANDIRANE

    2013-12-01

    Full Text Available Neogastropoda, highly diversed group of predatory marine snails, often been confused by shell colour and design pattern for identification. Gastropod resources which became economically important in India during the last decade are the whelk. The species Babylonia zeylanica of the family Babyloniidae began to be fished and exported from the country to China, Singapore, Thailand and Europe. This paper reports the molecular study of the group published to date with eight families of neogastropod taxa. For this study the 18S rRNA gene of B. zeylanica and other published data were collected from the GenBank. Kimura-2-Parameter genetic distance, nucleotide composition and neighbour joining analyses were conducted in all the eight families. The result clearly shows that Babyloniidae is clustered closely with Columbellidae of super family of Buccinoidea. Further additional gene data and increased sampling is warranted to give new insights into the phylogenetic relationships of Neogastropoda.

  1. discussion on validity of rana maoershanensis based on partial sequence of 16s rrna gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    rana maoershanensis found in mt.maoershan in guangxi,china was reported as a new species in 2007,but there was no molecular data for this frog.the partial sequences (543 bp) of 16s rrna gene from 12 specimens of 3 brown frog species (rana hanluica,r.maoershanensis and r.chensinensis) were analyzed with 17 specimens of 9 species from genbank.the nucleotide sequence divergence between r.maoershanensis and the other brown frog species were 4.5%-6.5%,with 22-30 nucleotide substitutions at this locus.the phylogenetic relationships based on mp,ml,and bayesian inference indicate that the brown frogs from southern china were diverged into three groups (clades a,b and c).r.maoershanensis was clustered together a well-supported subclade (b-l).it is suggested that r.maoershanensis is a valid species.

  2. Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA

    Science.gov (United States)

    Ciganda, Martin; Williams, Noreen

    2012-01-01

    P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC. PMID:22253864

  3. Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species.

    Science.gov (United States)

    Dey, S; Singh, V P; Kumar, A A; Sharma, B; Srivastava, S K; Singh, Nem

    2007-08-01

    The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).

  4. Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis).

    Science.gov (United States)

    Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

    2005-03-01

    Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.

  5. 16S rRNA gene sequencing as a tool to study microbial populations in foods and process environments

    DEFF Research Database (Denmark)

    Buschhardt, Tasja; Hansen, Tina Beck; Bahl, Martin Iain

    2015-01-01

    and their role in food safety. During method optimization, we have identified several factors which distort the characterization of microbial populations, including DNA extraction methods, DNA polymerases, and most importantly the analyzed fragment of the 16S rRNA gene. Methods: This study investigated microbial...... reference. Results: Taxonomic assignments and abundances of sequences in the total community and in the Enterobacteriaceae subpopulation were affected by the 16S rRNA gene variable region, DNA extraction methods, and polymerases chosen. However, community compositions were very reproducible when the same......Introduction: Methodological constraints during culturing and biochemical testing have left the true microbiological diversity of foods and process environments unexplored. Culture-independent molecular methods, such as 16S rRNA gene sequencing, may provide deeper insight into microbial communities...

  6. Characterization of a novel association between two trypanosome-specific proteins and 5S rRNA.

    Directory of Open Access Journals (Sweden)

    Martin Ciganda

    Full Text Available P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC.

  7. Characterization of a novel association between two trypanosome-specific proteins and 5S rRNA.

    Science.gov (United States)

    Ciganda, Martin; Williams, Noreen

    2012-01-01

    P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC.

  8. Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction

    DEFF Research Database (Denmark)

    Recht, M I; Douthwaite, S; Dahlquist, K D

    1999-01-01

    Decoding of genetic information occurs upon interaction of an mRNA codon-tRNA anticodon complex with the small subunit of the ribosome. The ribosomal decoding region is associated with highly conserved sequences near the 3' end of 16 S rRNA. The decoding process is perturbed by the aminoglycoside...... of universally conserved nucleotides at 1406 to 1408 and 1494 to 1495 in the decoding region of plasmid-encoded bacterial 16 S rRNA. Phenotypic changes range from the benign effect of U1406-->A or A1408-->G substitutions, to the highly deleterious 1406G and 1495 mutations that assemble into 30 S subunits...... but are defective in forming functional ribosomes. Changes in the local conformation of the decoding region caused by these mutations were identified by chemical probing of isolated 30 S subunits. Ribosomes containing 16 S rRNA with mutations at positions 1408, 1407+1494, or 1495 had reduced affinity...

  9. Characterization of the 18S rRNA gene for designing universal eukaryote specific primers.

    Science.gov (United States)

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

  10. Characterization of the 18S rRNA gene for designing universal eukaryote specific primers.

    Directory of Open Access Journals (Sweden)

    Kenan Hadziavdic

    Full Text Available High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

  11. Differential identification of Entamoeba spp. based on the analysis of 18S rRNA.

    Science.gov (United States)

    Santos, Helena Lúcia Carneiro; Bandea, Rebecca; Martins, Luci Ana Fernandes; de Macedo, Heloisa Werneck; Peralta, Regina Helena Saramago; Peralta, Jose Mauro; Ndubuisi, Mackevin I; da Silva, Alexandre J

    2010-03-01

    Entamoeba histolytica is known to cause intestinal and extra-intestinal disease while the other Entamoeba species are not considered to be pathogenic. However, all Entamoeba spp. should be reported when identified in clinical samples. Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features overlap. E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii are morphologically identical but can be differentiated using molecular tools. We developed a polymerase chain reaction (PCR) procedure followed by DNA sequencing of specific regions of 18S rRNA gene to differentiate the Entamoeba spp. commonly found in human stools. This approach was used to analyze 45 samples from cases evaluated for the presence of Entamoeba spp. by microscopy and a real-time PCR method capable of differential detection of E. histolytica and E. dispar. Our results demonstrated an agreement of approximately 98% (45/44) between the real-time PCR for E. histolytica and E. dispar and the 18S rRNA analysis described here. Five previously negative samples by microscopy revealed the presence of E. dispar, E. hartmanii, or E. coli DNA. In addition, we were able to detect E. hartmanii in a stool sample that had been previously reported as negative for Entamoeba spp. by microscopy. Further microscopic evaluation of this sample revealed the presence of E. hartmanii cysts, which went undetected during the first microscopic evaluation. This PCR followed by DNA sequencing will be useful to refine the diagnostic detection of Entamoeba spp. in stool and other clinical specimens.

  12. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing.

    Science.gov (United States)

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4-76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating.

  13. Characterization of the 18S rRNA Gene for Designing Universal Eukaryote Specific Primers

    Science.gov (United States)

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M.; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies. PMID:24516555

  14. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    Science.gov (United States)

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

  15. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

    Science.gov (United States)

    Olson, Nathan D; Lund, Steven P; Zook, Justin M; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B

    2015-03-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies.

  16. Phylogenetic relationships within the family Halomonadaceae based on comparative 23S and 16S rRNA gene sequence analysis.

    Science.gov (United States)

    de la Haba, Rafael R; Arahal, David R; Márquez, M Carmen; Ventosa, Antonio

    2010-04-01

    A phylogenetic study of the family Halomonadaceae was carried out based on complete 16S rRNA and 23S rRNA gene sequences. Several 16S rRNA genes of type strains were resequenced, and 28 new sequences of the 23S rRNA gene were obtained. Currently, the family includes nine genera (Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola and Zymobacter). These genera are phylogenetically coherent except Halomonas, which is polyphyletic. This genus comprises two clearly distinguished clusters: group 1 includes Halomonas elongata (the type species) and the species Halomonas eurihalina, H. caseinilytica, H. halmophila, H. sabkhae, H. almeriensis, H. halophila, H. salina, H. organivorans, H. koreensis, H. maura and H. nitroreducens. Group 2 comprises the species Halomonas aquamarina, H. meridiana, H. axialensis, H. magadiensis, H. hydrothermalis, H. alkaliphila, H. venusta, H. boliviensis, H. neptunia, H. variabilis, H. sulfidaeris, H. subterranea, H. janggokensis, H. gomseomensis, H. arcis and H. subglaciescola. Halomonas salaria forms a cluster with Chromohalobacter salarius and the recently described genus Salinicola, and their taxonomic affiliation requires further study. More than 20 Halomonas species are phylogenetically not within the core constituted by the Halomonas sensu stricto cluster (group 1) or group 2 and, since their positions on the different phylogenetic trees are not stable, they cannot be recognized as additional groups either. In general, there is excellent agreement between the phylogenies based on the two rRNA gene sequences, but the 23S rRNA gene showed higher resolution in the differentiation of species of the family Halomonadaceae.

  17. Defining reference sequences for Nocardia species by similarity and clustering analyses of 16S rRNA gene sequence data.

    Directory of Open Access Journals (Sweden)

    Manal Helal

    Full Text Available BACKGROUND: The intra- and inter-species genetic diversity of bacteria and the absence of 'reference', or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia. METHODS: A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization. RESULTS: The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52% corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as 'centroids' in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578. CONCLUSION: The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra

  18. SSU rRNA reveals a sequential increase in shell complexity among the euglyphid testate amoebae (Rhizaria: Euglyphida)

    DEFF Research Database (Denmark)

    Lara, Enrique; Heger, Thierry J; Mitchell, Edward A D;

    2007-01-01

    The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study...... aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows...

  19. Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

    Science.gov (United States)

    Burlibașa, Liliana; Suciu, Ilinca

    2015-12-01

    Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

  20. Escherichia coli Vertebral Osteomyelitis Diagnosed According to Broad-range 16S rRNA Gene Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Shibata, Satoshi; Tanizaki, Ryutaro; Watanabe, Koji; Makabe, Kenta; Shoda, Naoki; Kutsuna, Satoshi; Nagamatsu, Maki; Oka, Shinichi; Ohmagari, Norio

    2015-01-01

    Identifying the causative agent of pyogenic osteomyelitis is often challenging, especially when antibiotics are administered before a biopsy. We herein present a case of osteomyelitis in the cervical vertebrae presenting with progressive paralytic symptoms, in which we successfully identified Escherichia coli from a biopsy specimen using broad-range 16S rRNA gene polymerase chain reaction (PCR) even though sensitive antibiotics had been used for more than 50 days before the biopsy. Broad-range 16S rRNA gene PCR is a useful diagnostic method, especially when prebiopsy antibiotics are unavoidably used for a clinically unstable state.

  1. Changes in growth, rRNA content, and cell morphology of Listeria monocytogenes induced by CO2 up- and downshift

    DEFF Research Database (Denmark)

    Jydegaard-Axelsen, A.M.; Aaes-Jorgensen, A.; Koch, A.G.;

    2005-01-01

    Cell morphology, rRNA content, and growth were examined for Listeria monocytogenes LO28 and EGD, respectively, grown in brain-heart infusion (BHI) and on slices of sausage at 10degreesC in 100% CO2, 100% N-2, and air. In CO2, filamentous cells were formed by both strains on sausage slices and by L...... unchanged. On sausage slices, the number of colony forming units also increased rapidly for both strains in response to CO2 downshift. Large variations in rRNA content of individual cells were observed in the tested scenarios. The results demonstrate the risk of underestimating the number of infectious...

  2. Changes in growth, rRNA content, and cell morphology of Listeria monocytogenes induced by CO2 up- and downshift

    DEFF Research Database (Denmark)

    Jydegaard-Axelsen, A.M.; Aaes-Jorgensen, A.; Koch, A.G.

    2005-01-01

    Cell morphology, rRNA content, and growth were examined for Listeria monocytogenes LO28 and EGD, respectively, grown in brain-heart infusion (BHI) and on slices of sausage at 10degreesC in 100% CO2, 100% N-2, and air. In CO2, filamentous cells were formed by both strains on sausage slices and by L...... unchanged. On sausage slices, the number of colony forming units also increased rapidly for both strains in response to CO2 downshift. Large variations in rRNA content of individual cells were observed in the tested scenarios. The results demonstrate the risk of underestimating the number of infectious...

  3. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

    Science.gov (United States)

    Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martín, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco

    2013-01-01

    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  4. Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol.

    Directory of Open Access Journals (Sweden)

    Christian Milani

    Full Text Available Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.

  5. Concurrent speciation in the eastern woodland salamanders (Genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12s genes.

    Science.gov (United States)

    Highton, Richard; Hastings, Amy Picard; Palmer, Catherine; Watts, Richard; Hass, Carla A; Culver, Melanie; Arnold, Stevan J

    2012-05-01

    Salamanders of the North American plethodontid genus Plethodon are important model organisms in a variety of studies that depend on a phylogenetic framework (e.g., chemical communication, ecological competition, life histories, hybridization, and speciation), and consequently their systematics has been intensively investigated over several decades. Nevertheless, we lack a synthesis of relationships among the species. In the analyses reported here we use new DNA sequence data from the complete nuclear albumin gene (1818 bp) and the 12s mitochondrial gene (355 bp), as well as published data for four other genes (Wiens et al., 2006), up to a total of 6989 bp, to infer relationships. We relate these results to past systematic work based on morphology, allozymes, and DNA sequences. Although basal relationships show a strong consensus across studies, many terminal relationships remain in flux despite substantial sequencing and other molecular and morphological studies. This systematic instability appears to be a consequence of contemporaneous bursts of speciation in the late Miocene and Pliocene, yielding many closely related extant species in each of the four eastern species groups. Therefore we conclude that many relationships are likely to remain poorly resolved in the face of additional sequencing efforts. On the other hand, the current classification of the 45 eastern species into four species groups is supported. The Plethodon cinereus group (10 species) is the sister group to the clade comprising the other three groups, but these latter groups (Plethodon glutinosus [28 species], Plethodon welleri [5 species], and Plethodon wehrlei [2 species]) probably diverged from each other at approximately the same time. Copyright © 2012. Published by Elsevier Inc.

  6. Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

    DEFF Research Database (Denmark)

    Sahm, K.; MacGregor, BJ; Jørgensen, BB

    1999-01-01

    is a valuable tool for a more realistic assessment of SRB abundance in the natural environment. The distribution of SRB was investigated in a coastal marine sediment by hybridization of membrane-immobilized rRNA with oligonucleotide probes. As represented by general probe-target groups, SRB rRNA contributed...... between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulpho-bacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates...

  7. The participation of 5S rRNA in the co-translational formation of a eukaryotic 5S ribonucleoprotein complex

    OpenAIRE

    Lin, Elong; Lin, Sue-Wen; Lin, Alan

    2001-01-01

    The eukaryotic ribosomal 5S RNA–protein complex (5S rRNP) is formed by a co-translational event that requires 5S rRNA binding to the nascent peptide chain of eukaryotic ribosomal protein L5. Binding between 5S rRNA and the nascent chain is specific: neither the 5S rRNA nor the nascent chain of L5 protein can be substituted by other RNAs or other ribosomal proteins. The region responsible for binding 5S rRNA is located at positions 35–50 with amino acid sequence RLV...

  8. Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

    Science.gov (United States)

    Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

    2016-12-06

    The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development.

  9. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Science.gov (United States)

    Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping...

  10. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment

    Science.gov (United States)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  11. Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications

    DEFF Research Database (Denmark)

    Harrington, C.S.; On, Stephen L.W.

    1999-01-01

    Phylogenetic relationships of Campylobacter hyointestinalis subspecies were examined by means of 16S rRNA gene sequencing. Sequence similarities among C. hyointestinalis subsp. lawsonii strains exceeded 99.0 %, but values among C. hyointestinalis subsp. hyointestinalis strains ranged from 96.4...

  12. Campylobacter jejuni, an uncommon cause of splenic abscess diagnosed by 16S rRNA gene sequencing.

    Science.gov (United States)

    Seng, Piseth; Quenard, Fanny; Menard, Amélie; Heyries, Laurent; Stein, Andreas

    2014-12-01

    Splenic abscess is a rare disease that primarily occurs in patients with splenic trauma, endocarditis, sickle cell anemia, or other diseases that compromise the immune system. This report describes a culture-negative splenic abscess in an immunocompetent patient caused by Campylobacter jejuni, as determined by 16S rRNA gene sequencing.

  13. 16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, Human Papilloma Virus infection and surgical treatment

    NARCIS (Netherlands)

    Guerrero-Preston, Rafael; Godoy-Vitorino, Filipa; Jedlicka, Anne; Rodriguez-Hilario, Arnold; Gonzalez, Herminio; Bondy, Jessica; Lawson, Fahcina; Folawiyo, Oluwasina; Michailidi, Christina; Dziedzic, Amanda; Thangavel, Rajagowthamee; Hadar, Tal; Noordhuis, Maartje G.; Westra, William; Koch, Wayne; Sidransky, David

    2016-01-01

    Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropha

  14. Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

    Science.gov (United States)

    Bar-Yaacov, Dan; Frumkin, Idan; Yashiro, Yuka; Schlesinger, Orr; Bieri, Philipp; Greber, Basil; Ban, Nenad; Zarivach, Raz; Alfonta, Lital; Pilpel, Yitzhak; Suzuki, Tsutomu; Mishmar, Dan

    2016-01-01

    The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function. PMID:27631568

  15. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    Science.gov (United States)

    Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

    2013-12-01

    Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type.

  16. The secondary structure of large-subunit rRNA divergent domains, a marker for protist evolution

    DEFF Research Database (Denmark)

    Lenaers, G; Nielsen, Henrik; Engberg, J;

    1988-01-01

    ), Tetrahymena thermophila (ciliate), Physarum polycephalum and Dictyostelium discoideum (slime moulds), Crithidia fasciculata and Giardia lamblia (parasitic flagellates). The folding for the D3, D7a and D10 divergent domains has been refined and a consensus model for the protist 24-26S rRNA structure...

  17. Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1

    NARCIS (Netherlands)

    Fairley, Jennifer A.; Mitchell, Louise E.; Berg, Tracy; Kenneth, Niall S.; von Schubert, Conrad; Sillje, Herman H. W.; Medema, Rene H.; Nigg, Erich A.; White, Robert J.

    2012-01-01

    Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase Ill (pol Ill) through direct binding and phosphorylation of transcription factor Brit During interphase, Plk1 promotes

  18. Tomato (Solanum lycopersicum) variety discrimination and hybridization analysis based on the 5S rRNA region.

    Science.gov (United States)

    Sun, Yan-Lin; Kang, Ho-Min; Kim, Young-Sik; Baek, Jun-Pill; Zheng, Shi-Lin; Xiang, Jin-Jun; Hong, Soon-Kwan

    2014-05-04

    The tomato (Solanum lycopersicum) is a major vegetable crop worldwide. To satisfy popular demand, more than 500 tomato varieties have been bred. However, a clear variety identification has not been found. Thorough understanding of the phylogenetic relationship and hybridization information of tomato varieties is very important for further variety breeding. Thus, in this study, we collected 26 tomato varieties and attempted to distinguish them based on the 5S rRNA region, which is widely used in the determination of phylogenetic relations. Sequence analysis of the 5S rRNA region suggested that a large number of nucleotide variations exist among tomato varieties. These variable nucleotide sites were also informative regarding hybridization. Chromas sequencing of Yellow Mountain View and Seuwiteuking varieties indicated three and one variable nucleotide sites in the non-transcribed spacer (NTS) of the 5S rRNA region showing hybridization, respectively. Based on a phylogenetic tree constructed using the 5S rRNA sequences, we observed that 16 tomato varieties were divided into three groups at 95% similarity. Rubiking and Sseommeoking, Lang Selection Procedure and Seuwiteuking, and Acorn Gold and Yellow Mountain View exhibited very high identity with their partners. This work will aid variety authentication and provides a basis for further tomato variety breeding.

  19. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Science.gov (United States)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  20. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures

    NARCIS (Netherlands)

    S.A. Boers (Stefan A.); J.P. Hays (John P.); R. Jansen (Ruud)

    2015-01-01

    textabstract16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current

  1. Improved taxonomic assignment of human intestinal 16S rRNA sequences by a dedicated reference database

    NARCIS (Netherlands)

    Ritari, Jarmo; Salojärvi, Jarkko; Lahti, Leo; Vos, de Willem M.

    2015-01-01

    Background: Current sequencing technology enables taxonomic profiling of microbial ecosystems at high resolution and depth by using the 16S rRNA gene as a phylogenetic marker. Taxonomic assignation of newly acquired data is based on sequence comparisons with comprehensive reference databases to f

  2. 16S rRNA gene sequencing in routine identification of anaerobic bacteria isolated from blood cultures

    DEFF Research Database (Denmark)

    Justesen, Ulrik Stenz; Skov, Marianne Nielsine; Knudsen, Elisa;

    2010-01-01

    A comparison between conventional identification and 16S rRNA gene sequencing of anaerobic bacteria isolated from blood cultures in a routine setting was performed (n = 127). With sequencing, 89% were identified to the species level, versus 52% with conventional identification. The times...

  3. First report of neonatal bacteremia caused by "Haemophilus quentini" diagnosed by 16S rRNA gene sequencing, Italy.

    Science.gov (United States)

    Giufrè, Maria; Cardines, Rita; Degl'Innocenti, Roberto; Cerquetti, Marina

    2015-10-01

    We report the first case of neonatal bacteremia caused by a "Haemophilus quentini" isolate in Italy. The isolate was differentiated from H. influenzae by 16S rRNA sequencing and was characterized by comparison with the wild-type "H. quentini" CCUG 36167. Both isolates carried substitutions in penicillin-binding protein 3 but were susceptible to aminopenicillins.

  4. Direct 16S rRNA gene sequencing of polymicrobial culture-negative samples with analysis of mixed chromatograms

    DEFF Research Database (Denmark)

    Hartmeyer, Gitte N; Justesen, Ulrik S

    2010-01-01

    Two cases involving polymicrobial culture-negative samples were investigated by 16S rRNA gene sequencing, with analysis of mixed chromatograms. Fusobacterium necrophorum, Prevotella intermedia and Streptococcus constellatus were identified from pleural fluid in a patient with Lemierre's syndrome...

  5. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

    Science.gov (United States)

    LaFrentz, B R; Waldbieser, G C; Welch, T J; Shoemaker, C A

    2014-07-01

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

  6. Dynamics and persistence of Dead Sea microbial populations as shown by high-throughput sequencing of rRNA.

    Science.gov (United States)

    Rhodes, Matthew E; Oren, Aharon; House, Christopher H

    2012-04-01

    16S rRNA amplicon libraries from a haloarchaeal bloom in the hypersaline Dead Sea in 1992 were analyzed together with the 2007 residual population and simulated blooms in experimental mesocosms. Significant population shifts were observed during the bloom, and surprisingly a signature from the bloom was retained 15 years later.

  7. The final step in 5.8S rRNA processing is cytoplasmic in Saccharomyces cerevisiae.

    Science.gov (United States)

    Thomson, Emma; Tollervey, David

    2010-02-01

    The 18S rRNA component of yeast (Saccharomyces cerevisiae) 40S ribosomes undergoes cytoplasmic 3' cleavage following nuclear export, whereas exported pre-60S subunits were believed to contain only mature 5.8S and 25S rRNAs. However, in situ hybridization detected 3'-extended forms of 5.8S rRNA in the cytoplasm, which were lost when Crm1-dependent preribosome export was blocked by treatment with leptomycin B (LMB). LMB treatment rapidly blocked processing of 6S pre-rRNA to 5.8S rRNA, leading to TRAMP-dependent pre-rRNA degradation. The 6S pre-rRNA was coprecipitated with the 60S export adapter Nmd3 and cytoplasmic 60S synthesis factor Lsg1. The longer 5.8S+30 pre-rRNA (a form of 5.8S rRNA 3' extended by approximately 30 nucleotides) is processed to 6S by the nuclear exonuclease Rrp6, and nuclear pre-rRNA accumulated in the absence of Rrp6. In contrast, 6S to 5.8S processing requires the cytoplasmic exonuclease Ngl2, and cytoplasmic pre-rRNA accumulated in strains lacking Ngl2. We conclude that nuclear pre-60S particles containing the 6S pre-rRNA bind Nmd3 and Crm1 and are exported to the cytoplasm prior to final maturation by Ngl2.

  8. The Final Step in 5.8S rRNA Processing Is Cytoplasmic in Saccharomyces cerevisiae▿ †

    Science.gov (United States)

    Thomson, Emma; Tollervey, David

    2010-01-01

    The 18S rRNA component of yeast (Saccharomyces cerevisiae) 40S ribosomes undergoes cytoplasmic 3′ cleavage following nuclear export, whereas exported pre-60S subunits were believed to contain only mature 5.8S and 25S rRNAs. However, in situ hybridization detected 3′-extended forms of 5.8S rRNA in the cytoplasm, which were lost when Crm1-dependent preribosome export was blocked by treatment with leptomycin B (LMB). LMB treatment rapidly blocked processing of 6S pre-rRNA to 5.8S rRNA, leading to TRAMP-dependent pre-rRNA degradation. The 6S pre-rRNA was coprecipitated with the 60S export adapter Nmd3 and cytoplasmic 60S synthesis factor Lsg1. The longer 5.8S+30 pre-rRNA (a form of 5.8S rRNA 3′ extended by ∼30 nucleotides) is processed to 6S by the nuclear exonuclease Rrp6, and nuclear pre-rRNA accumulated in the absence of Rrp6. In contrast, 6S to 5.8S processing requires the cytoplasmic exonuclease Ngl2, and cytoplasmic pre-rRNA accumulated in strains lacking Ngl2. We conclude that nuclear pre-60S particles containing the 6S pre-rRNA bind Nmd3 and Crm1 and are exported to the cytoplasm prior to final maturation by Ngl2. PMID:20008552

  9. Analysis of the secondary structure of mitochondrial LSU rRNA of Peruvian land snails (Orthalicidae: Gastropoda

    Directory of Open Access Journals (Sweden)

    Jorge Ramirez Ramirez

    2011-05-01

    Full Text Available The alignment of ribosomal genes is difficult due to insertion and deletion events of nucleotides, making the alignment ambiguous. This can be overcome by using information from the secondary structure of ribosomal genes. The aim of this study was to evaluate the utility of the secondary structure in improving the alignment of the 16S rRNA gene in land snails of the family Orthalicidae. We assessed 10 Orthalicid species (five genera. Total DNA was isolated and the partial 16S rRNA gene was amplified and sequenced using internal primers. The sequences were aligned with ClustalX and manually corrected, in DCSE format, using the 16S rRNA secondary structure of Albinaria caerulea (Pulmonata: Clausiliidae. The sequences obtained ranged from 323 to 345 bp corresponding to parts of both domains IV and V of the 16S rRNA gene. The secondary structure was recovered by homology using RnaViz 2.0. Most stems are conserved, and in general the loops are more variable. The compensatory mutations in stems are related to maintenance of the structure. The absence of a bulge-stem-loop in domain V places the family Orthalicidae within the Heterobranchia.

  10. Spatial Distribution of Escherichia coli in the Mouse Large Intestine Inferred from rRNA In Situ Hybridization

    DEFF Research Database (Denmark)

    Poulsen, Lars Kærgaard; Lan, Fusheng; Sternberg, Claus

    1994-01-01

    Fluorescent oligonucleotide probes targeting rRNA were used to develop an in situ hybridization technique by which the spatial distribution of Escherichia coli in the large intestines of streptomycin-treated mice was determined. Single E. coli cells were identified in thin frozen sections from th...

  11. The antibiotics micrococcin and thiostrepton interact directly with 23S rRNA nucleotides 1067A and 1095A

    DEFF Research Database (Denmark)

    Rosendahl, G; Douthwaite, S

    1994-01-01

    The antibiotics thiostrepton and micrococcin bind to the GTPase region in domain II of 23S rRNA, and inhibit ribosomal A-site associated reactions. When bound to the ribosome, these antibiotics alter the accessibility of nucleotides 1067A and 1095A towards chemical reagents. Plasmid-coded Escheri...

  12. Molecular diagnosis of Kingella kingae pericarditis by amplification and sequencing of the 16S rRNA gene.

    Science.gov (United States)

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainardi, Jean-Luc

    2007-09-01

    Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing.

  13. The secondary structure of large-subunit rRNA divergent domains, a marker for protist evolution

    DEFF Research Database (Denmark)

    Lenaers, G; Nielsen, Henrik; Engberg, J

    1988-01-01

    ), Tetrahymena thermophila (ciliate), Physarum polycephalum and Dictyostelium discoideum (slime moulds), Crithidia fasciculata and Giardia lamblia (parasitic flagellates). The folding for the D3, D7a and D10 divergent domains has been refined and a consensus model for the protist 24-26S rRNA structure...

  14. Gradual reduction in rRNA transcription triggers p53 acetylation and apoptosis via MYBBP1A.

    Science.gov (United States)

    Kumazawa, Takuya; Nishimura, Kazuho; Katagiri, Naohiro; Hashimoto, Sayaka; Hayashi, Yuki; Kimura, Keiji

    2015-06-05

    The nucleolus, whose primary function is ribosome biogenesis, plays an essential role in p53 activation. Ribosome biogenesis is inhibited in response to cellular stress and several nucleolar proteins translocate from the nucleolus to the nucleoplasm, where they activate p53. In this study, we analysed precisely how impaired ribosome biogenesis regulates the activation of p53 by depleting nucleolar factors involved in rRNA transcription or rRNA processing. Nucleolar RNA content decreased when rRNA transcription was inhibited. In parallel with the reduced levels of nucleolar RNA content, the nucleolar protein Myb-binding protein 1 A (MYBBP1A) translocated to the nucleoplasm and increased p53 acetylation. The acetylated p53 enhanced p21 and BAX expression and induced apoptosis. In contrast, when rRNA processing was inhibited, MYBBP1A remained in the nucleolus and nonacetylated p53 accumulated, causing cell cycle arrest at the G1 phase by inducing p21 but not BAX. We propose that the nucleolus functions as a stress sensor to modulate p53 protein levels and its acetylation status, determining cell fate between cell cycle arrest and apoptosis by regulating MYBBP1A translocation.

  15. The antibiotics micrococcin and thiostrepton interact directly with 23S rRNA nucleotides 1067A and 1095A

    DEFF Research Database (Denmark)

    Rosendahl, G; Douthwaite, S

    1994-01-01

    The antibiotics thiostrepton and micrococcin bind to the GTPase region in domain II of 23S rRNA, and inhibit ribosomal A-site associated reactions. When bound to the ribosome, these antibiotics alter the accessibility of nucleotides 1067A and 1095A towards chemical reagents. Plasmid-coded Escheri...

  16. A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity

    NARCIS (Netherlands)

    Vos, M.; Quince, C.; Pijl, A.S.; De Hollander, M.; Kowalchuk, G.A.

    2012-01-01

    Background The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S r

  17. rRNA operons and genome size of 'Candidatus Liberibacter americanus', a bacterium associated with citrus huanglongbing in Brazil.

    Science.gov (United States)

    Wulff, N A; Eveillard, S; Foissac, X; Ayres, A J; Bové, J-M

    2009-08-01

    Huanglongbing is one of the most severe diseases of citrus worldwide and is associated with 'Candidatus (Ca.) Liberibacter africanus' in Africa, 'Ca. Liberibacter asiaticus' in Asia and the Americas (Brazil, USA and Cuba) and 'Ca. Liberibacter americanus' (Lam) in Brazil. In the absence of axenic cultures, genetic information on liberibacters is scarce. The sequences of the entire 23S rRNA and 5S rRNA genes from Lam have now been obtained, using a consensus primer designed on known tRNAMet sequences of rhizobia. The size of the Lam genome was determined by PFGE, using Lam-infected periwinkle plants for bacterial enrichment, and was found to be close to 1.31 Mbp. In order to determine the number of ribosomal operons on the Lam genome, probes designed to detect the 16S rRNA gene and the 3' end of the 23S rRNA gene were developed and used for Southern hybridization with I-CeuI-treated genomic DNA. Our results suggest that there are three ribosomal operons in a circular genome. Lam is the first liberibacter species for which such data are available.

  18. Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1

    NARCIS (Netherlands)

    Fairley, Jennifer A.; Mitchell, Louise E.; Berg, Tracy; Kenneth, Niall S.; von Schubert, Conrad; Sillje, Herman H. W.; Medema, Rene H.; Nigg, Erich A.; White, Robert J.

    2012-01-01

    Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase Ill (pol Ill) through direct binding and phosphorylation of transcription factor Brit During interphase, Plk1 promotes

  19. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures

    NARCIS (Netherlands)

    S.A. Boers (Stefan A.); J.P. Hays (John P.); R. Jansen (Ruud)

    2015-01-01

    textabstract16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitat

  20. Improving the microbial community reconstruction at the genus level by multiple 16S rRNA regions.

    Science.gov (United States)

    Wang, Shengqin; Sun, Beili; Tu, Jing; Lu, Zuhong

    2016-06-07

    16S rRNA genes have been widely used for phylogenetic reconstruction and the quantification of microbial diversity through the application of next-generation sequencing technology. However, long-read sequencing is still costly, while short-read sequencing carries less information for complex microbial community profiling; therefore, the applications of high throughput sequencing platforms still remain challenging in microbial community reconstruction analysis. Here, we developed a method to investigate the profile of aligned 16S rRNA gene sequences and to measure the proper region for microbial community reconstruction, as a step in creating a more efficient way to detect microorganism at the genus level. Finally, we found that each genus has its own preferential genus-specific amplicons for a genus assignment, which are not always located in hyper variable regions (HVRs). It was also noted that the rare genera should contribute less than dominant ones to the common profile of the aligned 16S rRNA sequences and have lower affinity to the common universal primer. Therefore, using multiple 16S rRNA regions rather than one "universal" region can significantly improve the ability of microbial community reconstruction. In addition, we found that a short fragment is suitable for most genera identifications, and the proper conserved regions used for primer design are larger than before. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. A new sequence data set of SSU rRNA gene for Scleractinia and its phylogenetic and ecological applications

    KAUST Repository

    Arrigoni, Roberto

    2016-11-27

    Scleractinian corals (i.e. hard corals) play a fundamental role in building and maintaining coral reefs, one of the most diverse ecosystems on Earth. Nevertheless, their phylogenies remain largely unresolved and little is known about dispersal and survival of their planktonic larval phase. The small subunit ribosomal RNA (SSU rRNA) is a commonly used gene for DNA barcoding in several metazoans, and small variable regions of SSU rRNA are widely adopted as barcode marker to investigate marine plankton community structure worldwide. Here, we provide a large sequence data set of the complete SSU rRNA gene from 298 specimens, representing all known extant reef coral families and a total of 106 genera. The secondary structure was extremely conserved within the order with few exceptions due to insertions or deletions occurring in the variable regions. Remarkable differences in SSU rRNA length and base composition were detected between and within acroporids (Acropora, Montipora, Isopora and Alveopora) compared to other corals. The V4 and V9 regions seem to be promising barcode loci because variation at commonly used barcode primer binding sites was extremely low, while their levels of divergence allowed families and genera to be distinguished. A time-calibrated phylogeny of Scleractinia is provided, and mutation rate heterogeneity is demonstrated across main lineages. The use of this data set as a valuable reference for investigating aspects of ecology, biology, molecular taxonomy and evolution of scleractinian corals is discussed.

  2. 16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, Human Papilloma Virus infection and surgical treatment

    NARCIS (Netherlands)

    Guerrero-Preston, Rafael; Godoy-Vitorino, Filipa; Jedlicka, Anne; Rodriguez-Hilario, Arnold; Gonzalez, Herminio; Bondy, Jessica; Lawson, Fahcina; Folawiyo, Oluwasina; Michailidi, Christina; Dziedzic, Amanda; Thangavel, Rajagowthamee; Hadar, Tal; Noordhuis, Maartje G.; Westra, William; Koch, Wayne; Sidransky, David

    2016-01-01

    Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropha

  3. Mapping important nucleotides in the peptidyl transferase centre of 23 S rRNA using a random mutagenesis approach

    DEFF Research Database (Denmark)

    Porse, B T; Garrett, R A

    1995-01-01

    assigned to the donor substrate binding site and a possible base-pairing interaction between the 3'-terminal sequence of the peptidyl-tRNA and the sequence psi/U-G-G2582, that is conserved in all the non-mitochondrial 23 S-like rRNA sequences, is proposed. Three sites that have been implicated in aminoacyl-tRNA...

  4. 23S rRNA gene mutations contributing to macrolide resistance in Campylobacter jejuni and Campylobacter coli

    Science.gov (United States)

    Operon specific 23S rRNA mutations affecting minimum inhibitory concentrations (MICs) of macrolides (erythromycin [ERY], azithromycin [AZM], tylosin [TYL]) and a lincosamide (clindamycin [CLI]) were examined in a collection of Campylobacter jejuni and C. coli isolates. The three copies of the Campy...

  5. Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.

    Directory of Open Access Journals (Sweden)

    Swee Hoe Ong

    Full Text Available The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90% in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.

  6. Bypassing rRNA methylation by RsmA/Dim1during ribosome maturation in the hyperthermophilic archaeon Nanoarchaeum equitans

    DEFF Research Database (Denmark)

    Seistrup, Kenneth H; Rose, Simon; Birkedal, Ulf

    2017-01-01

    In all free-living organisms a late-stage checkpoint in the biogenesis of the small ribosomal subunit involves rRNA modification by an RsmA/Dim1 methyltransferase. The hyperthermophilic archaeon Nanoarchaeum equitans, whose existence is confined to the surface of a second archaeon, Ignicoccus hos...

  7. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    Science.gov (United States)

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  8. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses.

    Directory of Open Access Journals (Sweden)

    Jiazhen Chen

    Full Text Available Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported.The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains.Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72% had intragenomic nucleotide polymorphisms (SNPs in multiple locations, and 13 strains (9.4% had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89 and 100% (19/19 of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3-100%. However, the inter-species similarities

  9. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

    Science.gov (United States)

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the

  10. Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.

    Science.gov (United States)

    Locati, Mauro D; Pagano, Johanna F B; Ensink, Wim A; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Zhu, Kongju; Spaink, Herman P; Girard, Geneviève; Rauwerda, Han; Jonker, Martijs J; Dekker, Rob J; Breit, Timo M

    2017-04-01

    5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci. © 2017 Locati et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  11. CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis.

    Science.gov (United States)

    Bai, Baoyan; Moore, Henna M; Laiho, Marikki

    2013-01-01

    CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.

  12. Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons

    Science.gov (United States)

    Pagano, Johanna F.B.; Ensink, Wim A.; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Zhu, Kongju; Spaink, Herman P.; Girard, Geneviève; Rauwerda, Han; Jonker, Martijs J.; Dekker, Rob J.

    2017-01-01

    5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci. PMID:28003516

  13. Efficient subtraction of insect rRNA prior to transcriptome analysis of Wolbachia-Drosophila lateral gene transfer

    Directory of Open Access Journals (Sweden)

    Kumar Nikhil

    2012-05-01

    Full Text Available Abstract Background Numerous methods exist for enriching bacterial or mammalian mRNA prior to transcriptome experiments. Yet there persists a need for methods to enrich for mRNA in non-mammalian animal systems. For example, insects contain many important and interesting obligate intracellular bacteria, including endosymbionts and vector-borne pathogens. Such obligate intracellular bacteria are difficult to study by traditional methods. Therefore, genomics has greatly increased our understanding of these bacteria. Efficient subtraction methods are needed for removing both bacteria and insect rRNA in these systems to enable transcriptome-based studies. Findings A method is described that efficiently removes >95% of insect rRNA from total RNA samples, as determined by microfluidics and transcriptome sequencing. This subtraction yielded a 6.2-fold increase in mRNA abundance. Such a host rRNA-depletion strategy, in combination with bacterial rRNA depletion, is necessary to analyze transcription of obligate intracellular bacteria. Here, transcripts were identified that arise from a lateral gene transfer of an entire Wolbachia bacterial genome into a Drosophila ananassae chromosome. In this case, an rRNA depletion strategy is preferred over polyA-based enrichment since transcripts arising from bacteria-to-animal lateral gene transfer may not be poly-adenylated. Conclusions This enrichment method yields a significant increase in mRNA abundance when poly-A selection is not suitable. It can be used in combination with bacterial rRNA subtraction to enable experiments to simultaneously measure bacteria and insect mRNA in vector and endosymbiont biology experiments.

  14. 16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.

    Directory of Open Access Journals (Sweden)

    Silvio D Brugger

    Full Text Available A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7 and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.

  15. Comparative metagenomic and rRNA microbial diversity characterization using archaeal and bacterial synthetic communities.

    Science.gov (United States)

    Shakya, Migun; Quince, Christopher; Campbell, James H; Yang, Zamin K; Schadt, Christopher W; Podar, Mircea

    2013-06-01

    Next-generation sequencing has dramatically changed the landscape of microbial ecology, large-scale and in-depth diversity studies being now widely accessible. However, determining the accuracy of taxonomic and quantitative inferences and comparing results obtained with different approaches are complicated by incongruence of experimental and computational data types and also by lack of knowledge of the true ecological diversity. Here we used highly diverse bacterial and archaeal synthetic communities assembled from pure genomic DNAs to compare inferences from metagenomic and SSU rRNA amplicon sequencing. Both Illumina and 454 metagenomic data outperformed amplicon sequencing in quantifying the community composition, but the outcome was dependent on analysis parameters and platform. New approaches in processing and classifying amplicons can reconstruct the taxonomic composition of the community with high reproducibility within primer sets, but all tested primers sets lead to significant taxon-specific biases. Controlled synthetic communities assembled to broadly mimic the phylogenetic richness in target environments can provide important validation for fine-tuning experimental and computational parameters used to characterize natural communities.

  16. Epigenetic silencing of nucleolar rRNA genes in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Maciej Pietrzak

    Full Text Available BACKGROUND: Ribosomal deficits are documented in mild cognitive impairment (MCI, which often represents an early stage Alzheimer's disease (AD, as well as in advanced AD. The nucleolar rRNA genes (rDNA, transcription of which is critical for ribosomal biogenesis, are regulated by epigenetic silencing including promoter CpG methylation. METHODOLOGY/PRINCIPAL FINDINGS: To assess whether CpG methylation of the rDNA promoter was dysregulated across the AD spectrum, we analyzed brain samples from 10 MCI-, 23 AD-, and, 24 age-matched control individuals using bisulfite mapping. The rDNA promoter became hypermethylated in cerebro-cortical samples from MCI and AD groups. In parietal cortex, the rDNA promoter was hypermethylated more in MCI than in advanced AD. The cytosine methylation of total genomic DNA was similar in AD, MCI, and control samples. Consistent with a notion that hypermethylation-mediated silencing of the nucleolar chromatin stabilizes rDNA loci, preventing their senescence-associated loss, genomic rDNA content was elevated in cerebrocortical samples from MCI and AD groups. CONCLUSIONS/SIGNIFICANCE: In conclusion, rDNA hypermethylation could be a new epigenetic marker of AD. Moreover, silencing of nucleolar chromatin may occur during early stages of AD pathology and play a role in AD-related ribosomal deficits and, ultimately, dementia.

  17. Dinoflagellate nuclear SSU rRNA phylogeny suggests multiple plastid losses and replacements.

    Science.gov (United States)

    Saldarriaga, J F; Taylor, F J; Keeling, P J; Cavalier-Smith, T

    2001-09-01

    Dinoflagellates are a trophically diverse group of protists with photosynthetic and non-photosynthetic members that appears to incorporate and lose endosymbionts relatively easily. To trace the gain and loss of plastids in dinoflagellates, we have sequenced the nuclear small subunit rRNA gene of 28 photosynthetic and four non-photosynthetic species, and produced phylogenetic trees with a total of 81 dinoflagellate sequences. Patterns of plastid gain, loss, and replacement were plotted onto this phylogeny. With the exception of the apparently early-diverging Syndiniales and Noctilucales, all non-photosynthetic dinoflagellates are very likely to have had photosynthetic ancestors with peridinin-containing plastids. The same is true for all dinoflagellates with plastids other than the peridinin-containing plastid: their ancestors have replaced one type of plastid for another, in some cases most likely through a non-photosynthetic intermediate. Eight independent instances of plastid loss and three of replacement can be inferred from existing data, but as more non-photosynthetic lineages are characterized these numbers will surely grow.

  18. Mitochondrial 16S rRNA sequence variations and phylogeny of the Chinese sisorid catfishes

    Institute of Scientific and Technical Information of China (English)

    GUO Xianguang; ZHANG Yaoguang; HE Shunping; CHEN Yiyu

    2004-01-01

    Partial sequences of mitochondrial 16S rRNA gene were obtained by PCR amplification for comparisons among nine species of glyptosternoid fishes and six species of non-glyptosternoids representing 10 sisorid genera. There are compositional biases in the A-rich unpaired regions and G-rich paired regions. A-G transitions are primarily responsible for the Ts/Tv bias in unpaired regions. The overall substitution rate in unpaired regions is almost two times higher than that in the paired regions. Saturation plots at comparable levels of sequence divergence demonstrate no saturation effects. Phylogenetic analyses using both maximum likelihood and Bayesian methods support the monophyly of Sisoridae. Chinese sisorid catfishes are composed of two major lineages, one represented by (Gagata (Bagarius, Glyptothorax)) and the other by "glyptosternoids + Pseudecheneis".The glyptosternoids may not be a monophyletic group. A previous hypothesis referring to Pseudecheneis as the sister group of monophyletic glyptosternoids, based on morphological evidence, is not supported by the molecular data.Pseudecheneis is shown to be a sister taxon of Glaridoglanis.Pareuchiloglanis might be paraphyletic with Pseudexostoma and Euchiloglanis. Our results also support the hypothesis that Pareuchiloglanis anteanalis might be considered as the synonyms of Pareuchiloglanis sinensis, and genus Euchiloglanis might have only one valid species, Euchiloglanis davidi.

  19. Pentamidine inhibits Coxiella burnetii growth and 23S rRNA intron splicing in vitro.

    Science.gov (United States)

    Minnick, Michael F; Hicks, Linda D; Battisti, James M; Raghavan, Rahul

    2010-10-01

    Coxiella burnetii is the bacterial agent of Q fever in humans. Acute Q fever generally manifests as a flu-like illness and is typically self-resolving. In contrast, chronic Q fever usually presents with endocarditis and is often life-threatening without appropriate antimicrobial therapy. Unfortunately, available options for the successful treatment of chronic Q fever are both limited and protracted (>18 months). Pentamidine, an RNA splice inhibitor used to treat fungal and protozoal infections, was shown to reduce intracellular growth of Coxiella by ca. 73% at a concentration of 1 microM (ca. 0.6 microg/mL) compared with untreated controls, with no detectable toxic effects on host cells. Bacterial targets of pentamidine include Cbu.L1917 and Cbu.L1951, two group I introns that disrupt the 23S rRNA gene of Coxiella, as demonstrated by the drug's ability to inhibit intron RNA splicing in vitro. Since both introns are highly conserved amongst all eight genotypes of the pathogen, pentamidine is predicted to be efficacious against numerous strains of C. burnetii. To our knowledge, this is the first report describing antibacterial activity for this antifungal/antiprotozoal agent.

  20. Phylogenetic Relationship of Phosphate Solubilizing Bacteria according to 16S rRNA Genes

    Directory of Open Access Journals (Sweden)

    Mohammad Bagher Javadi Nobandegani

    2015-01-01

    Full Text Available Phosphate solubilizing bacteria (PSB can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang oil palm field (University Putra Malaysia. Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer in an oil palm field.

  1. Formation of Tertiary Interactions during rRNA GTPase Center Folding.

    Science.gov (United States)

    Rau, Michael J; Welty, Robb; Tom Stump, W; Hall, Kathleen B

    2015-08-28

    The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg(2+) ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg(2+)-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg(2+) addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring from 5 ms to 4s and with global folding from 1 to 5s. Site-specific substitution with (15)N-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg(2+) and suggest that there are two initial sites of Mg(2+) ion association.

  2. Identification of sulfate reducers and Syntrophobacter sp. in anaerobic granular sludge by fatty-acid biomarkers and 16S rRNA probing

    NARCIS (Netherlands)

    Oude Elferink, S.J.W.H.; Boschker, H.T.S.; Stams, A.J.M.

    1998-01-01

    The sulfate-reducing bacterial sludge population in anaerobic bioreactors, treating different types of wastewater in the presence or absence of sulfate, was evaluated by polar-lipid fatty acid (PLFA) analyses, and by 16S rRNA dot-blot hybridizations using specific 16S rRNA- targeted oligonucleotide

  3. Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

    DEFF Research Database (Denmark)

    Sahm, K.; MacGregor, BJ; Jørgensen, BB

    1999-01-01

    between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulpho-bacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates...

  4. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Science.gov (United States)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  5. Mutations in 23S rRNA at the Peptidyl Transferase Center and Their Relationship to Linezolid Binding and Cross-Resistance

    DEFF Research Database (Denmark)

    Long, Katherine; Munck, Christian

    2010-01-01

    The oxazolidinone antibiotic linezolid targets the peptidyl transferase center (PTC) on the bacterial ribosome. Thirteen single and four double 23S rRNA mutations were introduced into a Mycobacterium smegmatis strain with a single rRNA operon. Converting bacterial base identity by single mutations...

  6. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  7. Identification of sulfate reducers and Syntrophobacter sp. in anaerobic granular sludge by fatty-acid biomarkers and 16S rRNA probing

    NARCIS (Netherlands)

    Oude Elferink, S.J.W.H.; Boschker, H.T.S.; Stams, A.J.M.

    1998-01-01

    The sulfate-reducing bacterial sludge population in anaerobic bioreactors, treating different types of wastewater in the presence or absence of sulfate, was evaluated by polar-lipid fatty acid (PLFA) analyses, and by 16S rRNA dot-blot hybridizations using specific 16S rRNA- targeted oligonucleotide

  8. Comparison of COBAS AMPLICOR Neisseria gonorrhoeae PCR, including confirmation with N. gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, D.S.; Bos, P.A.; van Zwet, A.A.; Voorst-Vader, P.C.; Schirm, J.

    2005-01-01

    : A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  9. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    2005-01-01

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased t

  10. The evaluation of an identification algorithm for Mycobacterium species using the 16S rRNA coding gene and rpoB

    Directory of Open Access Journals (Sweden)

    Yuko Kazumi

    2012-01-01

    Conclusions: The 16S rRNA gene identification is a rapid and prevalent method but still has some limitations. Therefore, the stepwise combination of rpoB with 16S rRNA gene analysis is an effective system for the identification of Mycobacterium species.

  11. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation

    DEFF Research Database (Denmark)

    Møller, Annette K.; Søborg, Ditte A.; Abu Al-Soud, Waleed;

    2013-01-01

    The bacterial community structures in High-Arctic snow over sea ice and an ice-covered freshwater lake were examined by pyrosequencing of 16S rRNA genes and 16S rRNA gene sequencing of cultivated isolates. Both the pyrosequence and cultivation data indicated that the phylogenetic composition...

  12. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    2005-01-01

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased t

  13. Comparison of COBAS AMPLICOR Neisseria gonorrhoeae PCR, including confirmation with N. gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, D.S.; Bos, P.A.; van Zwet, A.A.; Voorst-Vader, P.C.; Schirm, J.

    2005-01-01

    : A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  14. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    Science.gov (United States)

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  15. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation

    DEFF Research Database (Denmark)

    Møller, Annette; Søborg, Ditte Andreasen; Al-Soud, Waleed Abu

    2013-01-01

    The bacterial community structures in High-Arctic snow over sea ice and an ice-covered freshwater lake were examined by pyrosequencing of 16S rRNA genes and 16S rRNA gene sequencing of cultivated isolates. Both the pyrosequence and cultivation data indicated that the phylogenetic composition...

  16. Diversity of 18S rRNA Gene of 19 Wild Herbage Germplasms%19种野生牧草种质资源18S rRNA 基因的多态性

    Institute of Scientific and Technical Information of China (English)

    武玉祥; 田兵; 王啸; 陈彬; 冉雪琴; 王嘉福

    2014-01-01

    为了开发牧草资源,对贵州部分野生草本植物种质资源的遗传多样性进行研究。根据模式植物拟南芥18S rRNA 基因序列设计特异性引物,对贵州大学农场试验田自然生长的19种野生草本植物的18S rRNA 基因序列进行扩增、测序、构建进化树。结果表明:将获得的1000 bp 左右的 DNA 片段测序进行同源比对,共找到2280个碱基变异位点,分布在8个区段。据各样本18S rRNA 基因的遗传距离构建进化树推测,菊科、苋科和藜科之间存在较近的遗传相似性,豆科中三叶草属与豌豆属之间有较近的遗传距离。%In order to explore forage resource,the genetic diversity of 18 S rRNA gene in 19 kinds of wild herb germplasms were investigated,which were collected from the farm of Guizhou Unversity.The results showed that about 1000 bp fragments of 18 S rRNA genes were amplificated using specific primers based on the gene of Arabidopsis thaliana.After sequencing and homologous comparison,a total of 2 280 nucleotides were found out to be polymorphim sites.Phylogenetic tree of each family were constructed by similarity of 18S rRNA gene.The molecular classification of 19 kinds of wild herbs was consistent with its category based on morphological characteristics.Furthermore,the molecular classification could be useful to distinguish those similar species in morphology, and the genetic data suggested a close genetic relationship in three families,Compositae,Amaranthaceae and Chenopodiaceae.Trifolium and Pisum might share a high genetic similarty with each other.

  17. Nearly complete rRNA genes from 371 Animalia: updated structure-based alignment and detailed phylogenetic analysis.

    Science.gov (United States)

    Mallatt, Jon; Craig, Catherine Waggoner; Yoder, Matthew J

    2012-09-01

    This study presents a manually constructed alignment of nearly complete rRNA genes from most animal clades (371 taxa from ~33 of the ~36 metazoan phyla), expanded from the 197 sequences in a previous study. This thorough, taxon-rich alignment, available at http://www.wsu.edu/~jmallatt/research/rRNAalignment.html and in the Dryad Repository (doi: http://dx.doi.org/10.5061/dryad.1v62kr3q), is based rigidly on the secondary structure of the SSU and LSU rRNA molecules, and is annotated in detail, including labeling of the erroneous sequences (contaminants). The alignment can be used for future studies of the molecular evolution of rRNA. Here, we use it to explore if the larger number of sequences produces an improved phylogenetic tree of animal relationships. Disappointingly, the resolution did not improve, neither when the standard maximum-likelihood method was used, nor with more sophisticated methods that partitioned the rRNA into paired and unpaired sites (stem, loop, bulge, junction), or accounted for the evolution of the paired sites. For example, no doublet model of paired-site substitutions (16-state, 16A and 16B, 7A-F, or 6A-C models) corrected the placement of any rogue taxa or increased resolution. The following findings are from the simplest, standard, ML analysis. The 371-taxon tree only imperfectly supported the bilaterian clades of Lophotrochozoa and Ecdysozoa, and this problem remained after 17 taxa with unstably positioned sequences were omitted from the analysis. The problem seems to stem from base-compositional heterogeneity across taxa and from an overrepresentation of highly divergent sequences among the newly added taxa (e.g., sequences from Cephalopoda, Rotifera, Acoela, and Myxozoa). The rogue taxa continue to concentrate in two locations in the rRNA tree: near the base of Arthropoda and of Bilateria. The approximately uncertain (AU) test refuted the monophyly of Mollusca and of Chordata, probably due to long-branch attraction of the highly

  18. Molecular characterization of the full-length 23S and 5S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis.

    Science.gov (United States)

    Tazumi, Akihiro; Saito, Satoru; Sekizuka, Tsuyoshi; Murayama, Ohoshi; Takamiya, Shinzaburo; Moore, John E; Millar, B Cherie; Matsuda, Motoo

    2007-08-01

    An approximately 4.2 kbp region encoding 23S and 5S rRNA genes was identified when recombinant plasmid DNAs from two genomic DNA libraries and an inverse PCR product of Taylorella asinigenitalis UK-1 isolate were analyzed. Full-length genes of 23S rRNA (3,225 bp) and 5S rRNA (117 bp) of T. asinigenitalis are described. The present sequence analysis identified a non-coding hypothetically intrinsic transcription terminator region downstream of the 5S rRNA gene. The sequence, however, downstream of the 5S rRNA gene did not show any distal tRNA genes. Surprisingly, an intervening sequence (IVS) of 270 bp in length, including two specific tandem repeat units of 80 bp and one partial unit of 48 bp with unknown functions was identified in the first quarter of the 23S rRNA gene sequence. A second IVS of 70 bp in length was also identified in the central region of the 23S rRNA gene. In addition, by using PCR and sequencing procedures, two T. asinigenitalis isolates, UK-1 and UK-2, carried multiple IVSs in the first quarter and central regions. Moreover, the 23S rRNA fragmentation occurred in the UK-1 isolate. A phylogenetic analysis was first carried out based on the 23S rRNA sequence data from T. asinigenitalis UK-1 and 13 other beta-Proteobacteria. This is the first report of IVSs in the 23S rRNA gene from the beta-Proteobacteria.

  19. An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models

    Directory of Open Access Journals (Sweden)

    Shenkar Noa

    2009-08-01

    Full Text Available Abstract Background Tunicates have been recently revealed to be the closest living relatives of vertebrates. Yet, with more than 2500 described species, details of their evolutionary history are still obscure. From a molecular point of view, tunicate phylogenetic relationships have been mostly studied based on analyses of 18S rRNA sequences, which indicate several major clades at odds with the traditional class-level arrangements. Nonetheless, substantial uncertainty remains about the phylogenetic relationships and taxonomic status of key groups such as the Aplousobranchia, Appendicularia, and Thaliacea. Results Thirty new complete 18S rRNA sequences were acquired from previously unsampled tunicate species, with special focus on groups presenting high evolutionary rate. The updated 18S rRNA dataset has been aligned with respect to the constraint on homology imposed by the rRNA secondary structure. A probabilistic framework of phylogenetic reconstruction was adopted to accommodate the particular evolutionary dynamics of this ribosomal marker. Detailed Bayesian analyses were conducted under the non-parametric CAT mixture model accounting for site-specific heterogeneity of the evolutionary process, and under RNA-specific doublet models accommodating the occurrence of compensatory substitutions in stem regions. Our results support the division of tunicates into three major clades: 1 Phlebobranchia + Thaliacea + Aplousobranchia, 2 Appendicularia, and 3 Stolidobranchia, but the position of Appendicularia could not be firmly resolved. Our study additionally reveals that most Aplousobranchia evolve at extremely high rates involving changes in secondary structure of their 18S rRNA, with the exception of the family Clavelinidae, which appears to be slowly evolving. This extreme rate heterogeneity precluded resolving with certainty the exact phylogenetic placement of Aplousobranchia. Finally, the best fitting secondary-structure and CAT-mixture models

  20. Analysis of the Precursor rRNA Fractions of Rapidly Growing Mycobacteria: Quantification by Methods That Include the Use of a Promoter (rrnA P1) as a Novel Standard†

    OpenAIRE

    Menéndez, María del Carmen; Rebollo, María José; Núñez, María del Carmen; Cox, Robert A.; García, María Jesús

    2005-01-01

    Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their rrn operons. Mycobacterium chelonae and M. fortuitum are members of the rapidly growing mycobacterial group. They carry a total of five promoters each, encoded, respectively, by one and two rrn operons per genome. Quantification of precursor rrn transcriptional products (pre-rrn) has allowed detection of different promoter usage during cell growth. Bacteria gr...

  1. Sequence Analysis and Comparison of 16S rRNA, 23S rRNA and 16S/23S Intergenic Spacer Region of Greening Bacterium Associated with Yellowing Disease (Huanglongbing) of Kinnow Mandarin.

    Science.gov (United States)

    Gupta, K N; Baranwal, V K; Haq, Q M R

    2012-03-01

    High incidence (up to 40%) of symptoms of yellowing and yellow mottling was observed in 5-8 years old orchards of kinnow mandarin {Citrus reticulate Balanco ('King' × 'Willow mandarin')} in the Punjab state of India during a survey in January 2007. These symptoms are often confused with nutrient deficiency and other stress related disorders. However, a greening bacterium has been attributed to cause the disease. The disease was graft transmissible and sequencing of 16S rRNA, 16S/23S intergenic spacer region and 23S rRNA of the greening bacterium associated with yellowing disease in kinnow mandarin confirmed it to be Candidatus Liberibacter asiaticus ('Ca L. asiaticus') showing maximum identity of 95.9% with 'Ca L. asiaticus' from USA and Brazil in 16S rRNA. The study indicates definite association of 'Ca L. asiaticus' with yellowing/chlorotic mottling symptoms of greening disease of kinnow mandarin in Punjab state of India.

  2. Beyond 16S rRNA Community Profiling: Intra-Species Diversity in the Gut Microbiota

    Science.gov (United States)

    Ellegaard, Kirsten M.; Engel, Philipp

    2016-01-01

    Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities. PMID:27708630

  3. Distribution of DNA and localization of rRNA transcription in G2 phase nucleolus of Physarum polycephalum

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using electron microscopy, NAMA-Ur DNA selective staining and BrUTP incorporation, the nucleo lus ultrastructure, the distribution of DNA and the rRNA transcription sites in nucleolus of G2 phase Physarum poly cephalum were studied. The nucleolus was found to be different in structure from that of other plant cells. Fibrillar cen tern (FCs) were present in a large amount all over the nucleolus. DNA was distributed both in dense fibrillar components (DFC) and in FC. The DNA in the nucleolus was less condensed than that of the chromosome territory. These changes suggested that the transcription was active within the nucleolus. BrUTP incorporation localized the rRNA transcription in DFC and at the interface of FC and DFC, suggesting that the DNA in FC is in a storage form and only the rDNA in DFC is transcribed.

  4. Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus.

    Science.gov (United States)

    Dimarco, Eufrosina; Cascone, Eleonora; Bellavia, Daniele; Caradonna, Fabio

    2012-10-15

    We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus; this study, performed at DNA level only, lends itself as starting point to verify that these clusters could contain transcribed genes, then, to demonstrate the presence of heterogeneity at functional RNA level, also. In the present work we report in P. lividus ribosomes the existence of several transcribed variants of the 5S rRNA and we associate all transcribed variants to the cluster to which belong. Our finding is the first demonstration of the presence of high heterogeneity in functional 5S rRNA molecules in animal ribosomes, a feature that had been considered a peculiarity of some plants.

  5. Identification of new 18S rRNA strains of Babesia canis isolated from dogs with subclinical babesiosis.

    Science.gov (United States)

    Łyp, P; Adaszek, Ł; Furmaga, B; Winiarczyk, S

    2015-01-01

    In this study, we used PCR to detect and characterize B. canis from naturally infected dogs in Poland with subclinical babesiosis by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from ten dogs with subclinical babesiosis. A 559-bp fragment of the B. canis 18S rRNA gene was amplified by PCR. Sequencing of the PCR products led to the identification of a new variant of Babesia canis, differing from the previously detected protozoa genotypes (18S rRNA-A and 18S rRNA-B) with nucleotide substitutions in positions 150 and 151 of the tested gene fragment. The results indicate the emergence within the Polish territory of a new, previously unencountered Babesia canis genotype responsible for the development of subclinical babesiosis.

  6. Metagenomic data of fungal internal transcribed Spacer and 18S rRNA gene sequences from Lonar lake sediment, India.

    Science.gov (United States)

    Dudhagara, Pravin; Ghelani, Anjana; Bhavsar, Sunil; Bhatt, Shreyas

    2015-09-01

    The data in this article contains the sequences of fungal Internal Transcribed Spacer (ITS) and 18S rRNA gene from a metagenome of Lonar soda lake, India. Sequences were amplified using fungal specific primers, which amplified the amplicon lined between the 18S and 28S rRNA genes. Data were obtained using Fungal tag-encoded FLX amplicon pyrosequencing (fTEFAP) technique and used to analyze fungal profile by the culture-independent method. Primary analysis using PlutoF 454 pipeline suggests the Lonar lake mycobiome contained the 29 different fungal species. The raw sequencing data used to perform this analysis along with FASTQ file are located in the NCBI Sequence Read Archive (SRA) under accession No. SRX889598 (http://www.ncbi.nlm.nih.gov/sra/SRX889598).

  7. Analysis of rRNA Gene Methylation in Arabidopsis thaliana by CHEF-Conventional 2D Gel Electrophoresis.

    Science.gov (United States)

    Mohannath, Gireesha; Pikaard, Craig S

    2016-01-01

    Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb-9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes, and sub-chromosomal DNA fragments, etc. Here, we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes.

  8. Phylogeny of the cuttlefishes (Mollusca:Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

    Institute of Scientific and Technical Information of China (English)

    LIN Xiangzhi; ZHENG Xiaodong; XIAO Shu; WANG Rucai

    2004-01-01

    To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit I (COI) gene and partial 16S rRNA gene are sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method.Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea,which includes the families Sepiidae, Sepiolidae and Idiosepiidae, is not supported. From the two families of Sepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA and amino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, but do not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNA of cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at a higher taxonomic level (i.e., family).

  9. 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Weller, R.; Ward, D.M. (Montana State Univ., Bozeman (United States)); Weller, J.W. (Univ. of Montana, Missoula (United States))

    1991-04-01

    Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences form three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type.

  10. The Deinococcus-Thermus phylum and the effect of rRNA composition on phylogenetic tree construction

    Science.gov (United States)

    Weisburg, W. G.; Giovannoni, S. J.; Woese, C. R.

    1989-01-01

    Through comparative analysis of 16S ribosomal RNA sequences, it can be shown that two seemingly dissimilar types of eubacteria Deinococcus and the ubiquitous hot spring organism Thermus are distantly but specifically related to one another. This confirms an earlier report based upon 16S rRNA oligonucleotide cataloging studies (Hensel et al., 1986). Their two lineages form a distinctive grouping within the eubacteria that deserved the taxonomic status of a phylum. The (partial) sequence of T. aquaticus rRNA appears relatively close to those of other thermophilic eubacteria. e.g. Thermotoga maritima and Thermomicrobium roseum. However, this closeness does not reflect a true evolutionary closeness; rather it is due to a "thermophilic convergence", the result of unusually high G+C composition in the rRNAs of thermophilic bacteria. Unless such compositional biases are taken into account, the branching order and root of phylogenetic trees can be incorrectly inferred.

  11. Comparative analysis of mt LSU rRNA secondary structures of Odonates: structural variability and phylogenetic signal.

    Science.gov (United States)

    Misof, B; Fleck, G

    2003-12-01

    Secondary structures of the most conserved part of the mt 16S rRNA gene, domains IV and V, have been recently analysed in a comparative study. However, full secondary structures of the mt LSU rRNA molecule are published for only a few insect species. The present study presents full secondary structures of domains I, II, IV and V of Odonates and one representative of mayflies, Ephemera sp. The reconstructions are based on a comparative approach and minimal consensus structures derived from sequence alignments. The inferred structures exhibit remarkable similarities to the published Drosophila melanogaster model, which increases confidence in these structures. Structural variance within Odonates is homoplastic, and neighbour-joining trees based on tree edit distances do not correspond to any of the phylogenetically expected patterns. However, despite homoplastic quantitative structural variation, many similarities between Odonates and Ephemera sp. suggest promising character sets for higher order insect systematics that merit further investigations.

  12. Validation of a PCR Assay for Chlamydophila abortus rRNA gene detection in a murine model

    Directory of Open Access Journals (Sweden)

    Francielle Gibson da Silva-Zacarias

    2009-11-01

    Full Text Available Chlamydophila abortus (C. abortus is associated with reproductive problems in cattle, sheep, and goats. Diagnosis of C. abortus using embryonated chicken eggs or immortalized cell lines has a very low sensitivity. Polymerase chain reaction (PCR assays have been used to detect C. abortus infection in clinical specimens and organ fragments, such as placenta, fetal organs, vaginal secretions, and semen. The aim of this study was to develop a PCR assay for the amplification of an 856-bp fragment of the rRNA gene of the Chlamydiaceae family. The PCR assay was evaluated using organs from 15 mice experimentally infected with the S26/3 reference strain of C. abortus. The results of the rRNA PCR were compared to the results from another PCR system (Omp2 PCR that has been previously described for the Omp2 (outer major protein gene from the Chlamydiaceae family. From the 15 C. abortus-inoculated mice, 13 (K=0.84, standard error =0.20 tested positive using the rRNA PCR assay and 9 (K=0.55, standard error=0.18 tested positive using the Omp2 PCR assay. The detection limit, measured using inclusion-forming units (IFU, for C. abortus with the rRNA PCR (1.05 IFU was 100-fold lower than for the Omp2 PCR (105 IFU. The higher sensitivity of the rRNA PCR, as compared to the previously described PCR assay, and the specificity of the assay, demonstrated using different pathogenic microorganisms of the bovine reproductive system, suggest that the new PCR assay developed in this study can be used for the molecular diagnosis of C. abortus in abortion and other reproductive failures in bovines, caprines, and ovines.Chlamydophila abortus (C. abortus é frequentemente associada a distúrbios reprodutivos em bovinos, ovinos e caprinos. Para o diagnóstico, os métodos de cultivo em ovo embrionado de galinha e em células de linhagem contínua apresentam baixa sensibilidade. A reação em cadeia da polimerase (PCR tem sido utilizada em placenta, órgãos fetais, secre

  13. Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

    DEFF Research Database (Denmark)

    Nielsen, Jeppe Lund; Schramm, Andreas; Bernhard, Anne E.

    2004-01-01

      FLOW CYTOMETRY-ASSISTED CLONING OF SPECIFIC SEQUENCE MOTIFS FROM COMPLEX 16S RRNA GENE LIBRARIES Jeppe L. Nielsen,1 Andreas Schramm,1,2 Anne E. Bernhard,1 Gerrit J. van den Engh,3 and David A. Stahl1* Department of Civil and Environmental Engineering, University of Washington,1 and Institute...... for Systems Biology,3 Seattle, Washington, and Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany2 A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting......-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.  ...

  14. rRNA and Poly-β-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast and Famine Cycles

    Science.gov (United States)

    Frigon, Dominic; Muyzer, Gerard; van Loosdrecht, Mark; Raskin, Lutgarde

    2006-01-01

    Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-β-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological

  15. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform.

    Directory of Open Access Journals (Sweden)

    Hideyuki Tamaki

    Full Text Available BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1, after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

  16. Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing

    OpenAIRE

    Jarvis, Karen G.; White, James R; Grim, Christopher J.; Ewing, Laura; Ottesen, Andrea R; Beaubrun, Junia Jean-Gilles; Pettengill, James B.; Brown, Eric; Hanes, Darcy E.

    2015-01-01

    Background Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective...

  17. Detection and quantification of Dehalogenimonas and "Dehalococcoides" populations via PCR-based protocols targeting 16S rRNA genes.

    Science.gov (United States)

    Yan, Jun; Rash, Brian A; Rainey, Fred A; Moe, William M

    2009-12-01

    Members of the haloalkane dechlorinating genus Dehalogenimonas are distantly related to "Dehalococcoides" but share high homology in some variable regions of their 16S rRNA gene sequences. In this study, primers and PCR protocols intended to uniquely target Dehalococcoides were reevaluated, and primers and PCR protocols intended to uniquely target Dehalogenimonas were developed and tested. Use of the genus-specific primers revealed the presence of both bacterial groups in groundwater at a Louisiana Superfund site.

  18. Typification of virulent and low virulence Babesia bigemina clones by 18S rRNA and rap-1c.

    Science.gov (United States)

    Thompson, C; Baravalle, M E; Valentini, B; Mangold, A; Torioni de Echaide, S; Ruybal, P; Farber, M; Echaide, I

    2014-06-01

    The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones.

  19. The Identification of Discriminating Patterns from 16S rRNA Gene to Generate Signature for Bacillus Genus.

    Science.gov (United States)

    More, Ravi P; Purohit, Hemant J

    2016-08-01

    The 16S ribosomal RNA (16S rRNA) gene has been widely used for the taxonomic classification of bacteria. A molecular signature is a set of nucleotide patterns, which constitute a regular expression that is specific to each particular taxon. Our main goal was to identify discriminating nucleotide patterns in 16S rRNA gene and then to generate signatures for taxonomic classification. To demonstrate our approach, we used the phylum Firmicutes as a model using representative taxa Bacilli (class), Bacillales (order), Bacillaceae (family), and Bacillus (genus), according to their dominance at each hierarchical taxonomic level. We applied combined composite vector and multiple sequence alignment approaches to generate gene-specific signatures. Further, we mapped all the patterns into the different hypervariable regions of 16S rRNA gene and confirmed the most appropriate distinguishing region as V3-V4 for targeted taxa. We also examined the evolution in discriminating patterns of signatures across taxonomic levels. We assessed the comparative classification accuracy of signatures with other methods (i.e., RDP Classifier, KNN, and SINA). Results revealed that the signatures for taxa Bacilli, Bacillales, Bacillaceae, and Bacillus could correctly classify isolate sequences with sensitivity of 0.99, 0.97, 0.94, and 0.89, respectively, and specificity close to 0.99. We developed signature-based software DNA Barcode Identification (DNA BarID) for taxonomic classification that is available at website http://www.neeri.res.in/DNA_BarID.htm . This pattern-based study provides a deeper understanding of taxon-specific discriminating patterns in 16S rRNA gene with respect to taxonomic classification.

  20. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    Science.gov (United States)

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J; Vaulot, Daniel

    2011-04-28

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  1. Structural diversity of eukaryotic 18S rRNA and its impact on alignment and phylogenetic reconstruction.

    Science.gov (United States)

    Xie, Qiang; Lin, Jinzhong; Qin, Yan; Zhou, Jianfu; Bu, Wenjun

    2011-02-01

    Ribosomal RNAs are important because they catalyze the synthesis of peptides and proteins. Comparative studies of the secondary structure of 18S rRNA have revealed the basic locations of its many length-conserved and length-variable regions. In recent years, many more sequences of 18S rDNA with unusual lengths have been documented in GenBank. These data make it possible to recognize the diversity of the secondary and tertiary structures of 18S rRNAs and to identify the length-conserved parts of 18S rDNAs. The longest 18S rDNA sequences of almost every known eukaryotic phylum were included in this study. We illustrated the bioinformatics-based structure to show that, the regions that are more length-variable, regions that are less length-variable, the splicing sites for introns, and the sites of A-minor interactions are mostly distributed in different parts of the 18S rRNA. Additionally, this study revealed that some length-variable regions or insertion positions could be quite close to the functional part of the 18S rRNA of Foraminifera organisms. The tertiary structure as well as the secondary structure of 18S rRNA can be more diverse than what was previously supposed. Besides revealing how this interesting gene evolves, it can help to remove ambiguity from the alignment of eukaryotic 18S rDNAs and to improve the performance of 18S rDNA in phylogenetic reconstruction. Six nucleotides shared by Archaea and Eukaryota but rarely by Bacteria are also reported here for the first time, which might further support the supposed origin of eukaryote from archaeans.

  2. Deep sequencing of 16S rRNA gene to efficiently assess bacterial richness and the rare biosphere in soils

    OpenAIRE

    Terrat, Sébastien; Dequiedt, Samuel; Bachar, Dipankar; Christen, Richard; Mougel, Christophe; Lelievre, Mélanie; Maron, Pierre-Alain; Plassart, Pierre; Wincker, Patrick; Cruaud, C; Jolivet, Claudy; Arrouays, Dominique; Bispo, Antonio; Lemanceau, Philippe; Ranjard, Lionel

    2012-01-01

    Soil is one of the most important reservoirs of microbiological diversity on our planet and, above all, one of the last bastions of such biodiversity. Since two decades, numerous molecular tools have been developed to assess accurately this huge diversity directly from soil DNA. In this context, 16S rRNA gene is widely used to study microbial community taxonomic diversity, as it can be easily amplified by PCR, and large databases are available relating obtained sequences to bacteria...

  3. Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    Science.gov (United States)

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa

    2016-05-01

    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.

  4. 16s rRNA Identification of Pediococcus spp. from Broiler and Studies of Adherence Ability on Immobilized Mucus

    OpenAIRE

    Ema Damayanti; Lies Mira Yusiati; Achmad Dinoto

    2015-01-01

    The objectives of this research were to study taxonomical status of lactic acid bacteria (LAB) isolated from broiler and adherence ability on mucus in vitro. Molecular analysis was performed by analyzing 16S rRNA gene using universal primer. The adherence assay on mucus was carried out using microplate method with total plate count (TPC), absorbance (A550) and confirmed by scanning electron microscopy (SEM). The results of this studies revealed that three of LAB isolates have closed relation ...

  5. Molecular characterization of Sarcocystis species from Polish roe deer based on ssu rRNA and cox1 sequence analysis.

    Science.gov (United States)

    Kolenda, Rafał; Ugorski, Maciej; Bednarski, Michał

    2014-08-01

    Sarcocysts from four Polish roe deer were collected and examined by light microscopy, small subunit ribosomal RNA (ssu rRNA), and the subunit I of cytochrome oxidase (cox1) sequence analysis. This resulted in identification of Sarcocystis gracilis, Sarcocystis oviformis, and Sarcocystis silva. However, we were unable to detect Sarcocystis capreolicanis, the fourth Sarcocystis species found previously in Norwegian roe deer. Polish sarcocysts isolated from various tissues differed in terms of their shape and size and were larger than the respective Norwegian isolates. Analysis of ssu rRNA gene revealed the lack of differences between Sarcocystis isolates belonging to one species and a very low degree of genetic diversity between Polish and Norwegian sarcocysts, ranging from 0.1% for Sarcocystis gracilis and Sarcocystis oviformis to 0.44% for Sarcocystis silva. Contrary to the results of the ssu rRNA analysis, small intraspecies differences in cox1 sequences were found among Polish Sarcocystis gracilis and Sarcocystis silva isolates. The comparison of Polish and Norwegian cox1 sequences representing the same Sarcocystis species revealed similar degree of sequence identity, namely 99.72% for Sarcocystis gracilis, 98.76% for Sarcocystis silva, and 99.85% for Sarcocystis oviformis. Phylogenetic reconstruction and genetic population analyses showed an unexpected high degree of identity between Polish and Norwegian isolates. Moreover, cox1 gene sequences turned out to be more accurate than ssu rRNA when used to reveal phylogenetic relationships among closely related species. The results of our study revealed that the same Sarcocystis species isolated from the same hosts living in different geographic regions show a very high level of genetic similarity.

  6. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    OpenAIRE

    de los Reyes, F L; Ritter, W; Raskin, L.

    1997-01-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacteri...

  7. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    Directory of Open Access Journals (Sweden)

    Xiao Li Shi

    Full Text Available The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX, which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  8. Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene

    OpenAIRE

    2012-01-01

    Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from...

  9. Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

    Science.gov (United States)

    Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

    2015-09-01

    Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

  10. Diversity of Archaea in Icelandic hot springs based on 16S rRNA and chaperonin genes.

    Science.gov (United States)

    Mirete, Salvador; de Figueras, Carolina G; González-Pastor, Jose E

    2011-07-01

    The diversity of archaeal communities growing in four hot springs (65-90 °C, pH 6.5) was assessed with 16S rRNA gene primers specific for the domain Archaea. Overall, mainly uncultured members of the Desulfurococcales, the Thermoproteales and the Korarchaeota, were identified. Based on this diversity, a set of chaperonin heat-shock protein (Hsp60) gene sequences from different archaeal species were aligned to design two degenerate primer sets for the amplification of the chaperonin gene: Ths and Kor (which can also detect the korarchaeotal chaperonin gene from one of the samples). A phylogenetic tree was constructed using the chaperonin sequences retrieved and other sequences from cultured representatives. The Alpha and Beta paralogs of the chaperonin gene were observed within the main clades and orthologs among them. Cultivated representatives from these clades were assigned to either paralog in the chaperonin tree. Uncultured representatives observed in the 16S rRNA gene analysis were found to be related to the Desulfurococcales. The topologies of the 16S rRNA gene and chaperonin phylogenetic trees were compared, and similar phylogenetic relationships were observed. Our results suggest that the chaperonin Hsp60 gene may be used as a phylogenetic marker for the clades found in this extreme environment.

  11. TaxCollector: Modifying Current 16S rRNA Databases for the Rapid Classification at Six Taxonomic Levels

    Directory of Open Access Journals (Sweden)

    Eric W. Triplett

    2010-07-01

    Full Text Available The high level of conservation of 16S ribosomal RNA gene (16S rRNA in all Prokaryotes makes this gene an ideal tool for the rapid identification and classification of these microorganisms. Databases such as the Ribosomal Database Project II (RDP-II and the Greengenes Project offer access to sets of ribosomal RNA sequence databases useful in identification of microbes in a culture-independent analysis of microbial communities. However, these databases do not contain all of the taxonomic levels attached to the published names of the bacterial and archaeal sequences. TaxCollector is a set of scripts developed in Python language that attaches taxonomic information to all 16S rRNA sequences in the RDP-II and Greengenes databases. These modified databases are referred to as TaxCollector databases, which when used in conjunction with BLAST allow for rapid classification of sequences from any environmental or clinical source at six different taxonomic levels, from domain to species. The TaxCollector database prepared from the RDP-II database is an important component of a new 16S rRNA pipeline called PANGEA. The usefulness of TaxCollector databases is demonstrated with two very different datasets obtained using samples from a clinical setting and an agricultural soil. The six TaxCollector scripts are freely available on http://taxcollector.sourceforge.net and on http://www.microgator.org.

  12. A conserved alternative splicing event in plants reveals an ancient exonization of 5S rRNA that regulates TFIIIA.

    Science.gov (United States)

    Barbazuk, W Brad

    2010-01-01

    Uncovering conserved alternative splicing (AS) events can identify AS events that perform important functions. This is especially useful for identifying premature stop codon containing (PTC) AS isoforms that may regulate protein expression by being targets for nonsense mediated decay. This report discusses the identification of a PTC containing splice isoform of the TFIIIA gene that is highly conserved in land plants. TFIIIA is essential for RNA Polymerase III-based transcription of 5S rRNA in eukaryotes. Two independent groups have determined that the PTC containing alternative exon is ultraconserved and is coupled with nonsense-mediated mRNA decay. The alternative exon appears to have been derived by the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA. This provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized non-coding element.

  13. The B chromosomes of the African cichlid fish Haplochromis obliquidens harbour 18S rRNA gene copies

    Directory of Open Access Journals (Sweden)

    Martins Cesar

    2010-01-01

    Full Text Available Abstract Background Diverse plant and animal species have B chromosomes, also known as accessory, extra or supernumerary chromosomes. Despite being widely distributed among different taxa, the genomic nature and genetic behavior of B chromosomes are still poorly understood. Results In this study we describe the occurrence of B chromosomes in the African cichlid fish Haplochromis obliquidens. One or two large B chromosome(s occurring in 39.6% of the analyzed individuals (both male and female were identified. To better characterize the karyotype and assess the nature of the B chromosomes, fluorescence in situ hybridization (FISH was performed using probes for telomeric DNA repeats, 18S and 5S rRNA genes, SATA centromeric satellites, and bacterial artificial chromosomes (BACs enriched in repeated DNA sequences. The B chromosomes are enriched in repeated DNAs, especially non-active 18S rRNA gene-like sequences. Conclusion Our results suggest that the B chromosome could have originated from rDNA bearing subtelo/acrocentric A chromosomes through formation of an isochromosome, or by accumulation of repeated DNAs and rRNA gene-like sequences in a small proto-B chromosome derived from the A complement.

  14. Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

    Science.gov (United States)

    Fairley, Jennifer A; Mitchell, Louise E; Berg, Tracy; Kenneth, Niall S; von Schubert, Conrad; Silljé, Herman H W; Medema, René H; Nigg, Erich A; White, Robert J

    2012-02-24

    Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences.

    Science.gov (United States)

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-07-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences.

  16. Clinical Fusobacterium mortiferum Isolates Cluster with Undifferentiated Clostridium rectum Species Based on 16S rRNA Gene Phylogenetic Analysis.

    Science.gov (United States)

    Lee, Yangsoon; Eun, Chang Soo; Han, Dong Soo

    2016-05-01

    The most commonly encountered clinical Fusobacterium species are F. nucleatum and F. necrophorum; other Fusobacteria, such as F. mortiferum and F. varium, have occasionally been isolated from human specimens. Clostridium rectum is a gram-positive species characterized as a straight bacillus with oval sub-terminal spores. The close 16S rRNA gene sequence relationship of C. rectum with the genus Fusobacterium is unexpected given their very different phenotypic characteristics. Between 2014 and 2015, a total of 19 Fusobacterium isolates were recovered from the colonic tissue of 10 patients at a university hospital. All isolates were identified based on 16S rRNA gene sequencing. The phylogenetic relationship among these isolates was estimated using the neighbor-joining method and the Molecular Evolutionary Genetic Analysis (MEGA) version 6. Based on phylogenetic analysis, the F. mortiferum isolates clustered into two groups - F. mortiferum DSM 19809 (group I) and F. mortiferum ATCC 25557 (group II) - even though they are of the same species. Furthermore, the F. mortiferum DSM 19809 (group I) showed a close phylogenetic relationship with C. rectum, even though C. rectum is classified as a gram-positive spore-producing bacillus. C. rectum is clearly unrelated to the genus Clostridium as it shows highest 16S rRNA gene sequence similarity with species from the genus Fusobacterium Therefore, additional methods such as Gram staining and other biochemical methods should be performed for Fusobacterium identification.

  17. Direct evidence for redundant segmental replacement between multiple 18S rRNA genes in a single Prototheca strain.

    Science.gov (United States)

    Ueno, Ryohei; Huss, Volker A R; Urano, Naoto; Watabe, Shugo

    2007-11-01

    Informational genes such as those encoding rRNAs are related to transcription and translation, and are thus considered to be rarely subject to lateral gene transfer (LGT) between different organisms, compared to operational genes having metabolic functions. However, several lines of evidence have suggested or confirmed the occurrence of LGT of DNA segments encoding evolutionarily variable regions of rRNA genes between different organisms. In the present paper, we show, for the first time to our knowledge, that variable regions of the 18S rRNA gene are segmentally replaced by multiple copies of different sequences in a single strain of the green microalga Prototheca wickerhamii, resulting in at least 17 genotypes, nine of which were actually transcribed. Recombination between different 18S rRNA genes occurred in seven out of eight variable regions (V1-V5 and V7-V9) of eukaryotic small subunit (SSU) rRNAs. While no recombination was observed in V1, one to three different recombination loci were demonstrated for the other regions. Such segmental replacement was also implicated for helix H37, which is defined as V6 of prokaryotic SSU rRNAs. Our observations provide direct evidence for redundant recombination of an informational gene, which encodes a component of mature ribosomes, in a single strain of one organism.

  18. IMNGS: A comprehensive open resource of processed 16S rRNA microbial profiles for ecology and diversity studies.

    Science.gov (United States)

    Lagkouvardos, Ilias; Joseph, Divya; Kapfhammer, Martin; Giritli, Sabahattin; Horn, Matthias; Haller, Dirk; Clavel, Thomas

    2016-09-23

    The SRA (Sequence Read Archive) serves as primary depository for massive amounts of Next Generation Sequencing data, and currently host over 100,000 16S rRNA gene amplicon-based microbial profiles from various host habitats and environments. This number is increasing rapidly and there is a dire need for approaches to utilize this pool of knowledge. Here we created IMNGS (Integrated Microbial Next Generation Sequencing), an innovative platform that uniformly and systematically screens for and processes all prokaryotic 16S rRNA gene amplicon datasets available in SRA and uses them to build sample-specific sequence databases and OTU-based profiles. Via a web interface, this integrative sequence resource can easily be queried by users. We show examples of how the approach allows testing the ecological importance of specific microorganisms in different hosts or ecosystems, and performing targeted diversity studies for selected taxonomic groups. The platform also offers a complete workflow for de novo analysis of users' own raw 16S rRNA gene amplicon datasets for the sake of comparison with existing data. IMNGS can be accessed at www.imngs.org.

  19. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua; (NCI)

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  20. A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.

    Directory of Open Access Journals (Sweden)

    Michiel Vos

    Full Text Available BACKGROUND: The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria. METHODOLOGY/PRINCIPAL FINDINGS: Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster. CONCLUSIONS/SIGNIFICANCE: The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.

  1. Partial Sequencing of 16S rRNA Gene of Selected Staphylococcus aureus Isolates and its Antibiotic Resistance

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    Harsi Dewantari Kusumaningrum

    2016-08-01

    Full Text Available The choice of primer used in 16S rRNA sequencing for identification of Staphylococcus species found in food is important. This study aimed to characterize Staphylococcus aureus isolates by partial sequencing based on 16S rRNA gene employing primers 16sF, 63F or 1387R. The isolates were isolated from milk, egg dishes and chicken dishes and selected based on the presence of sea gene that responsible for formation of enterotoxin-A. Antibiotic susceptibility of the isolates towards six antibiotics was also tested. The use of 16sF resulted generally in higher identity percentage and query coverage compared to the sequencing by 63F or 1387R. BLAST results of all isolates, sequenced by 16sF, showed 99% homology to complete genome of four S. aureus strains, with different characteristics on enterotoxin production and antibiotic resistance. Considering that all isolates were carrying sea gene, indicated by the occurence of 120 bp amplicon after PCR amplification using primer SEA1/SEA2,  the isolates were most in agreeing to S. aureus subsp. aureus ST288. This study indicated that 4 out of 8 selected isolates were resistant towards streptomycin. The 16S rRNA gene sequencing using 16sF is useful for identification of S. aureus. However, additional analysis such as PCR employing specific gene target, should give a valuable supplementary information, when specific characteristic is expected.

  2. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras.

    Science.gov (United States)

    Ashelford, Kevin E; Chuzhanova, Nadia A; Fry, John C; Jones, Antonia J; Weightman, Andrew J

    2006-09-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

  3. RAP, the Sole Octotricopeptide Repeat Protein in Arabidopsis, Is Required for Chloroplast 16S rRNA Maturation[W

    Science.gov (United States)

    Kleinknecht, Laura; Wang, Fei; Stübe, Roland; Philippar, Katrin; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2014-01-01

    The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5′ region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis. PMID:24585838

  4. RAP, the sole octotricopeptide repeat protein in Arabidopsis, is required for chloroplast 16S rRNA maturation.

    Science.gov (United States)

    Kleinknecht, Laura; Wang, Fei; Stübe, Roland; Philippar, Katrin; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2014-02-01

    The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5' region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

  5. Selective expression of two types of 28S rRNA paralogous genes in the chaetognath Spadella cephaloptera.

    Science.gov (United States)

    Barthélémy, R-M; Casanova, J-P; Grino, M; Faure, E

    2007-09-13

    Significant intra-individual variation in the sequences of the ribosomal RNA (rRNA) genes is highly unusual in animal genomes; however, two classes of both 18S and 28S rRNA gene sequences have been detected in chaetognaths, a small phylum of marine invertebrates. One species, Spadella cephaloptera Busch, 1851, is well-suited to the methods of in situ analysis of gene expression, since it is totally transparent. To test our hypothesis of a possible functional division of the two classes of genes, we carried out in situ hybridization. Our results indicated that 28S class II genes are expressed intensively in the oocytes of chaetognaths. In contrast, hybridization using an heterologous probe of 28S class I genes revealed only a single and relatively weak signal in a distinct area of intestinal cells. Our results suggest that the S. cephaloptera genome contains at least three different types of rRNA 28S genes; however, those which are expressed during housekeeping conditions could not be detected in our experiments.

  6. VITCOMIC: visualization tool for taxonomic compositions of microbial communities based on 16S rRNA gene sequences

    Directory of Open Access Journals (Sweden)

    Kurokawa Ken

    2010-06-01

    Full Text Available Abstract Background Understanding the community structure of microbes is typically accomplished by sequencing 16S ribosomal RNA (16S rRNA genes. These community data can be represented by constructing a phylogenetic tree and comparing it with other samples using statistical methods. However, owing to high computational complexity, these methods are insufficient to effectively analyze the millions of sequences produced by new sequencing technologies such as pyrosequencing. Results We introduce a web tool named VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community that can analyze millions of bacterial 16S rRNA gene sequences and calculate the overall taxonomic composition for a microbial community. The 16S rRNA gene sequences of genome-sequenced strains are used as references to identify the nearest relative of each sample sequence. With this information, VITCOMIC plots all sequences in a single figure and indicates relative evolutionary distances. Conclusions VITCOMIC yields a clear representation of the overall taxonomic composition of each sample and facilitates an intuitive understanding of differences in community structure between samples. VITCOMIC is freely available at http://mg.bio.titech.ac.jp/vitcomic/.

  7. Isolation, crystallization, and investigation of ribosomal protein S8 complexed with specific fragments of rRNA of bacterial or archaeal origin.

    Science.gov (United States)

    Tishchenko, S V; Vassilieva, J M; Platonova, O B; Serganov, A A; Fomenkova, N P; Mudrik, E S; Piendl, W; Ehresmann, C; Ehresmann, B; Garber, M B

    2001-09-01

    The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8-RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.

  8. Description of an unusual Neisseria meningitidis isolate containing and expressing Neisseria gonorrhoeae-Specific 16S rRNA gene sequences.

    Science.gov (United States)

    Walcher, Marion; Skvoretz, Rhonda; Montgomery-Fullerton, Megan; Jonas, Vivian; Brentano, Steve

    2013-10-01

    An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination.

  9. The tumor suppressive role of eIF3f and its function in translation inhibition and rRNA degradation.

    Directory of Open Access Journals (Sweden)

    Fushi Wen

    Full Text Available Deregulated translation plays an important role in human cancer. We previously reported decreased eukaryotic initiation factor 3 subunit f (eIF3f expression in pancreatic cancer. Whether decreased eIF3f expression can transform normal epithelial cells is not known. In our current study, we found evidence that stable knockdown of eIF3f in normal human pancreatic ductal epithelial cells increased cell size, nuclear pleomorphism, cytokinesis defects, cell proliferation, clonogenicity, apoptotic resistance, migration, and formation of 3-dimensional irregular masses. Our findings support the tumor suppressive role of eIF3f in pancreatic cancer. Mechanistically, we found that eIF3f inhibited both cap-dependent and cap-independent translation. An increase in the ribosomal RNA (rRNA level was suggested to promote the generation of cancer. The regulatory mechanism of rRNA degradation in mammals is not well understood. We demonstrated here that eIF3f promotes rRNA degradation through direct interaction with heterogeneous nuclear ribonucleoprotein (hnRNP K. We showed that hnRNP K is required for maintaining rRNA stability: under stress conditions, eIF3f dissociates hnRNP K from rRNA, thereby preventing it from protecting rRNA from degradation. We also demonstrated that rRNA degradation occurred in non-P body, non-stress granule cytoplasmic foci that contain eIF3f. Our findings established a new mechanism of rRNA decay regulation mediated by hnRNP K/eIF3f and suggest that the tumor suppressive function of eIF3f may link to impaired rRNA degradation and translation.

  10. [Detection of bacterial signal of 16S rRNA gene in prostate tissues obtained by perineal approach from patient with chronic pelvic pain syndrome].

    Science.gov (United States)

    Zhu, Qing-Feng; Xie, Hui; Weng, Zhi-Liang; Yang, Yi-Rong; Chen, Bi-Cheng

    2009-03-31

    To explore the role of bacteria in etiology of chronic pelvic pain syndrome (CPPS), i.e., chronic prostatitis and the correlation between presence of bacterial signal of 16S ribosomal RNA (16S rRNA) gene and the response to antibiotics. Samples of prostatic and subcutaneous tissues were obtained by biopsy via perineal approach from 112 CPPS patients, aged 20 - 48. Polymerase chain reaction was conducted to detect the 16S rRNA gene of bacteria. The patients were treated with gatifloxacin 0.4 g once a day for 4 weeks and then 4 weeks later the effects of treatment were assessed by the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI). PCR was completed in 94 of the 112 patients, and 18 were excluded because their subcutaneous biopsies were positive for 16S rRNA, showing the possible contamination of their prostatic tissues. The total positive rate of bacterial 16S rRNA gene was 63.8% (60/94). The positive rate of bacterial 16S rRNA gene in the patients with IIIa CPPS and IIIb CPPS were 68.3% and 60.3% respectively. The total gatifloxacin effective rate of positive bacterial signal group after the was 55.0%, significantly higher than that of the negative bacterial signal group (14.7%, P 6S rRNA positive IIIa CPPS patients was 75%, significantly higher than that of the 6S rRNA negative IIIa CPPS patients (23.1%, P 6S rRNA positive IIIb CPPS patients was 37.5%, significantly higher than that of the 6S rRNA negative IIIb CPPS patients (9.5%, P < 0.05). Bacterial infection is related to CPPS in part of the patients. Bacterial signal detection helps predict the effect of antimicrobial therapy.

  11. The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA

    DEFF Research Database (Denmark)

    Liu, M; Kirpekar, F; Van Wezel, G P

    2000-01-01

    tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic-producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two...... Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification...

  12. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    Science.gov (United States)

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

  13. Phylogenetic relationships of Chilean leptodactylids: a molecular approach based on mitochondrial genes 12S and 16S Relaciones filogenéticas de los leptodactílidos chilenos: una aproximación molecular basada en los genes mitocondriales 12S y 16S

    Directory of Open Access Journals (Sweden)

    CLAUDIO CORREA

    2006-12-01

    Full Text Available Most Chilean amphibians belong to the subfamily Telmatobiinae (Anura, Leptodactylidae. Several phylogenetic studies of Leptodactylidae and Telmatobiinae, based principally on morphological characters, have implicitly suggested closer relationships of some species of the Telmatobiinae with members of other subfamilies of leptodactylids, including the leptodactyline genus Pleurodema which is present in Chile. Furthermore, a growing number of molecular studies suggest a non monophyletic status for Telmatobiinae, although none of these studies have investigated the phylogenetic relationships of this subfamily. We compared partial sequences of the ribosomal mitochondrial genes 12S and 16S to determine the phylogenetic relationships of Chilean leptodactylids and its position within the modern anurans (Neobatrachia. We included 22 species from nine of the 10 genera of telmatobiines present in Chile (Alsodes, Atelognathus, Batrachyla, Caudiverbera, Eupsophus, Hylorina, Insuetophrynus, Telmatobufo and Telmatobius, two species of the genus Pleurodema, and one species of Rhinodermatidae, which is considered a leptodactylid derivative family by some authors. We also included 51 species representing most of the families that compose Neobatrachia. Phylogenetic reconstructions were performed using the methods of maximum parsimony, maximum likelihood and Bayesian inference. The topologies obtained in all the analyses indicate that Telmatobiinae is a polyphyletic assemblage, composed by species belonging to Hyloidea (most of the genera and species more related to Australasian taxa (the clade Caudiverbera + Telmatobufo, defined as the tribe Calyptocephalellini. These molecular data support groups based on other kinds of evidence (Caudiverbera + Telmatobufo, Alsodes + Eupsophus and Batrachyla + Hylorina and raise new phylogenetic hypotheses for several genera of telmatobiines (Atelognathus with Batrachyla and Hylorina, Insuetophrynus + Rhinoderma. The phylogenetic

  14. Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

    Science.gov (United States)

    Kalliala, Ilkka; Nieminen, Pekka; Salonen, Anne

    2017-01-01

    Background Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species. Results The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001). Conclusions The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal

  15. Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis.

    Science.gov (United States)

    Virtanen, Seppo; Kalliala, Ilkka; Nieminen, Pekka; Salonen, Anne

    2017-01-01

    Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species. The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001). The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal microbiota studies employing different

  16. Paternity testing with VNTR DNA systems. II. Evaluation of 271 cases of disputed paternity with the VNTR systems D2S44, D5S43, D7S21, D7S22, and D12S11

    DEFF Research Database (Denmark)

    Hansen, Hanna Elsebeth; Morling, N

    1993-01-01

    Paternity testing was carried out in 271 cases of disputed paternity using the 5 VNTR systems D2S44 (YNH24), D5S43 (MS8), D7S21 (MS31), D7S22 (g3), and D12S11 (MS43a), and 10-15 conventional marker systems including the HLA-A,B system. By means of the matching criteria for the VNTR systems...

  17. Paternity testing with VNTR DNA systems. I. Matching criteria and population frequencies of the VNTR systems D2S44, D5S43, D7S21, D7S22, and D12S11 in Danes

    DEFF Research Database (Denmark)

    Morling, N; Hansen, Hanna Elsebeth

    1993-01-01

    -systems D2S44 (YNH24), D5S43 (MS8), D7S21 (MS31), D7S22 (g3), and D12S11 (MS43a). The intra gel variability of 970 duplicate investigations on the same gel of DNA from 122 individuals showed no differences exceeding 1.25 mm between the positions of the corresponding DNA fragments. The comparison of 1...

  18. Use of 1 6S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies[white triangle down

    National Research Council Canada - National Science Library

    Rebecca J Case; Yan Boucher; Ingela Dahllöf; Carola Holmström

    2007-01-01

      Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology...

  19. Thousands of primer-free, high-quality, full-length SSU rRNA sequences from all domains of life

    DEFF Research Database (Denmark)

    Karst, Soeren M; Dueholm, Morten S; McIlroy, Simon J

    2016-01-01

    Ribosomal RNA (rRNA) genes are the consensus marker for determination of microbial diversity on the planet, invaluable in studies of evolution and, for the past decade, high-throughput sequencing of variable regions of ribosomal RNA genes has become the backbone of most microbial ecology studies...... (SSU) rRNA genes and synthetic long read sequencing by molecular tagging, to generate primer-free, full-length SSU rRNA gene sequences from all domains of life, with a median raw error rate of 0.17%. We generated thousands of full-length SSU rRNA sequences from five well-studied ecosystems (soil, human...... gut, fresh water, anaerobic digestion, and activated sludge) and obtained sequences covering all domains of life and the majority of all described phyla. Interestingly, 30% of all bacterial operational taxonomic units were novel, compared to the SILVA database (less than 97% similarity...

  20. Molecular characterization of gap region in 28S rRNA molecules in brine shrimp Artemia parthenogenetica and planarian Dugesia japonica.

    Science.gov (United States)

    Sun, Shuhong; Xie, Hui; Sun, Yan; Song, Jing; Li, Zhi

    2012-04-01

    In most insects and some other protostomes, a small stretch of nucleotides can be removed from mature 28S rRNA molecules, which could create two 28S rRNA subunits (28Sα and 28Sβ). Thus, during electrophoresis, the rRNA profiles of these organisms may differ significantly from the standard benchmark since the two subunits co-migrate with the 18S rRNA. To understand the structure and mechanism of the atypical 28S rRNA molecule, partial fragments of 28Sα and 28Sβ in brine shrimp Artemia parthenogenetica and planarian Dugesia japonica were cloned using a modified technology based on terminal transferase. Alignment with the corresponding sequences of 28S rDNAs indicates that there are 41 nucleotides in A. parthenogenetica and 42 nucleotides in D. japonica absent from the mature rRNAs. The AU content of the gap sequences of D. japonica and A. parthenogenetica is high. Both the gaps may form stem-loop structure. In D. japonica a UAAU cleavage signal is identified in the loop, but it is absent in A. parthenogenetica. Thus, it is proposed that the gap processing of 28S rRNA was a late enzyme-dependent cleavage event in the rRNA maturational process based on the AU rich gap sequence and the formation of the stem-loop structure to expose the processing segment, while the deletion of the gap region would not affect the structure and function of the 28S rRNA molecule.

  1. Mild Glucose Starvation Induces KDM2A-Mediated H3K36me2 Demethylation through AMPK To Reduce rRNA Transcription and Cell Proliferation.

    Science.gov (United States)

    Tanaka, Yuji; Yano, Hirohisa; Ogasawara, Sachiko; Yoshioka, Sho-Ichi; Imamura, Hiromi; Okamoto, Kengo; Tsuneoka, Makoto

    2015-12-01

    Environmental conditions control rRNA transcription. Previously, we found that serum and glucose deprivation induces KDM2A-mediated H3K36me2 demethylation in the rRNA gene (rDNA) promoter and reduces rRNA transcription in the human breast cancer cell line MCF-7. However, the molecular mechanism and biological significance are still unclear. In the present study, we found that glucose starvation alone induced the KDM2A-dependent reduction of rRNA transcription. The treatment of cells with 2-deoxy-d-glucose, an inhibitor of glycolysis, reduced rRNA transcription and H3K36me2 in the rDNA promoter, both of which were completely dependent on KDM2A in low concentrations of 2-deoxy-d-glucose, that is, mild starvation conditions. The mild starvation induced these KDM2A activities through AMP-activated kinase (AMPK) but did not affect another AMPK effector of rRNA transcription, TIF-IA. In the triple-negative breast cancer cell line MDA-MB-231, the mild starvation also reduced rRNA transcription in a KDM2A-dependent manner. We detected KDM2A in breast cancer tissues irrespective of their estrogen receptor, progesterone receptor, and HER2 status, including triple-negative cancer tissues. In both MCF-7 and MDA-MB-231 cells, mild starvation reduced cell proliferation, and KDM2A knockdown suppressed the reduction of cell proliferation. These results suggest that under mild glucose starvation AMPK induces KDM2A-dependent reduction of rRNA transcription to control cell proliferation.

  2. Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

    OpenAIRE

    Eisen, J. A.; Smith, S W; Cavanaugh, Colleen Marie

    1992-01-01

    The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and b...

  3. Maturation of the 5S rRNA 5' end is catalyzed in vitro by the endonuclease tRNase Z in the archaeon H. volcanii.

    Science.gov (United States)

    Hölzle, Annette; Fischer, Susan; Heyer, Ruth; Schütz, Stefanie; Zacharias, Martin; Walther, Paul; Allers, Thorsten; Marchfelder, Anita

    2008-05-01

    Ribosomal RNA molecules are synthesized as precursors that have to undergo several processing steps to generate the functional rRNA. The 5S rRNA in the archaeon Haloferax volcanii is transcribed as part of a multicistronic transcript containing both large rRNAs and one or two tRNAs. Release of the 5S rRNA from the precursor requires two endonucleolytic cleavages by enzymes as yet not identified. Here we report the first identification of an archaeal 5S rRNA processing endonuclease. The enzyme tRNase Z, which was initially identified as tRNA processing enzyme, generates not only tRNA 3' ends but also mature 5S rRNA 5' ends in vitro. Interestingly, the sequence upstream of the 5S rRNA can be folded into a mini-tRNA, which might explain the processing of this RNA by tRNase Z. The endonuclease is active only at low salt concentrations in vitro, which is in contrast to the 2-4 M KCl concentration present inside the cell in vivo. Electron microscopy studies show that there are no compartments inside the Haloferax cell that could provide lower salt environments. Processing of the 5S rRNA 5' end is not restricted to the haloarchaeal tRNase Z since tRNase Z enzymes from a thermophilic archaeon, a lower and a higher eukaryote, are as well able to cleave the tRNA-like structure 5' of the 5S rRNA. Knock out of the tRNase Z gene in Haloferax volcanii is lethal, showing that the protein is essential for the cell.

  4. Comparative analysis of the 5S rRNA and its associated proteins reveals unique primitive rather than parasitic features in Giardia lamblia.

    Science.gov (United States)

    Feng, Jin-Mei; Sun, Jun; Xin, De-Dong; Wen, Jian-Fan

    2012-01-01

    5S rRNA is a highly conserved ribosomal component. Eukaryotic 5S rRNA and its associated proteins (5S rRNA system) have become very well understood. Giardia lamblia was thought by some researchers to be the most primitive extant eukaryote while others considered it a highly evolved parasite. Previous reports have indicated that some aspects of its 5S rRNA system are simpler than that of common eukaryotes. We here explore whether this is true to its entire system, and whether this simplicity is a primitive or parasitic feature. By collecting and confirming pre-existing data and identifying new data, we obtained almost complete datasets of the system of three isolates of G. lamblia, two other parasitic excavates (Trichomonas vaginalis, Trypanosoma cruzi), and one free-living one (Naegleria gruberi). After comprehensively comparing each aspect of the system among these excavates and also with those of archaea and common eukaryotes, we found all the three Giardia isolates to harbor a same simplified 5S rRNA system, which is not only much simpler than that of common eukaryotes but also the simplest one among those of these excavates, and is surprisingly very similar to that of archaea; we also found among these excavates the system in parasitic species is not necessarily simpler than that in free-living species, conversely, the system of free-living species is even simpler in some respects than those of parasitic ones. The simplicity of Giardia 5S rRNA system should be considered a primitive rather than parasitically-degenerated feature. Therefore, Giardia 5S rRNA system might be a primitive system that is intermediate between that of archaea and the common eukaryotic model system, and it may reflect the evolutionary history of the eukaryotic 5S rRNA system from the archaeal form. Our results also imply G. lamblia might be a primitive eukaryote with secondary parasitically-degenerated features.

  5. Phylogeny of intestinal ciliates, including Charonina ventriculi, and comparison of microscopy and 18S rRNA gene pyrosequencing for rumen ciliate community structure analysis.

    Science.gov (United States)

    Kittelmann, Sandra; Devente, Savannah R; Kirk, Michelle R; Seedorf, Henning; Dehority, Burk A; Janssen, Peter H

    2015-04-01

    The development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups.

  6. Identificación de Yarrowia lipolytica (Ascomycota: Hemiascomycetes como contaminante en la obtención de amplificados del gen 28S rRNA de moluscos

    Directory of Open Access Journals (Sweden)

    Jenny Chirinos

    2011-05-01

    Full Text Available En el presente trabajo se identifica una secuencia de DNA no esperada proveniente de los amplificados del gen 28S rRNA de moluscos terrestres. Las extracciones de DNA se realizaron del tejido del pie de caracoles terrestres por el método del CTAB modificado. Las PCRs fueron llevadas a cabo con primers universales para el gen COI e iniciadores diseñados para moluscos, para el marcador 16S rRNA, 28S rRNA y la región ITS-2. Los tamaños aproximados de las bandas de los amplificados de moluscos fueron de 706 pb para el COI, 330 pb para el 16S rRNA, 900 pb para el ITS-2 y 583 pb para el 28S rRNA; un amplificado del último marcador fue de una longitud inesperada, ~340 pb. Las secuencias de DNA fueron comparadas con la base de datos del GenBank mediante el programa BLASTn y la muestra con la banda de tamaño inesperado resultó en un 100% de identidad y cobertura del 99% con el gen 26S rRNA de la levadura Yarrowia lipolytica. El análisis filogenético con Neighbour-Joining y los valores de divergencia confirmaron la identificación, proporcionando resultados que apoyan la ubicación taxonómica de la especie dentro del clado de los Hemiascomycetes.

  7. A minor class of 5S rRNA genes in Saccharomyces cerevisiae X2180-1B, one member of which lies adjacent to a Ty transposable element.

    OpenAIRE

    Piper, P W; Lockheart, A; Patel, N.

    1984-01-01

    In Saccharomyces cerevisiae the majority of the genes for 5S rRNA lie within a 9kb rDNA sequence that is present as 100-200 tandemly-repeated copies on Chromosome XII. Following our observations that about 10% of yeast 5S rRNA exists as minor variant sequences, we screened a collection of yeast DNA fragments cloned in lambda gt for 5S rRNA genes whose flanking sequences differed from those adjacent to 5S rRNA genes of the rDNA repeat. Three variant 5S rRNA genes were isolated on the basis of ...

  8. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

    Directory of Open Access Journals (Sweden)

    Lee SH

    2016-04-01

    Full Text Available Sin Hang Lee,1,21Pathology Department, Milford Hospital, Milford, CT, USA; 2Milford Molecular Diagnostics, Milford, CT, USA Abstract: Lyme disease (LD, the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. Keywords: Lyme disease, Borrelia burgdorferi, homeologous 16S rRNA genes, DNA sequencing

  9. Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing.

    Science.gov (United States)

    Jarvis, Karen G; White, James R; Grim, Christopher J; Ewing, Laura; Ottesen, Andrea R; Beaubrun, Junia Jean-Gilles; Pettengill, James B; Brown, Eric; Hanes, Darcy E

    2015-08-12

    Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective pre-enrichment culture step commonly used by laboratory analysts to resuscitate microorganisms in foods suspected of contamination with pathogens. Cilantro samples were processed for Salmonella detection according to the method in the United States Food and Drug Administration Bacteriological Analytical Manual. Genomic DNA was extracted from culture supernatants prior to and after a 24-hour nonselective pre-enrichment step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen the libraries for Salmonella, as some samples were known to be culture positive. Additionally, culture positive cilantro samples were examined for the presence of Salmonella using shotgun metagenomics on the Illumina MiSeq. Time zero uncultured samples had an abundance of Proteobacteria while the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes. Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples revealed variable degrees of Salmonella contamination among the sequenced samples. Our cilantro study demonstrates the use of high-throughput sequencing to reveal the microbiome of cilantro, and how the microbiome changes during the culture-based protocols employed by food safety laboratories to detect foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to a predominance of Firmicutes suggests that changing our culture-based methods will improve

  10. Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing

    Directory of Open Access Journals (Sweden)

    Chia Sing eChan

    2015-03-01

    Full Text Available The Sungai Klah (SK hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-meter-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0 to 9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC. In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3−V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream and geochemical parameters (broad temperature and pH range. It is speculated that symbiotic relationships occur between the members of the community.

  11. Microbial Dark Matter: Unusual intervening sequences in 16S rRNA genes of candidate phyla from the deep subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Jarett, Jessica; Stepanauskas, Ramunas; Kieft, Thomas; Onstott, Tullis; Woyke, Tanja

    2014-03-17

    The Microbial Dark Matter project has sequenced genomes from over 200 single cells from candidate phyla, greatly expanding our knowledge of the ecology, inferred metabolism, and evolution of these widely distributed, yet poorly understood lineages. The second phase of this project aims to sequence an additional 800 single cells from known as well as potentially novel candidate phyla derived from a variety of environments. In order to identify whole genome amplified single cells, screening based on phylogenetic placement of 16S rRNA gene sequences is being conducted. Briefly, derived 16S rRNA gene sequences are aligned to a custom version of the Greengenes reference database and added to a reference tree in ARB using parsimony. In multiple samples from deep subsurface habitats but not from other habitats, a large number of sequences proved difficult to align and therefore to place in the tree. Based on comparisons to reference sequences and structural alignments using SSU-ALIGN, many of these ?difficult? sequences appear to originate from candidate phyla, and contain intervening sequences (IVSs) within the 16S rRNA genes. These IVSs are short (39 - 79 nt) and do not appear to be self-splicing or to contain open reading frames. IVSs were found in the loop regions of stem-loop structures in several different taxonomic groups. Phylogenetic placement of sequences is strongly affected by IVSs; two out of three groups investigated were classified as different phyla after their removal. Based on data from samples screened in this project, IVSs appear to be more common in microbes occurring in deep subsurface habitats, although the reasons for this remain elusive.

  12. The structure of the archaebacterial ribosomal protein S7 and its possible interaction with 16S rRNA.

    Science.gov (United States)

    Hosaka, H; Yao, M; Kimura, M; Tanaka, I

    2001-11-01

    Ribosomal protein S7 is one of the ubiquitous components of the small subunit of the ribosome. It is a 16S rRNA-binding protein positioned close to the exit of the tRNA, and it plays a role in initiating assembly of the head of the 30S subunit. Previous structural analyses of eubacterial S7 have shown that it has a stable alpha-helix core and a flexible beta-arm. Unlike these eubacterial proteins, archaebacterial or eukaryotic S7 has an N-terminal extension of approximately 60 residues. The crystal structure of S7 from archaebacterium Pyrococcus horikoshii (PhoS7) has been determined at 2.1 A resolution. The final model of PhoS7 consists of six major alpha-helices, a short 3(10)-helix and two beta-stands. The major part (residues 18-45) of the N-terminal extension of PhoS7 reinforces the alpha-helical core by well-extended hydrophobic interactions, while the other part (residues 46-63) is not visible in the crystal and is possibly fixed only by interacting with 16S rRNA. These differences in the N-terminal extension as well as in the insertion (between alpha1 and alpha2) of the archaebacterial S7 structure from eubacterial S7 are such that they do not necessitate a major change in the structure of the currently available eubacterial 16S rRNA. Some of the inserted chains might pass through gaps formed by helices of the 16S rRNA.

  13. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    Science.gov (United States)

    Tang, Xianghai; Yu, Rencheng; Zhou, Mingjiang; Yu, Zhigang

    2012-03-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

  14. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

    Science.gov (United States)

    Schloss, Patrick D; Jenior, Matthew L; Koumpouras, Charles C; Westcott, Sarah L; Highlander, Sarah K

    2016-01-01

    Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  15. 16S-23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads.

    Science.gov (United States)

    Tokajian, Sima; Issa, Nahla; Salloum, Tamara; Ibrahim, Joe; Farah, Maya

    2016-01-01

    Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S-23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S-23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2-57.9 mol%. Five distinct ITS types were identified: ITS(none) (without tRNA genes), ITS(Ala(TGC)), ITS(Ala(TGC)+Ile(GAT)), ITS(Ile(GAT)+Ala(TGC)), and ITS (Ile(GAT)+Pseudo). All of the identified tRNA(Ala(TGC)) molecules consisted of 73 bases, and all of the tRNA(Ile(GAT)) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S-23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence.

  16. Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

    Science.gov (United States)

    Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

    2011-01-01

    To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

  17. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    Science.gov (United States)

    de los Reyes, F L; Ritter, W; Raskin, L

    1997-03-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein- and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems.

  18. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

    Directory of Open Access Journals (Sweden)

    Patrick D. Schloss

    2016-03-01

    Full Text Available Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  19. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    Institute of Scientific and Technical Information of China (English)

    TANG Xianghai; YU Rencheng; ZHOU Mingjiang; YU Zhigang

    2012-01-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs).This species consists of many strains that differ in their ability to produce toxins but have similar morphology,making identification difficult.In this study,species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A.minutum from two phylogenetic clades.We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes.Three ribotype-specific probes,M-GC-1,M-PC-2,and M-PC-3,were designed.The former is specific for the GC clade (“Global clade”) of A.minutum,the majority of which have been found non-toxic,and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade (“Pacific clade”).The specificity of these three probes was confirmed by FISH.All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions.However,the accessibility of rRNA molecules in ribosomes varied among the probe binding positions.Thus,there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1,M-PC-3),or just nucleolus (M-PC-2).Our results provide a methodological basis for studying the biogeography and population dynamics of A.minutum,and providing an early warning of toxic HABs.

  20. Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA.

    Science.gov (United States)

    Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M; Villegas, Leopoldo; Escalante, Ananias A; Kachur, S Patrick; Barnwell, John W; Peterson, David S; Udhayakumar, Venkatachalam; Kissinger, Jessica C

    2011-07-01

    Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.

  1. The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics

    DEFF Research Database (Denmark)

    Long, K. S.; Poehlsgaard, Jacob; Kehrenberg, C.

    2006-01-01

    A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin...... drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind...

  2. Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples.

    Directory of Open Access Journals (Sweden)

    Jennifer J Barb

    Full Text Available There is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology.This paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9 processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY. Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies and their sequencing data is subjected to a novel analytical pipeline.Results are presented at family and genus level. The Kullback-Leibler divergence (DKL, a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst average DKL while the V4 gave the lowest (best average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria.The results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at different time points

  3. Terminal restriction fragment length polymorphism (T-RFLP) profiling of bacterial 16S rRNA genes.

    Science.gov (United States)

    Osborne, Catherine A

    2014-01-01

    T-RFLP profiling is a very effective method for comparing many samples in an environmental microbiology study, because fingerprints of microbial diversity can be generated in a sensitive, reproducible, and cost-effective manner. This protocol describes the steps required to generate T-RFLP profiles of the dominant members of a bacterial community, by PCR amplification of the bacterial 16S rRNA genes and three restriction endonuclease digests to generate three different profiles for each sample. The generation of multiple profiles per sample provides enough information to confidently differentiate rich environmental bacterial communities.

  4. Sampling of intestinal microbiota and targeted amplification of bacterial 16S rRNA genes for microbial ecologic analysis.

    Science.gov (United States)

    Tong, Maomeng; Jacobs, Jonathan P; McHardy, Ian H; Braun, Jonathan

    2014-11-03

    Dysbiosis of host-associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Accurate analysis of microbial composition and functional state in humans or mice requires appropriate collection and pre-processing of biospecimens. Methods to sample luminal and mucosal microbiota from human or mouse intestines and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using the methods in this unit can be used for downstream quantitative analysis of microbial ecology.

  5. Cyanobacterial endobionts within a major marine planktonic calcifier (Globigerina bulloides, Foraminifera) revealed by 16S rRNA metabarcoding

    Science.gov (United States)

    Bird, Clare; Darling, Kate F.; Russell, Ann D.; Davis, Catherine V.; Fehrenbacher, Jennifer; Free, Andrew; Wyman, Michael; Ngwenya, Bryne T.

    2017-02-01

    We investigated the possibility of bacterial symbiosis in Globigerina bulloides, a palaeoceanographically important, planktonic foraminifer. This marine protist is commonly used in micropalaeontological investigations of climatically sensitive subpolar and temperate water masses as well as wind-driven upwelling regions of the world's oceans. G. bulloides is unusual because it lacks the protist algal symbionts that are often found in other spinose species. In addition, it has a large offset in its stable carbon and oxygen isotopic compositions compared to other planktonic foraminifer species, and also that predicted from seawater equilibrium. This is suggestive of novel differences in ecology and life history of G. bulloides, making it a good candidate for investigating the potential for bacterial symbiosis as a contributory factor influencing shell calcification. Such information is essential to evaluate fully the potential response of G. bulloides to ocean acidification and climate change. To investigate possible ecological interactions between G. bulloides and marine bacteria, 18S rRNA gene sequencing, fluorescence microscopy, 16S rRNA gene metabarcoding and transmission electron microscopy (TEM) were performed on individual specimens of G. bulloides (type IId) collected from two locations in the California Current. Intracellular DNA extracted from five G. bulloides specimens was subjected to 16S rRNA gene metabarcoding and, remarkably, 37-87 % of all 16S rRNA gene sequences recovered were assigned to operational taxonomic units (OTUs) from the picocyanobacterium Synechococcus. This finding was supported by TEM observations of intact Synechococcus cells in both the cytoplasm and vacuoles of G. bulloides. Their concentrations were up to 4 orders of magnitude greater inside the foraminifera than those reported for the California Current water column and approximately 5 % of the intracellular Synechococcus cells observed were undergoing cell division. This suggests

  6. Molecular phylogenetics of subclass Peritrichia (Ciliophora: Oligohymenophorea) based on expanded analyses of 18S rRNA sequences.

    Science.gov (United States)

    Utz, Laura R P; Eizirik, Eduardo

    2007-01-01

    Phylogenetic relationships among peritrich ciliates remain unclear in spite of recent progress. To expand the analyses performed in previous studies, and to statistically test hypotheses of monophyly, we analyzed a broad sample of 18s rRNA sequences (including 15 peritrich genera), applying a conservative alignment strategy and several phylogenetic approaches. The main results are that: (i) the monophyly of Peritrichia cannot be rejected; (ii) the two main clades of Sessilida do not correspond to formally recognized taxa; (iii) the monophyly of genera Vorticella and Epistylis is significantly rejected; and (iv) morphological structures commonly used in peritrich taxonomy may be evolutionarily labile.

  7. Metabolism of 18S rRNA in rat liver cells in different functional states of protein-synthesizing apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Chirkov, G.P.; Druzhinina, M.K.; Todorov, I.N.

    1986-04-10

    The ratio of the absolute radioactivities of 28S and 18S RNAs in the fractions of membrane-bound and free polysomes and the fraction of free rat liver ribosomes was studied under conditions of inhibition of translation by cycloheximide, insulin, and cAMP. It was found that insulin and cAMP, in contrast to cycloheximide, do not induce selective degradation of 18S rRNA. The results are discussed from the standpoint of the possible role of the phosphorylation of protein S6 in the degradation of the 40S ribosomal subunit.

  8. Molecular phylogenetics of the spider family Micropholcommatidae (Arachnida: Araneae) using nuclear rRNA genes (18S and 28S).

    Science.gov (United States)

    Rix, Michael G; Harvey, Mark S; Roberts, J Dale

    2008-03-01

    The spider family Micropholcommatidae is an enigmatic taxon of uncertain limits and uncertain affinities. Various phylogenetic hypotheses have been proposed for the family, but these hypotheses have never been tested with a robust phylogenetic analysis. The existence of similar Australasian and New World taxa, the possibility of morphological convergence associated with extreme 'smallness', and the apparent paucity of synapomorphic morphological characters, have all clouded generic relationships in this group. We used fragments from two nuclear ribosomal RNA genes (18S and 28S) to test the monophyly and phylogenetic position of the Micropholcommatidae. The analyses incorporated 50 ingroup spider species, including 23 micropholcommatid species and representatives from 14 other spider families. Ribosomal RNA secondary structures were inferred for the V3-V5 region of the 18S rRNA gene, and Domain II of the 28S rRNA gene of Hickmania troglodytes [Higgins, E.T., Petterd, W.F., 1883. Description of a new cave-inhabiting spider, together with notes on mammalian remains from a recently discovered cave in the Chudleigh district. Pap. Proc. R. Soc. Tasman. 1882, 191-192]. These secondary structures were used to guide multiple sequence alignments, and determine the position and nature of indels in different taxa. Secondary structure information was also incorporated into a structurally partitioned rRNA analysis in MrBayes Version 3.1.2, using a doublet model of nucleotide substitution. This structurally partitioned rRNA analysis provided a less resolved but more conservative and informative estimate of phylogeny than an otherwise identical, unpartitioned rDNA analysis. With the exception of the Chilean species Teutoniella cekalovici [Platnick, N.I., Forster, R.R., 1986. On Teutoniella, an American genus of the spider family Micropholcommatidae (Araneae, Palpimanoidea). Am. Mus. Novit. 2854, 1-9], the family Micropholcommatidae was found to be monophyletic with three

  9. Linear programming model to construct phylogenetic network for 16S rRNA sequences of photosynthetic organisms and influenza viruses.

    Science.gov (United States)

    Mathur, Rinku; Adlakha, Neeru

    2014-06-01

    Phylogenetic trees give the information about the vertical relationships of ancestors and descendants but phylogenetic networks are used to visualize the horizontal relationships among the different organisms. In order to predict reticulate events there is a need to construct phylogenetic networks. Here, a Linear Programming (LP) model has been developed for the construction of phylogenetic network. The model is validated by using data sets of chloroplast of 16S rRNA sequences of photosynthetic organisms and Influenza A/H5N1 viruses. Results obtained are in agreement with those obtained by earlier researchers.

  10. Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera

    Science.gov (United States)

    2008-01-01

    Background The cytoplasmic ribosomal small subunit (SSU, 18S) ribosomal RNA (rRNA) is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. Information about secondary structure also supports the process of aligning rRNA sequences across taxa. Both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa. Here, we explore SSU rRNA secondary structures from the three extant classes of Phylum Porifera (Grant, 1836), a pivotal, but largely unresolved taxon of early branching Metazoa. This is the first phylogenetic study of poriferan SSU rRNA data to date that includes detailed comparative secondary structure information for all three sponge classes. Results We found base compositional and structural differences in SSU rRNA among Demospongiae, Hexactinellida (glass sponges) and Calcarea (calcareous sponges). We showed that analyses of primary rRNA sequences, including secondary structure-specific evolutionary models, in combination with reconstruction of the evolution of unusual structural features, reveal a substantial amount of additional information. Of special note was the finding that the gene tree topologies of marine haplosclerid demosponges, which are inconsistent with the current morphology-based classification, are supported by our reconstructed evolution of secondary structure features. Therefore, these features can provide alternative support for sequence-based topologies and give insights into the evolution of the molecule itself. To encourage and facilitate the application of rRNA models in phylogenetics of early metazoans, we present 52 SSU rRNA

  11. Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera

    Directory of Open Access Journals (Sweden)

    Wörheide Gert

    2008-02-01

    Full Text Available Abstract Background The cytoplasmic ribosomal small subunit (SSU, 18S ribosomal RNA (rRNA is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. Information about secondary structure also supports the process of aligning rRNA sequences across taxa. Both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa. Here, we explore SSU rRNA secondary structures from the three extant classes of Phylum Porifera (Grant, 1836, a pivotal, but largely unresolved taxon of early branching Metazoa. This is the first phylogenetic study of poriferan SSU rRNA data to date that includes detailed comparative secondary structure information for all three sponge classes. Results We found base compositional and structural differences in SSU rRNA among Demospongiae, Hexactinellida (glass sponges and Calcarea (calcareous sponges. We showed that analyses of primary rRNA sequences, including secondary structure-specific evolutionary models, in combination with reconstruction of the evolution of unusual structural features, reveal a substantial amount of additional information. Of special note was the finding that the gene tree topologies of marine haplosclerid demosponges, which are inconsistent with the current morphology-based classification, are supported by our reconstructed evolution of secondary structure features. Therefore, these features can provide alternative support for sequence-based topologies and give insights into the evolution of the molecule itself. To encourage and facilitate the application of rRNA models in phylogenetics of early

  12. Molecular evolution of rDNA in early diverging Metazoa: first comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera.

    Science.gov (United States)

    Voigt, Oliver; Erpenbeck, Dirk; Wörheide, Gert

    2008-02-27

    The cytoplasmic ribosomal small subunit (SSU, 18S) ribosomal RNA (rRNA) is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. Information about secondary structure also supports the process of aligning rRNA sequences across taxa. Both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa.Here, we explore SSU rRNA secondary structures from the three extant classes of Phylum Porifera (Grant, 1836), a pivotal, but largely unresolved taxon of early branching Metazoa. This is the first phylogenetic study of poriferan SSU rRNA data to date that includes detailed comparative secondary structure information for all three sponge classes. We found base compositional and structural differences in SSU rRNA among Demospongiae, Hexactinellida (glass sponges) and Calcarea (calcareous sponges). We showed that analyses of primary rRNA sequences, including secondary structure-specific evolutionary models, in combination with reconstruction of the evolution of unusual structural features, reveal a substantial amount of additional information. Of special note was the finding that the gene tree topologies of marine haplosclerid demosponges, which are inconsistent with the current morphology-based classification, are supported by our reconstructed evolution of secondary structure features. Therefore, these features can provide alternative support for sequence-based topologies and give insights into the evolution of the molecule itself. To encourage and facilitate the application of rRNA models in phylogenetics of early metazoans, we present 52 SSU rRNA secondary

  13. Identification of forensically important beetles (Coleoptera: Histeridae) in China based on 16S rRNA and Cyt b.

    Science.gov (United States)

    Su, R N; Guo, Y D; Xie, D; Peng, Y L; Cai, J F; Hua, F; Sheng, L H

    2013-09-01

    Exact identification of an insect sample is usually the first essential step in a forensic entomological analysis. However, the morphological similarity of beetles in the level of species usually poses a challenge for forensic scientists within their routine work. As a supplementary to traditional morphological method, molecular genetics identification turns out to be simple and time-saving. A molecular identification method involving a 288-bp segment of the 16S ribosomal RNA (16S rRNA) gene and a 334-bp segment of the cytochrome b (Cyt b) gene from 23 histerid beetles specimens, collected from 7 locations in 6 Chinese provinces, was evaluated. The 16S rRNA and Cyt b genes are sequenced to examine the ability of the region, resolve species identities and enrich the local databases. The monophyletic branches of the phylogenetic tree showed the potential of the markers in identifying beetles within families. Combined analysis is a more accurate approach for species identication than independent analysis.

  14. COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae).

    Science.gov (United States)

    Xia, Yun; Gu, Hai-Feng; Peng, Rui; Chen, Qin; Zheng, Yu-Chi; Murphy, Robert W; Zeng, Xiao-Mao

    2012-01-01

    The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.

  15. New mutation points in 23S rRNA gene associated with Helicobacter Pylori resistance to clarithromycin in northeast China

    Institute of Scientific and Technical Information of China (English)

    Qing Hao; Yan Li; Zhi-Jie Zhang; Yong Liu; Hong Gao

    2004-01-01

    AIM: To investigate the resistance rate of Helicobacter pylori (Hpylori) to clarithromycin, metronidazole, amoxicillin and tetracycline to guide clinical practice, and to study the mechanism of H pyloriresistant to clarithromycin.METHODS: Thirty H pyloristrains were isolated from the mucosa of peptic ulcer, gastric tumor and chronic gastritis patients, then the minimal inhibitory concentration (MIC) to clarithromycin, metronidazole, amoxicillin and tetracycline was evaluated by E-test method. The sequence analysis of PCR fragments was conducted in 23S rRNA gene of H pylori resistant to clarithromycin to get the resistance mechanism of the bacteria.RESULTS: Among 30 H pyloristrains, 7 cases were resistant to clarithromycin, 12 to metronidazole, 2 to tetracycline and no strain was found to be resistant to amoxicillin. The resistance rates were 23.3%, 40%, 6.7% and 0%,respectively. Three new mutation points were found to be related to the clarithromycin resistance in H pyloriisolates,which were G2224A, C2245T and T2289C.CONCLUSION: In northeast China, H pylorishows high resistance to metronidazole, while sensitive to amoxicillin.The mechanism of resistance to clarithromycin may be related to the mutation of G2224A, C2245T and T2289C in the 23S rRNA gene.

  16. Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling.

    Directory of Open Access Journals (Sweden)

    Ilario Ferrocino

    Full Text Available In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE. The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.

  17. Identification and phylogeny of Arabian snakes: Comparison of venom chromatographic profiles versus 16S rRNA gene sequences

    Science.gov (United States)

    Al Asmari, Abdulrahman; Manthiri, Rajamohammed Abbas; Khan, Haseeb Ahmad

    2014-01-01

    Identification of snake species is important for various reasons including the emergency treatment of snake bite victims. We present a simple method for identification of six snake species using the gel filtration chromatographic profiles of their venoms. The venoms of Echis coloratus, Echis pyramidum, Cerastes gasperettii, Bitis arietans, Naja arabica, and Walterinnesia aegyptia were milked, lyophilized, diluted and centrifuged to separate the mucus from the venom. The clear supernatants were filtered and chromatographed on fast protein liquid chromatography (FPLC). We obtained the 16S rRNA gene sequences of the above species and performed phylogenetic analysis using the neighbor-joining method. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks. The dendrograms generated from similarity matrix based on the presence/absence of particular chromatographic peaks clearly differentiated Elapids from Viperids. Molecular cladistics using 16S rRNA gene sequences resulted in jumping clades while separating the members of these two families. These findings suggest that chromatographic profiles of snake venoms may provide a simple and reproducible chemical fingerprinting method for quick identification of snake species. However, the validation of this methodology requires further studies on large number of specimens from within and across species. PMID:25313278

  18. Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

    Science.gov (United States)

    Moazed, D; Noller, H F

    1991-05-01

    Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

  19. Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling.

    Science.gov (United States)

    Ferrocino, Ilario; Di Cagno, Raffaella; De Angelis, Maria; Turroni, Silvia; Vannini, Lucia; Bancalari, Elena; Rantsiou, Kalliopi; Cardinali, Gianluigi; Neviani, Erasmo; Cocolin, Luca

    2015-01-01

    In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.

  20. 16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea, Echinodermata)

    Institute of Scientific and Technical Information of China (English)

    MA Haiyan; JIANG Guoliang; WU Zhiqiang; WANG Xin

    2009-01-01

    Cultured Apostichopusjaponicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopusjaponicus individuals.

  1. Genetic relationship between Neobenedenia girellae and N.melleni inferred from 28S rRNA sequences

    Institute of Scientific and Technical Information of China (English)

    WANG Jun; ZHANG Wen; SU Yongquan; DING Shaoxiong

    2004-01-01

    The fragments of 350 bp in 28S rRNA from the closely related monogenea of trematoda, Neobenedenia girellae and N. melleni are obtained by polymerase chain reaction (PCR) amplified using a couple of special primers and then sequenced. The results show that the comparison of 28S rRNA sequences, with only a base varying in 337bp accounting for 0.3% genetic difference, from the relative species N. girellae and N. melleni parasitized on the different fishes in different farms displays that they possess a very high genetic similarity of 99.7%, higher than that of 99.41% for the single species N. melleni sampled in different areas, and the intraspecific divergence of N.melleni is 0.59%. Meanwhile, the interspecific differences between the two Neobenedenia and three Benedenia (i.e., B. lutjani, B. rohdei and B. seriolae) range from 2.08% to11.73%. In addition, UPGMA and MP molecular phylogenetic trees are constructed and proved to be consistent with each other. Though the morphological characteristics and the results of genetic diversity for the two Neobenedenia show a high similarity, whether they belong to a single species or not are still undefined, and the more genes of them should be further investigated, in combination with the systematical and detailed morphological study.

  2. Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach.

    Science.gov (United States)

    Ambardar, Sheetal; Sangwan, Naseer; Manjula, A; Rajendhran, J; Gunasekaran, P; Lal, Rup; Vakhlu, Jyoti

    2014-10-01

    Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm-root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P < 0.05) from each other. This is the first report on the identification of bacteria associated with rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach.

  3. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    Science.gov (United States)

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  4. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    Directory of Open Access Journals (Sweden)

    Ilias Lagkouvardos

    2017-01-01

    Full Text Available The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  5. Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides.

    Science.gov (United States)

    Almutairi, Mashal M; Park, Sung Ryeol; Rose, Simon; Hansen, Douglas A; Vázquez-Laslop, Nora; Douthwaite, Stephen; Sherman, David H; Mankin, Alexander S

    2015-10-20

    Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous rRNA methylase genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate, respectively, the N6 amino group in 23S rRNA nucleotide A2058. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high-level resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism.

  6. 16s rRNA Identification of Pediococcus spp. from Broiler and Studies of Adherence Ability on Immobilized Mucus

    Directory of Open Access Journals (Sweden)

    Ema Damayanti

    2015-11-01

    Full Text Available The objectives of this research were to study taxonomical status of lactic acid bacteria (LAB isolated from broiler and adherence ability on mucus in vitro. Molecular analysis was performed by analyzing 16S rRNA gene using universal primer. The adherence assay on mucus was carried out using microplate method with total plate count (TPC, absorbance (A550 and confirmed by scanning electron microscopy (SEM. The results of this studies revealed that three of LAB isolates have closed relation to Pediococcus acidilactici (99.9% species.Three isolates of P. acidilactici have adherence ability on broiler mucus higher than that on porcine mucin with an adherence percentage of 55.5% versus 50.8% and absorbance A550 of 0.061 versus 0.051, respectively. The highest adherence ability showed by P. acidilactici R02 with adherence percentage was 59.3% and absorbance A550 = 0.068. Adherence on mucus were affected by the addition of 3 g/l of gastric juice and 0.3% (b/v of bile salt. Adherence analysis using SEM also showed that the adherence on broiler mucus was higher than the adherence on porcine mucin. Altogether this adherence studies, suggest that three isolates of P. acidilactici LAB were capable of colonizing host intestinal mucus in vitro as important property to be promising probiotic bacteria for broiler.Key words : adherence, broiler, Pediococcus, mucus, 16S rRNA

  7. Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica.

    Science.gov (United States)

    Christensen, H; Møller, P L; Vogensen, F K; Olsen, J E

    2000-01-01

    The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z4,z23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382-384 bases in length, which were 98.4-99.7% similar to each other and three long fragments of 505 bases with 98.0-99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.

  8. lncRNA-Induced Nucleosome Repositioning Reinforces Transcriptional Repression of rRNA Genes upon Hypotonic Stress

    Directory of Open Access Journals (Sweden)

    Zhongliang Zhao

    2016-03-01

    Full Text Available The activity of rRNA genes (rDNA is regulated by pathways that target the transcription machinery or alter the epigenetic state of rDNA. Previous work has established that downregulation of rRNA synthesis in quiescent cells is accompanied by upregulation of PAPAS, a long noncoding RNA (lncRNA that recruits the histone methyltransferase Suv4-20h2 to rDNA, thus triggering trimethylation of H4K20 (H4K20me3 and chromatin compaction. Here, we show that upregulation of PAPAS in response to hypoosmotic stress does not increase H4K20me3 because of Nedd4-dependent ubiquitinylation and proteasomal degradation of Suv4-20h2. Loss of Suv4-20h2 enables PAPAS to interact with CHD4, a subunit of the chromatin remodeling complex NuRD, which shifts the promoter-bound nucleosome into the transcriptional “off” position. Thus, PAPAS exerts a “stress-tailored” dual function in rDNA silencing, facilitating either Suv4-20h2-dependent chromatin compaction or NuRD-dependent changes in nucleosome positioning.

  9. Analysis of the cystic fibrosis lung microbiota via serial Illumina sequencing of bacterial 16S rRNA hypervariable regions.

    Directory of Open Access Journals (Sweden)

    Heather Maughan

    Full Text Available The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq: a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.

  10. Application of 16S rRNA metagenomics to analyze bacterial communities at a respiratory care centre in Taiwan.

    Science.gov (United States)

    Tang, Chuan Yi; Yiu, Siu-Ming; Kuo, Han-Yueh; Tan, Te-Sheng; Liao, Ki-Hok; Liu, Chih-Chin; Hon, Wing-Kai; Liou, Ming-Li

    2015-03-01

    In this study, we applied a 16S ribosomal RNA (rRNA) metagenomics approach to survey inanimate hospital environments (IHEs) in a respiratory care center (RCC). A total of 16 samples, including 9 from medical devices and 7 from workstations, were analyzed. Besides, clinical isolates were retrospectively analyzed during the sampling period in the RCC. A high amount of microbial diversity was detected, with an average of 1,836 phylotypes per sample. In addition to Acinetobacter, more than 60 % of the bacterial communities present among the top 25 abundant genera were dominated by skin-associated bacteria. Differences in bacterial profiles were restricted to individual samples. Furthermore, compliance with hand hygiene guidelines may be unsatisfactory among hospital staff according to a principal coordinate analysis that indicated clustering of bacterial communities between devices and workstations for most of the sampling sites. Compared to the high incidence of clinical isolates in the RCC, only Staphylococcus and Acinetobacter were highly abundant in the IHEs. Despite Acinetobacter was the most abundant genus present in IHEs of the RCC, potential pathogens, e.g., Acinetobacter baumannii, might remain susceptible to carbapenem. This study is the first in Taiwan to demonstrate a high diversity of human-associated bacteria in the RCC via 16S rRNA metagenomics, which allows for new assessment of potential health risks in RCCs, aids in the evaluation of existing sanitation protocols, and furthers our understanding of the development of healthcare-associated infections.

  11. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis.

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    Shuai Wang

    2016-05-01

    Full Text Available Plants have varying abilities to tolerate chilling (low but not freezing temperatures, and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.

  12. Removal of ribosomal subunits (and rRNA) from cytoplasmic extracts before solubilization with SDS and deproteinization.

    Science.gov (United States)

    Rio, Donald C; Ares, Manuel; Hannon, Gregory J; Nilsen, Timothy W

    2010-06-01

    More than 95% of total RNA is composed of ribosomal RNAs (rRNAs) (28S, 18S, 5.8S, and 5S). Here, we present a method that is effective in removing rRNA before extraction and purification of total RNA. If you choose to prepare cytoplasmic RNA and wish to analyze any RNA other than rRNA, it is desirable to eliminate the rRNAs by taking advantage of the fact that the ribosomal subunits are very large (40S and 60S). Few, if any, other cellular RNAs are present in such large macromolecular complexes. The vast majority of rRNAs can be removed by sedimentation. Of course, steps must be taken to avoid co-sedimentation of desired RNAs. Co-sedimentation can be greatly reduced by first dissociating ribosomes into their respective subunits by EDTA treatment. The subunits are then "cleaned" by treatment with high salt and nonionic detergent. Ribosomal subunits remain intact under these conditions and can be sedimented free of other RNAs. Subsequently, the remaining RNAs (messenger RNAs [mRNAs] and all other RNAs) can be purified and analyzed by a variety of methods.

  13. IDENTIFICATION OF Staphylococcus sp. STRAINS ISOLATED FROM POSITIVE WIDAL BLOOD BASED ON 16S rRNA GENE SEQUENCES

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    Sri Darmawati

    2015-12-01

    Full Text Available The purpose of this study is to identify 8 strains of Staphylococcus genus members isolated from positive Widal blood (4 strains of Staphylococcus saprophyticus, 1 strain of Staphylococcus warneri, 2 strains of Staphylococcus hominis, 1 strain of Staphylococcus xylosus and 1 strain of Staphylococcus capitis based on 16S rRNA gene sequences. The methods used in this study are conducting PCR of 16S rRNA gene, cloning genes using T-Vector pMD20 which is transformed to E. coli DH5α, sequencing. The results show that four strains (BA 47.4, BA 19.2, KD 29.5 and TG 09.1 are identified as Stap. Saprophyticus strains of Stap. saprophyticus members of ATCC 15305T (99.01-100% similarity. The strain of TG 01.3 is identified as Stap. Warneri strain of TG 01.3 of Stap. Warneri members of ATCC 27836T (99.74-99.93% similarity, 2strains (KT 29.2 and KD 35.1 are identified as of Stap. hominis members of DSM 20328T (99.4-99.67% similarity. The strain of KT 30.5 is not identified

  14. 5S rRNA and accompanying proteins in gonads: powerful markers to identify sex and reproductive endocrine disruption in fish.

    Science.gov (United States)

    Diaz de Cerio, Oihane; Rojo-Bartolomé, Iratxe; Bizarro, Cristina; Ortiz-Zarragoitia, Maren; Cancio, Ibon

    2012-07-17

    In anuran ovaries, 5S rDNA is regulated transcriptionally by transcription factor IIIA (TFIIIA), which upon transcription, binds 5S rRNA, forming 7S RNP. 5S rRNA can be stockpiled also in the form of 42S RNP bound to 42sp43. The aim of the present study was to assess the differential transcriptional regulation of 5S rRNA and associated proteins in thicklip gray mullet (Chelon labrosus) gonads. Up to 75% of the total RNA from mullet ovaries was 5S rRNA. qPCR quantification of 5S rRNA expression, in gonads of histologically sexed individuals from different geographical areas, successfully sexed animals. All males had expression levels that were orders of magnitude below expression levels in females, throughout an annual reproductive cycle, with the exception of two individuals: one in November and one in December. Moreover, intersex mullets from a polluted harbor had expression levels between both sexes. TFIIIA and 42sp43 were also very active transcriptionally in gonads of female and intersex mullets, in comparison to males. Nucleocytoplasmatic transport is important in this context and we also analyzed transcriptional levels of importins-α1, -α2, and -β2 and different exportins. Importin-αs behaved similarly to 5S rRNA. Thus, 5S rRNA and associated proteins constitute very powerful molecular markers of sex and effects of xenosterogens in fish gonads, with potential technological applications in the analysis of fish stock dynamics and reproduction as well as in environmental health assessment.

  15. hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0

    Science.gov (United States)

    Tomecki, Rafal; Labno, Anna; Drazkowska, Karolina; Cysewski, Dominik; Dziembowski, Andrzej

    2015-01-01

    Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5′-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site. PMID:26237581

  16. Second generation sequencing of three STRs D3S1358, D12S391 and D21S11 in Danes and a new nomenclature for sequenced STR alleles

    DEFF Research Database (Denmark)

    Gelardi, Chiara; Rockenbauer, Eszter; Dalsgaard, Sigrun

    2014-01-01

    Second generation sequencing (SGS) may revolutionize the field of forensic STR typing. Two of the essential requirements for implementation of an SGS based approach for forensic investigations are (1) establishment of adequate frequency databases and (2) adoption of a new STR nomenclature. We...... report the STR sequences and allele frequencies of three STR loci: D3S1358, D12S391 and D21S11 in 197 unrelated Danes. We used a new STR nomenclature that depicts the locus name used in forensic genetics, the length of the repeat region divided by the repeat length (typically 4 nucleotides) and detailed...

  17. Complete modification maps for the cytosolic small and large subunit rRNAs of Euglena gracilis: functional and evolutionary implications of contrasting patterns between the two rRNA components.

    Science.gov (United States)

    Schnare, Murray N; Gray, Michael W

    2011-10-14

    In the protist Euglena gracilis, the cytosolic small subunit (SSU) rRNA is a single, covalently continuous species typical of most eukaryotes; in contrast, the large subunit (LSU) rRNA is naturally fragmented, comprising 14 separate RNA molecules instead of the bipartite (28S+5.8S) eukaryotic LSU rRNA typically seen. We present extensively revised secondary structure models of the E. gracilis SSU and LSU rRNAs and have mapped the positions of all of the modified nucleosides in these rRNAs (88 in SSU rRNA and 262 in LSU rRNA, with only 3 LSU rRNA modifications incompletely characterized). The relative proportions of ribose-methylated nucleosides and pseudouridine (∼60% and ∼35%, respectively) are closely similar in the two rRNAs; however, whereas the Euglena SSU rRNA has about the same absolute number of modifications as its human counterpart, the Euglena LSU rRNA has twice as many modifications as the corresponding human LSU rRNA. The increased levels of rRNA fragmentation and modification in E. gracilis LSU rRNA are correlated with a 3-fold increase in the level of mispairing in helical regions compared to the human LSU rRNA. In contrast, no comparable increase in mispairing is seen in helical regions of the SSU rRNA compared to its homologs in other eukaryotes. In view of the reported effects of both ribose-methylated nucleoside and pseudouridine residues on RNA structure, these correlations lead us to suggest that increased modification in the LSU rRNA may play a role in stabilizing a 'looser' structure promoted by elevated helical mispairing and a high degree of fragmentation.

  18. Molecular evolution inferred from small subunit rRNA sequences: what does it tell us about phylogenetic relationships and taxonomy of the parabasalids?

    Science.gov (United States)

    Viscogliosi, E.; Edgcomb, V. P.; Gerbod, D.; Noel, C.; Delgado-Viscogliosi, P.; Sogin, M. L. (Principal Investigator)

    1999-01-01

    The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.

  19. Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections.

    Science.gov (United States)

    Fenollar, Florence; Roux, Véronique; Stein, Andréas; Drancourt, Michel; Raoult, Didier

    2006-03-01

    The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis, Comamonas terrigena, and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis, dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection.

  20. Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities.

    Science.gov (United States)

    Huys, Geert; Vanhoutte, Tom; Joossens, Marie; Mahious, Amal S; De Brandt, Evie; Vermeire, Severine; Swings, Jean

    2008-06-01

    The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V3, V3-V5, and V6-V8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V3 and V3-V5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.

  1. Phylogeny of some mycoplasmas from ruminants based on 16S rRNA sequences and definition of a new cluster within the hominis group.

    Science.gov (United States)

    Pettersson, B; Uhlén, M; Johansson, K E

    1996-10-01

    Almost complete (> 96%) 16S rRNA sequences from nine ruminant mycoplasmas have been determined by solid-phase DNA sequencing. Polymorphisms were found in four of the 16S rRNA sequences, which indicated the existence of two different rRNA operons. Seven polymorphisms were found in Mycoplasma agalatiae, three were found in Mycoplasma bovis, one was found in Mycoplasma alkalescens, and one was found in Mycoplasma bovirhinis. The sequence data were used for construction of phylogenetic trees. All but one of the ruminant mycoplasmas sequenced in this work clustered in the hominis group. A close relationship was found between M. agalactiae and M. bovis, with a 99% nucleotide similarity between their 16S rRNA sequences. They were also found to be members of the Mycoplasma lipophilum cluster of the hominis group. Furthermore, the 16S rRNA comparisons showed that Mycoplasma alkalescens and Mycoplasma canadense are closely related (> 98.5%), and these species were found to cluster in the Mycoplasma hominis cluster of the hominis group. Interestingly, M. bovirhinis grouped in a new phylogenetic cluster of the hominis group. The new cluster, which was supported by bootstrap percentage values, signature nucleotide analysis, and higher-order structural elements, was named the Mycoplasma synoviae cluster. Mycoplasma bovoculi, Mycoplasma conjunctivae, and Mycoplasma ovipneumoniae clustered in the Mycoplasma neurolyticum cluster of the hominis group. Mycoplasma alvi clustered with Mycoplasma pirum in the M. pneumoniae cluster of the pneumoniae group.

  2. 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells

    Directory of Open Access Journals (Sweden)

    Kuchipudi Suresh V

    2012-10-01

    Full Text Available Abstract Background One requisite of quantitative reverse transcription PCR (qRT-PCR is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA, ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9 (ATP5G1] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. Results The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs, pig tracheal epithelial cells (PTECs, and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells. Conclusions Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH were highly affected by influenza virus infection and

  3. Evolutionary relationships among Chlamydophila abortus variant strains inferred by rRNA secondary structure-based phylogeny.

    Science.gov (United States)

    Siarkou, Victoria I; Stamatakis, Alexandros; Kappas, Ilias; Hadweh, Paul; Laroucau, Karine

    2011-01-01

    The evolutionary relationships among known Chlamydophila abortus variant strains including the LLG and POS, previously identified as being highly distinct, were investigated based on rRNA secondary structure information. PCR-amplified overlapping fragments of the 16S, 16S-23S intergenic spacer (IS), and 23S domain I rRNAs were subjected to cloning and sequencing. Secondary structure analysis revealed the presence of transitional single nucleotide variations (SNVs), two of which occurred in loops, while seven in stem regions that did not result in compensatory substitutions. Notably, only two SNVs, in 16S and 23S, occurred within evolutionary variable regions. Maximum likelihood and Bayesian phylogeny reconstructions revealed that C. abortus strains could be regarded as representing two distinct lineages, one including the "classical" C. abortus strains and the other the "LLG/POS variant", with the type strain B577(T) possibly representing an intermediate of the two lineages. The two C. abortus lineages shared three unique (apomorphic) characters in the 23S domain I and 16S-23S IS, but interestingly lacked synapomorphies in the 16S rRNA. The two lineages could be distinguished on the basis of eight positions; four of these comprised residues that appeared to be signature or unique for the "classical" lineage, while three were unique for the "LLG/POS variant". The U277 (E. coli numbering) signature character, corresponding to a highly conserved residue of the 16S molecule, and the unique G681 residue, conserved in a functionally strategic region also of 16S, are the most pronounced attributes (autapomorphies) of the "classical" and the "LLG/POS variant" lineages, respectively. Both lineages were found to be descendants of a common ancestor with the Prk/Daruma C. psittaci variant. Compared with the "classical", the "LLG/POS variant" lineage has retained more ancestral features. The current rRNA secondary structure-based analysis and phylogenetic inference reveal new

  4. Evolutionary relationships among Chlamydophila abortus variant strains inferred by rRNA secondary structure-based phylogeny.

    Directory of Open Access Journals (Sweden)

    Victoria I Siarkou

    Full Text Available The evolutionary relationships among known Chlamydophila abortus variant strains including the LLG and POS, previously identified as being highly distinct, were investigated based on rRNA secondary structure information. PCR-amplified overlapping fragments of the 16S, 16S-23S intergenic spacer (IS, and 23S domain I rRNAs were subjected to cloning and sequencing. Secondary structure analysis revealed the presence of transitional single nucleotide variations (SNVs, two of which occurred in loops, while seven in stem regions that did not result in compensatory substitutions. Notably, only two SNVs, in 16S and 23S, occurred within evolutionary variable regions. Maximum likelihood and Bayesian phylogeny reconstructions revealed that C. abortus strains could be regarded as representing two distinct lineages, one including the "classical" C. abortus strains and the other the "LLG/POS variant", with the type strain B577(T possibly representing an intermediate of the two lineages. The two C. abortus lineages shared three unique (apomorphic characters in the 23S domain I and 16S-23S IS, but interestingly lacked synapomorphies in the 16S rRNA. The two lineages could be distinguished on the basis of eight positions; four of these comprised residues that appeared to be signature or unique for the "classical" lineage, while three were unique for the "LLG/POS variant". The U277 (E. coli numbering signature character, corresponding to a highly conserved residue of the 16S molecule, and the unique G681 residue, conserved in a functionally strategic region also of 16S, are the most pronounced attributes (autapomorphies of the "classical" and the "LLG/POS variant" lineages, respectively. Both lineages were found to be descendants of a common ancestor with the Prk/Daruma C. psittaci variant. Compared with the "classical", the "LLG/POS variant" lineage has retained more ancestral features. The current rRNA secondary structure-based analysis and phylogenetic

  5. Mitochondrial rRNA secondary structures and genome arrangements distinguish chelicerates: comparisons with a harvestman (Arachnida: Opiliones: Phalangium opilio).

    Science.gov (United States)

    Masta, Susan E

    2010-01-01

    Arachnids are a highly diverse group of arthropods, and many of the mitochondrial genomes that have been sequenced from arachnids possess unusual features in their inferred gene structures and genome organization. The first complete sequence of a mitochondrial genome from the arachnid order Opiliones (harvestmen) is presented here. Secondary structures of the two mitochondrial ribosomal subunits of Phalangium opilio are inferred and compared to mitochondrial rRNA structures of a hexapod and a chelicerate. The large subunit rRNA of P. opilio is found to have more helices conserved than in other arthropods, while the small subunit rRNA shows a complexity similar to that of other arthropods. These comparisons suggest that a reduction in rRNA complexity occurred in Pancrustacea after the divergence of Pancrustacea and Chelicerata from a common ancestor. The gene arrangement of the mitochondrial genome of P. opilio is compared with the gene order of taxa from all seven other orders of arachnids for which representative mitochondrial genomes have been sequenced. Taxa from five of these seven orders possess gene arrangements identical to that of Limulus polyphemus, and P. opilio is found to have a similar arrangement. However, in P. opilio, some genes near the putative control region are rearranged, with the suite of genes encoding tRNA(Gln), the control region, and tRNA(Ile) located downstream of the two ribosomal RNA genes, and upstream of where they are typically located in chelicerates. The genome encodes only 21 of the typical 22 mitochondrial tRNA genes and lacks the gene for tRNA(Leu(CUN)). The protein-coding genes in the mitochondrial genome of P. opilio show a significantly decreased use of codons recognized by tRNA(Leu(CUN)), likely due to selection to utilize the more specific tRNA(Leu(UUR)) anticodon. The gene arrangement and lack of a tRNA(Leu(CUN)) gene in P. opilio is most parsimoniously explained by the occurrence of at least two translocation events, one

  6. The origin of modern 5S rRNA: a case of relating models of structural history to phylogenetic data.

    Science.gov (United States)

    Sun, Feng-Jie; Caetano-Anollés, Gustavo

    2010-07-01

    Evolutionary models of molecular structures must incorporate molecular information at different levels of structural complexity and must be phrased within a phylogenetic perspective. In this regard, phylogenetic trees of substructures that are reconstructed from molecular features that contribute to order and thermodynamic stability show that a gradual model of evolution of 5S rRNA structure is more parsimonious than models that invoke large segmental duplications of the molecule. The search for trees of substructures that are most parsimonious, by their very nature, defines an objective strategy to select models of molecular change that best fit structural data. When combined with additional data, such as the age of protein domains that interact with RNA substructures, these trees can be used to falsify unlikely hypotheses.

  7. Molecular diversity of eukaryotes in municipal wastewater treatment processes as revealed by 18S rRNA gene analysis.

    Science.gov (United States)

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

    Eukaryotic communities involved in sewage treatment processes have been investigated by morphological identification, but have not yet been well-characterized using molecular approaches. In the present study, eukaryotic communities were characterized by constructing 18S rRNA gene clone libraries. The phylogenetic affiliations of a total of 843 clones were Alveolata, Fungi, Rhizaria, Euglenozoa, Stramenopiles, Amoebozoa, and Viridiplantae as protozoans and Rotifera, Gastrotricha, and Nematoda as metazoans. Sixty percent of the clones had <97% sequence identity to described eukaryotes, indicating the greater diversity of eukaryotes than previously recognized. A core OTU closely related to Epistylis chrysemydis was identified, and several OTUs were shared by 4-8 libraries. Members of the uncultured lineage LKM11 in Cryptomycota were predominant fungi in sewage treatment processes. This comparative study represents an initial step in furthering understanding of the diversity and role of eukaryotes in sewage treatment processes.

  8. Detection of Borrelia-specific 16S rRNA sequence in total RNA extracted from Ixodes ricinus ticks

    Directory of Open Access Journals (Sweden)

    Ž. Radulović

    2010-08-01

    Full Text Available A reverse transcriptase - polymerase chain reaction based assay for Borrelia species detection in ticks was developed. The method was based on amplification of 552 nucleotide bases long sequence of 16S rRNA, targeted by Borrelia specific primers. In the present study, total RNA extracted from Ixodes ricinus ticks was used as template. The results showed higher sensitivity for Borrelia detection as compared to standard dark-field microscopy. Method specificity was confirmed by cloning and sequencing of obtained 552 base pairs long amplicons. Phylogenetic analysis of obtained sequences showed that they belong to B. lusitaniae and B. afzelii genospecies. RT-PCR based method presented in this paper could be very useful as a screening test for detecting pathogen presence, especially when in investigations is required extraction of total RNA from ticks.

  9. Phylogenetic relationship of 16 Oedipodidae species (Insecta: Orthoptera) based on the 16S rRNA gene sequences

    Institute of Scientific and Technical Information of China (English)

    HUI-MENG LU; YUAN HUANG

    2006-01-01

    The sequences of the mitochondrial 16S rRNA gene of 16 Oedipodidae species were amplified and sequenced. All sequences were aligned and analyzed and the phyloge netic relationships were inferred. The properties of 16S gene in Oedipodidae showed typical patterns of many insects such as a high A+T content and variable distance-dependent transition/transversion ratios. The 0.2 weight for sites of loops may be advisable for phylogeny reconstruction using the maximum parsimony method. The phylogenetic analysis results do not support the current subfamily classification systems of Oedipodidae. Bryodemellinae and Bryodeminae are closely related and should be merged as one subfamily. The status of Oedipodinae and Locustinae is also problematic.

  10. Rapid Identification of Clinically Relevant Nocardia Species to Genus Level by 16S rRNA Gene PCR

    Science.gov (United States)

    Laurent, Frederic J.; Provost, Frederique; Boiron, Patrick

    1999-01-01

    Two regions of the gene coding for 16S rRNA in Nocardia species were selected as genus-specific primer sequences for a PCR assay. The PCR protocol was tested with 60 strains of clinically relevant Nocardia isolates and type strains. It gave positive results for all strains tested. Conversely, the PCR assay was negative for all tested species belonging to the most closely related genera, including Dietzia, Gordona, Mycobacterium, Rhodococcus, Streptomyces, and Tsukamurella. Besides, unlike the latter group of isolates, all Nocardia strains exhibited one MlnI recognition site but no SacI restriction site. This assay offers a specific and rapid alternative to chemotaxonomic methods for the identification of Nocardia spp. isolated from pathogenic samples. PMID:9854071

  11. Confidence intervals for similarity values determined for clonedSSU rRNA genes from environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Fields, M.W.; Schryver, J.C.; Brandt, C.C.; Yan, T.; Zhou, J.Z.; Palumbo, A.V.

    2007-04-02

    The goal of this research was to investigate the influenceof the error rate of sequence determination on the differentiation ofcloned SSU rRNA gene sequences for assessment of community structure. SSUrRNA cloned sequences from groundwater samples that represent differentbacterial divisions were sequenced multiple times with the samesequencing primer. From comparison of sequence alignments with unediteddata, confidence intervals were obtained from both a adouble binomial Tmodel of sequence comparison and by non-parametric methods. The resultsindicated that similarity values below 0.9946 arelikely derived fromdissimilar sequences at a confidence level of 0.95, and not sequencingerrors. The results confirmed that screening by direct sequencedetermination could be reliably used to differentiate at the specieslevel. However, given sequencing errors comparable to those seen in thisstudy, sequences with similarities above 0.9946 should be treated as thesame sequence if a 95 percent confidence is desired.

  12. MSClust: A Multi-Seeds Based Clustering Algorithm for microbiome profiling using 16S rRNA Sequence

    Science.gov (United States)

    Chen, Wei; Cheng, Yongmei; Zhang, Clarence; Zhang, Shaowu; Zhao, Hongyu

    2013-01-01

    Recent developments of next generation sequencing technologies have led to rapid accumulation of 16s rRNA sequences for microbiome profiling. One key step in data processing is to cluster short sequences into operational taxonomic units (OTUs). Although many methods have been proposed for OTU inferences, a major challenge is the balance between inference accuracy and computational efficiency, where inference accuracy is often sacrificed to accommodate the need to analyze large numbers of sequences. Inspired by the hierarchical clustering method and a modified greedy network clustering algorithm, we propose a novel multi-seeds based heuristic clustering method, named MSClust, for OTU inference. MSClust first adaptively selects multi-seeds instead of one seed for each candidate cluster, and the reads are then processed using a greedy clustering strategy. Through many numerical examples, we demonstrate that MSClust enjoys less memory usage, and better biological accuracy compared to existing heuristic clustering methods while preserving efficiency and scalability. PMID:23899776

  13. The crystal structure of Mtr4 reveals a novel arch domain required for rRNA processing

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, R.N.; Robinson, H.; Klauer, A. A.; Hintze, B. J.; van Hoof, A.; Johnson, S. J.

    2010-07-01

    The essential RNA helicase, Mtr4, performs a critical role in RNA processing and degradation as an activator of the nuclear exosome. The molecular basis for this vital function is not understood and detailed analysis is significantly limited by the lack of structural data. In this study, we present the crystal structure of Mtr4. The structure reveals a new arch-like domain that is specific to Mtr4 and Ski2 (the cytosolic homologue of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8S rRNA processing, and suggest that the arch functions independently of canonical helicase activity. In addition, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2-like helicases.

  14. The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase

    DEFF Research Database (Denmark)

    Vester, B; Hansen, L H; Douthwaite, S

    1995-01-01

    by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058. Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformation of this region in 23S rRNA was probed with chemical reagents...... of this region to the chemical reagents. The data indicate that a less-exposed conformation at A2058 leads to reduction in methylation by ErmE. Nucleotide G2057 and its interaction with C2611 maintain the conformation at A2058, and are thus important in forming the structural motif that is recognized by the Erm...

  15. Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences.

    Science.gov (United States)

    Yan, Ying; Xu, Yuan; Yi, Zhenzhen; Warren, Alan

    2013-09-01

    Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

  16. The sequence of Methanospirillum hungatei 23S rRNA confirms the specific relationship between the extreme halophiles and the Methanomicrobiales

    Science.gov (United States)

    Burggraf, S.; Ching, A.; Stetter, K. O.; Woese, C. R.

    1991-01-01

    We have determined the sequence of the 23S rRNA from the methanogenic archaeon Methanospirillum hungatei. This is the first such sequence from a member of the Methanomicrobiales. Moreover, it brings additional evidence to bear on the possible specific relationship between this particular group of methanogens and the extreme halophiles. Such evidence is critical in that several new (and relatively untested) methods of phylogenetic inference have lead to the controversial conclusion that the extreme halophiles are either not related to the archaea, or are only peripherally so. Analysis of the Methanospirillum hungatei 23S rRNA sequence shows the Methanomicrobiales are indeed a sister group of the extreme halophiles, further strengthening the conclusions reached from analysis of 16S rRNA sequences.

  17. Highly purified spermatozoal RNA obtained by a novel method indicates an unusual 28S/18S rRNA ratio and suggests impaired ribosome assembly.

    Science.gov (United States)

    Cappallo-Obermann, Heike; Schulze, Wolfgang; Jastrow, Holger; Baukloh, Vera; Spiess, Andrej-Nikolai

    2011-11-01

    Human spermatozoal RNA features special characteristics such as a significantly reduced quantity within spermatozoa compared with somatic cells is described as being devoid of ribosomal RNAs and is difficult to isolate due to a massive excess of genomic DNA in the lysates. Using a novel two-round column-based protocol for human ejaculates delivering highly purified spermatozoal RNA, we uncovered a heterogeneous, but specific banding pattern in microelectrophoresis with 28S ribosomal RNA being indicative for the amount of round cell contamination. Ejaculates with different round cell quantities and density-purified spermatozoa revealed that 18S rRNA but not 28S rRNA is inherent to a pure spermatozoal fraction. Transmission electron microscopy showed monoribosomes and polyribosomes in spermatozoal cytoplasm, while immunohistochemical results suggest the presence of proteins from small and large ribosomal subunits in retained spermatozoal cytoplasm irrespective of 28S rRNA absence.

  18. Anaerobic, non-sporulating, Gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing.

    Science.gov (United States)

    Lau, Susanna K P; Woo, Patrick C Y; Fung, Ami M Y; Chan, King-man; Woo, Gibson K S; Yuen, Kwok-yung

    2004-12-01

    Owing to the difficulties in identifying anaerobic, non-sporulating, Gram-positive bacilli in clinical microbiology laboratories, the epidemiology and clinical spectrum of disease of many of these bacteria have been poorly understood. The application of 16S rRNA gene sequencing in characterizing bacteraemia due to anaerobic, non-sporulating Gram-positive bacilli during a 4-year period is described. The first case of Olsenella uli bacteraemia, in a patient with acute cholangitis, is also reported. Among 165 blood culture isolates of anaerobic, Gram-positive bacilli, 75 were identified as Propionibacterium acnes by phenotypic tests and 21 as members of other anaerobic, non-sporulating Gram-positive bacilli by 16S rRNA gene sequencing. Of these 96 isolates, 16 (17 %) were associated with cases of clinically significant bacteraemia, among which 10 (63 %) were caused by Eggerthella, four (25 %) by Lactobacillus and one (6 %) by each of Eubacterium tenue and O. uli. Five of the 10 Eggerthella isolates were Eggerthella lenta, whereas the other five belonged to two novel Eggerthella species, with Eggerthella hongkongensis being almost as prevalent as Eggerthella lenta. Underlying disease in the gastrointestinal tract, isolation of Eggerthella and Lactobacillus, and monomicrobial bacteraemia were associated with clinically significant bacteraemia, whereas isolation of P. acnes and polymicrobial bacteraemia were associated with pseudobacteraemia. Most patients with clinically significant bacteraemia had underlying diseases, with diseases in the gastrointestinal tract being most common. The overall mortality rate was 31 %. Immunocompromised patients with clinically significant bacteraemia due to anaerobic, non-sporulating, Gram-positive bacilli other than P. acnes should be treated with appropriate antibiotics. The unexpected frequency of isolation of Eggerthella from blood cultures and its association with clinically significant disease suggest that this genus is probably of

  19. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing

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    Loreto Abusleme

    2014-04-01

    Full Text Available Background and objective: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Design: Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Results: Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. Conclusion: DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the

  20. Sequence heterogeneity in the 18S rRNA gene in Theileria equi from horses presented in Switzerland.

    Science.gov (United States)

    Liu, Qin; Meli, Marina L; Zhang, Yi; Meili, Theres; Stirn, Martina; Riond, Barbara; Weibel, Beatrice; Hofmann-Lehmann, Regina

    2016-05-15

    A reverse line blot (RLB) hybridization assay was adapted and applied for equine blood samples collected at the animal hospital of the University of Zurich to determine the presence of piroplasms in horses in Switzerland. A total of 100 equine blood samples were included in the study. The V4 hypervariable region of the 18S rRNA gene was amplified by polymerase chain reaction and analyzed using the RLB assay. Samples from seven horses hybridized to a Theileria/Babesia genus-specific and a Theileria genus-specific probe. Of these, two hybridized also to the Theileria equi-specific probe. The other five positive samples did not hybridize to any of the species-specific probes, suggesting the presence of unrecognized Theileria variants or genotypes. The 18S rRNA gene of the latter five samples were sequenced and found to be closely related to T. equi isolated from horses in Spain (AY534822) and China (KF559357) (≥98.4% identity). Four of the seven horses that tested positive had a documented travel history (France, Italy, and Spain) or lived abroad (Hungary). The present study adds new insight into the presence and sequence heterogeneity of T. equi in Switzerland. The results prompt that species-specific probes must be designed in regions of the gene unique to T. equi. Of note, none of the seven positive horses were suspected of having Theileria infection at the time of presentation to the clinic. Clinicians should be aware of the possibility of equine piroplasma infections outside of endemic areas and in horses without signs of piroplasmosis.