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Sample records for artificial chromosome clone

  1. Complete Genome Sequence of a Human Cytomegalovirus Strain AD169 Bacterial Artificial Chromosome Clone

    Science.gov (United States)

    Ostermann, Eleonore; Spohn, Michael; Indenbirken, Daniela

    2016-01-01

    The complete sequence of the human cytomegalovirus strain AD169 (variant ATCC) cloned as a bacterial artificial chromosome (AD169-BAC, also known as HB15 or pHB15) was determined. The viral genome has a length of 230,290 bp and shows 52 nucleotide differences compared to a previously sequenced AD169varATCC clone. PMID:27034483

  2. Bacterial Artificial Chromosome Clones of Viruses Comprising the Towne Cytomegalovirus Vaccine

    OpenAIRE

    Xiaohong Cui; Adler, Stuart P.; Davison, Andrew J.; Larry Smith; EL-Sayed E. Habib; McVoy, Michael A.

    2012-01-01

    Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. I...

  3. Complete Genomes of Classical Swine Fever Virus Cloned into Bacterial Artificial Chromosomes

    OpenAIRE

    Rasmussen, Thomas Bruun; Reimann, I; Uttenthal, Åse; De Beer, M.

    2011-01-01

    Complete genome amplification of viral RNA provides a new tool for the generation of modified pestiviruses. We have used our full-genome amplification strategy for generation of amplicons representing complete genomes of classical swine fever virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome (BAC) generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived. Our strategy allows construction of stable infec...

  4. Drought-tolerant rice germplasm developed from an Oryza officinalis transformation-competent artificial chromosome clone.

    Science.gov (United States)

    Liu, R; Zhang, H H; Chen, Z X; Shahid, M Q; Fu, X L; Liu, X D

    2015-10-29

    Oryza officinalis has proven to be a natural gene reservoir for the improvement of domesticated rice as it carries many desirable traits; however, the transfer of elite genes to cultivated rice by conventional hybridization has been a challenge for rice breeders. In this study, the conserved sequence of plant stress-related NAC transcription factors was selected as a probe to screen the O. officinalis genomic transformation-competent artificial chromosome library by Southern blot; 11 positive transformation-competent artificial chromosome clones were subsequently detected. By Agrobacterium-mediated transformation, an indica rice variety, Huajingxian 74 (HJX74), was transformed with a TAC clone harboring a NAC gene-positive genomic fragment from O. officinalis. Molecular analysis revealed that the O. officinalis genomic fragment was integrated into the genome of HJX74. The transgenic lines exhibited high tolerance to drought stress. Our results demonstrate that the introduction of stress-related transformation-competent artificial chromosome clones, coupled with a transgenic validation approach, is an effective method of transferring agronomically important genes from O. officinalis to cultivated rice.

  5. A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes.

    OpenAIRE

    Bradshaw, M S; Bollekens, J A; Ruddle, F H

    1995-01-01

    The functional analysis of genes frequently requires manipulation of large genomic regions embedded in yeast artificial chromosomes (YACs). We have designed a yeast-bacteria shuttle vector, pClasper, that can be used to clone specific regions of interest from YACs by homologous recombination. The important feature of pClasper is the presence of the mini-F factor replicon. This leads to a significant increase in the size of the plasmid inserts that can be maintained in bacteria after cloning b...

  6. Cloning the simian varicella virus genome in E. coli as an infectious bacterial artificial chromosome

    OpenAIRE

    Gray, Wayne L.; Zhou, Fuchun; Noffke, Juliane; Tischer, B Karsten

    2011-01-01

    Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectiou...

  7. Pathogenicity of a Very Virulent Strain of Marek's Disease Herpesvirus Cloned as Infectious Bacterial Artificial Chromosomes

    Directory of Open Access Journals (Sweden)

    Lorraine P. Smith

    2011-01-01

    Full Text Available Bacterial artificial chromosome (BAC vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130 of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones.

  8. Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates

    OpenAIRE

    Borenstein, Ronen; Frenkel, Niza

    2009-01-01

    Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constr...

  9. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    OpenAIRE

    Kalpana Dulal; Benjamin Silver; Hua Zhu

    2012-01-01

    Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination syste...

  10. Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome

    Science.gov (United States)

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek’s disease virus (MDV) is a highly oncogenic herpe...

  11. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    Directory of Open Access Journals (Sweden)

    Kalpana Dulal

    2012-01-01

    Full Text Available Bacterial artificial chromosome (BAC technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV. The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

  12. Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

    OpenAIRE

    Roth Swaantje J; Höper Dirk; Beer Martin; Feineis Silke; Tischer B Karsten; Osterrieder Nikolaus

    2011-01-01

    Abstract Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows mainte...

  13. Complete Genome Sequence of Cell Culture-Attenuated Guinea Pig Cytomegalovirus Cloned as an Infectious Bacterial Artificial Chromosome

    OpenAIRE

    Yang, Dongmei; Alam, Zohaib; Cui, Xiaohong; Chen, Michael; Sherrod, Carly J.; McVoy, Michael A.; Schleiss, Mark R.; Dittmer, Dirk P

    2014-01-01

    The complete genome sequence of attenuated guinea pig cytomegalovirus cloned as bacterial artificial chromosome N13R10 was determined. Comparison to pathogenic salivary gland-derived virus revealed 13 differences, 1 of which disrupted overlapping open reading frames encoding GP129 and GP130. Attenuation of N13R10 may arise from an inability to express GP129 and/or GP130.

  14. Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Although the centromeres of some plants have been investigated previously, our knowledge of the wheat centromere is still very limited. To understand the structure and function of the wheat centromere, we used two centromeric repeats (RCS1 and CCS1-5ab) to obtain some centromere-associated bacterial artificial chromosome (BAC) clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat (Triticum boeoticum Boiss.; 2n=2x=14) BAC library. Southern hybridization results indicated that, of the 32 candidates,there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization (FISH) analysis indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromeric regions in hexaploid wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or D-genome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific (or rich) sequences dispersing on chromosome arms in the BAC clone TbBAC5. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation in the period before metaphase.

  15. Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone.

    OpenAIRE

    Ketner, G; Spencer, F; Tugendreich, S; C. Connelly; Hieter, P

    1994-01-01

    A yeast artificial chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into mammalian cells. The adenovirus YAC could be manipulated efficiently using homologous recombination-based methods in the yeast host, and mutant viruses, including a variant that expresses the human analog of the Saccharomyces cerevisiae CDC27 gene, were readily recovered from modified derivatives of the Y...

  16. Generalized Gap Model for Bacterial Artificial Chromosome Clone Fingerprint Mapping and Shotgun Sequencing

    OpenAIRE

    Wendl, Michael C; Robert H Waterston

    2002-01-01

    We develop an extension to the Lander-Waterman theory for characterizing gaps in bacterial artificial chromosome fingerprint mapping and shotgun sequencing projects. It supports a larger set of descriptive statistics and is applicable to a wider range of project parameters. We show that previous assertions regarding inconsistency of the Lander-Waterman theory at higher coverages are incorrect and that another well-known but ostensibly different model is in fact the same. The apparent paradox ...

  17. Recovery of infectious virus from full-length cowpox virus (CPXV DNA cloned as a bacterial artificial chromosome (BAC

    Directory of Open Access Journals (Sweden)

    Roth Swaantje J

    2011-01-01

    Full Text Available Abstract Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV. We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR as a bacterial artificial chromosome (BAC in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.

  18. Highly selective isolation of human DNAs from rodent–human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning

    OpenAIRE

    Larionov, Vladimir; Kouprina, Natalya; Graves, Joan; Resnick, Michael A.

    1996-01-01

    Transformation-associated recombination (TAR) can be exploited in yeast to clone human DNAs. TAR cloning was previously accomplished using one or two telomere-containing vectors with a common human repeat(s) that could recombine with human DNA during transformation to generate yeast artificial chromosomes (YACs). On basis of the proposal that broken DNA ends are more recombinogenic than internal sequences, we have investigated if TAR cloning could be applied to the...

  19. Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.

    Science.gov (United States)

    Reddy, Sanjay M; Sun, Aijun; Khan, Owais A; Lee, Lucy F; Lupiani, Blanca

    2013-06-01

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.

  20. Sequential cloning of chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1991-12-31

    A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  1. Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes

    OpenAIRE

    Kouprina, N.; Ebersole, T.; Koriabine, M.; Pak, E; Rogozin, I. B.; Katoh, M; Oshimura, M; Ogi, K; Peredelchuk, M.; Solomon, G; Brown, W.; Barrett, J. C.; Larionov, V

    2003-01-01

    Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used...

  2. A Self-Excisable Infectious Bacterial Artificial Chromosome Clone of Varicella-Zoster Virus Allows Analysis of the Essential Tegument Protein Encoded by ORF9▿

    OpenAIRE

    Tischer, B. Karsten; Kaufer, Benedikt B; Sommer, Marvin; Wussow, Felix; Ann M Arvin; Osterrieder, Nikolaus

    2007-01-01

    In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recov...

  3. Artifically inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes

    Science.gov (United States)

    The long terminal repeat (LTR) sequence of reticuloendotheliosis virus (REV) was inserted into the very virulent Marek’s disease virus (MDV) Md5 bacterial artificial chromosome clone. The insertion site was nearly identical to the REV LTR that was naturally inserted into the JM/102W strain of MDV fo...

  4. Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system

    OpenAIRE

    Nah, Hee-Ju; Woo, Min-Woo; Choi, Si-Sun; Kim, Eung-Soo

    2015-01-01

    Background Direct cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-si...

  5. Complete Genomes of Classical Swine Fever Virus Cloned into Bacterial Artificial Chromosomes

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, I.; Uttenthal, Åse;

    Complete genome amplification of viral RNA provides a new tool for the generation of modified pestiviruses. We have used our full-genome amplification strategy for generation of amplicons representing complete genomes of classical swine fever virus. The amplicons were cloned directly into a stable...

  6. Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo

    OpenAIRE

    Tanaka, Michiko; Kagawa, Hiroyuki; Yamanashi, Yuji; Sata, Tetsutaro; Kawaguchi, Yasushi

    2003-01-01

    In recent years, several laboratories have reported on the cloning of herpes simplex virus type 1 (HSV-1) genomes as bacterial artificial chromosomes (BACs) in Escherichia coli and on procedures to manipulate these genomes by using the bacterial recombination machinery. However, the HSV-BACs reported so far are either replication incompetent or infectious, with a deletion of one or more viral genes due to the BAC vector insertion. For use as a multipurpose clone in research on HSV-1, we attem...

  7. Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

    OpenAIRE

    Larionov, V.; Kouprina, N; Graves, J.; Chen, X N; Korenberg, J R; Resnick, M A

    1996-01-01

    DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized v...

  8. Cloning and Mutagenesis of the Murine Gammaherpesvirus 68 Genome as an Infectious Bacterial Artificial Chromosome

    OpenAIRE

    Adler, Heiko; Messerle, Martin; Wagner, Markus; Koszinowski, Ulrich H.

    2000-01-01

    Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacteria...

  9. Bacterial Artificial Chromosome Mutagenesis Using Recombineering

    OpenAIRE

    Kumaran Narayanan; Qingwen Chen

    2011-01-01

    Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented...

  10. A high-throughput strategy for screening of bacterial artificial chromosome libraries and anchoring of clones on a genetic map constructed with single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Deal Karin R

    2009-01-01

    Full Text Available Abstract Background Current techniques of screening bacterial artificial chromosome (BAC libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate™ assay. Results To test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 Aegilops tauschii BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an Ae. tauschii genetic map. Of 1384 allele-informative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted Ae. tauschii BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the Ae. tauschii genetic map. For 41 (95% assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5% assays, positive clones were in two different contigs and the relationships of these contigs to loci on the Ae. tauschii genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools. Conclusion The highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high

  11. Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA)

    DEFF Research Database (Denmark)

    Cottingham, Matthew G; Andersen, Rikke F; Spencer, Alexandra J;

    2008-01-01

    -length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A...

  12. Artificially inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes.

    Science.gov (United States)

    Kim, Taejoong; Mays, Jody; Fadly, Aly; Silva, Robert F

    2011-06-01

    Researchers reported that co-cultivating the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in an REV long terminal repeat (LTR) being inserted into the internal repeat short (IRS) region of JM/102W. When the resulting recombinant virus was serially passed in cell culture, the initial LTR was duplicated and a second LTR spontaneously appeared in the terminal repeat short (TRS) region of the MDV genome. The virus, designated RM1, was significantly attenuated but still induced severe bursal and thymic atrophy (Isfort et al. PNAS 89:991-995). To determine whether the altered phenotype was due solely to the LTR, we cloned the LTR from the RM1 IRS region and inserted it into the IRS region of a very virulent bacterial artificial clone (BAC) of the Md5 strain of MDV, which we designated rMd5-RM1-LTR. During blind passage in duck embryo fibroblast cultures, the initial LTR in the rMd5-RM1-LTR was also duplicated, with LTRs appearing in both IRS and TRS regions of the MDV genome. The inserted LTR sequences and transcripts associated with the MDV open reading frames MDV085, MDV086, SORF2, US1, and US10 were molecularly characterized. The parental Md5 BAC contains a family of transcripts of 3, 2, and 1 kb that all terminate at the end of the US10 gene. The rMd5-RM1-LTR and RM1 viruses both express an additional 4 kb transcript that originates in the LTR and also terminates after US10. Collectively, the data suggest that our engineered rMd5-RM1-LTR virus very closely resembles the RM1 virus in its structure and transcription patterns.

  13. A five-fold pig bacterial artificial chromosome library:a resource for positional cloning and physical mapping

    Institute of Scientific and Technical Information of China (English)

    LIU Wei; LI Ning; ZHANG Ying; LIU Zhaoliang; GUO Li; WANG Xiaobo; FEI Jing; FENG Jidong; ZHAO Rui; HU Xiaoxiang

    2006-01-01

    A pig BAC library was constructed with genomic DNA from a male Erhualian pig. After partial digestion with Hind Ⅲ or BamH I the fragments obtained were cloned into the pBeloBAC11 vector. The library consists of 184320 clones which stored in 480pieces 384-well plates (20 plates per superpool). A two-step 4-dimension PCR screening system was established to screen the positive clones. An average insert size of 128 kb was estimated from 105 randomly isolated clones, which indicates that the library is more than five times of genomic coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 10single-copy genes were screened out and the positive clones were yielded between 1 and 8 with an average of 3.6. Positive superpools were obtained for 32 microsatellite markers selected from different regions of pig genome. The number of positive superpools for each marker varies from 1 to 9 with an average of 4.78. This BAC library provides an additional resource for pig physical mapping and gene identification.

  14. Bacterial Artificial Chromosome Mutagenesis Using Recombineering

    Directory of Open Access Journals (Sweden)

    Kumaran Narayanan

    2011-01-01

    Full Text Available Gene expression from bacterial artificial chromosome (BAC clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo.

  15. Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi▿

    Science.gov (United States)

    Costes, B.; Fournier, G.; Michel, B.; Delforge, C.; Raj, V. Stalin; Dewals, B.; Gillet, L.; Drion, P.; Body, A.; Schynts, F.; Lieffrig, F.; Vanderplasschen, A.

    2008-01-01

    Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines. PMID:18337580

  16. Next Generation Sequencing of Classical Swine Fever Virus and Border Disease virus cloned in Bacterial Artificial Chromosomes

    DEFF Research Database (Denmark)

    Fahnøe, Ulrik; Höper, Dirk; Beer, martin;

    2012-01-01

    be rescued only from some of our BAC constructs whereas others are not replication competent. To further analyze this discrepancy we have completely sequenced selected pestivirus BAC DNAs using a 454 Genome Sequencer FLX to evaluate the number/kind of deviations in the cloned genome sequences. In addition......, we have sequenced the full genome cDNA fragments used for the BACs by the same approach. This enables us to evaluate in more detail the nature of nucleotide changes in the pestivirus BACs that lead to lack of replicationcompetence and/or virus rescue. Additionally, detailed knowledge of the genomic...

  17. Screening and chromosome localization of two cotton BAC clones.

    Science.gov (United States)

    Cui, Xinglei; Liu, Fang; Liu, Yuling; Zhou, Zhongli; Wang, Chunying; Yanyan Zhao; Meng, Fei; Wang, Xingxing; Cai, Xiaoyan; Wang, Yuhong; Peng, Renhai; Wang, Kunbo

    2016-01-01

    Two bacterial artificial chromosome (BAC) clones (350B21 and 299N22) of Pima 90-53 cotton [Gossypium barbadense Linnaeus, 1753 (2n=4x=52)] were screened from a BAC library using SSR markers. Strong hybridization signals were detected at terminal regions of all A genome (sub-genome) chromosomes, but were almost absent in D genome (sub-genome) chromosomes with BAC clone 350B21 as the probe. The results indicate that specific sequences, which only exist at the terminal parts of A genome (sub-genome) chromosomes with a huge repeat number, may be contained in BAC clone 350B21. When utilizing FISH with the BAC clone 299N22 as probe, a pair of obvious signals was detected on chromosome 13 of D genome (sub-genome), while strong dispersed signals were detected on all A genome (sub-genome) chromosomes. The results showed that peculiar repetitive sequence, which was distributed throughout all A genome (sub-genome) chromosomes, may exist in BAC clone 299N22. The absence of the repetitive sequences, which exist in the two BAC clones, in D genome may account for the genome-size variation between A and D genomes. In addition, the microcolinearity analysis of the clone 299N22 and its homologous region on Gossypium raimondii Ulbrich, 1932 chromosome 13 (D513) indicated that the clone 299N22 might come from A sub-genome of sea island cotton (Gossypium barbadense), and a huge number of small deletions, illegitimate recombination, translocation and rearrangements may have occurred during the genus evolution. The two BAC clones studied here can be used as cytological markers but will be also be helpful to research in cotton genome evolution and comparative genomics. PMID:27186333

  18. Insertion of reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of a very virulent Marek's disease virus alters its pathogenicity.

    Science.gov (United States)

    Mays, Jody K; Silva, Robert F; Kim, Taejoong; Fadly, Aly

    2012-01-01

    Co-cultivation of the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in the generation of a recombinant MDV containing the REV long terminal repeat (LTR) named the RM1 strain of MDV, a strain that was highly attenuated for oncogenicity but induced severe bursal and thymic atrophy. We hypothesize that the phenotypic changes were solely due to the LTR insertion. Furthermore, we hypothesize that insertion of REV LTR into an analogous location in a different MDV would result in a similar phenotypic change. To test these hypotheses, we inserted the REV LTR into a bacterial artificial chromosome (BAC) clone of a very virulent strain of MDV, Md5, and designated the virus rMd5-RM1-LTR. The rMd5-RM1-LTR virus and the rMd5 virus were passaged in duck embryo fibroblast cells for up to 40 passages before pathogenicity studies. Susceptible chickens were inoculated intra-abdominally at hatch with the viruses rMd5-RM1-LTR, rMd5 BAC parental virus, wild-type strain Md5, or strain RM1 of MDV. The rMd5-RM1-LTR virus was attenuated at cell culture passage 40, whereas the rMd5 BAC without RM1 LTR retained its pathogenicity at cell culture passage 40. Using polymerase chain analysis, the RM1 LTR insert was detected in MDV isolated from buffy coat cells collected from chickens inoculated with rMd5-RM1-LTR, but only at 1 week post inoculation. The data suggest that the presence of the RM1 LTR insert within MDV genome for 1 week post inoculation with virus at hatch is sufficient to cause a reduction in pathogenicity of strain Md5 of MDV.

  19. Construction of a yeast artificial chromosome contig encompassing the human acidic fibroblast growth factor (FGF1) gene: Toward the cloning of the ANLL/MDS tumor-suppressor gene

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Ing-Ming; Gilmore, E.C.; Liu, Yang; Payson, R.A. (Ohio State Univ., Columbus, OH (United States))

    1994-02-01

    The region surrounding the human acidic fibroblast growth factor (FGF1) locus on chromosome 5q31 is of particular interest since it represents a critical region consistently lost in acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS) patients who have a demonstrable deletion of the distal portion of the long arm of chromosome 5. It is proposed that an ANLL/MDS leukemia suppressor gene resides on 5q31. The authors have previously shown that the gene is most likely localized between FGF1 and PDGFRB/CSF1R loci. The region has also been linked to at least four other genetic diseases, Treacher Collins syndrome, diastrophic dysplasia, limb-girdle muscular dystrophy, and an autosomal dominant deafness, by linkage analysis. Here, they describe yeast artificial chromosomes (YAC) spanning 450 kb around the FGF1 gene. Six YAC clones were isolated from a human YAC library and their restriction enzyme maps were determined. The overlap of the clones with each other and with FGF1 cosmid and phage clones was characterized. Three of the YAC clones were found to contain the entire FGF1 gene, which spans more than 100 kb. Proximal and distal ends of several of these YAC clones were isolated for further overlap cloning. The proximal ends of both Y2 and Y4 were localized to previously isolated FGF1 DNA by sequence analysis. The distal ends of these two clones also hybridized to a human-hamster hybrid containing chromosome 5 as the only human genetic material. These results suggest that these YAC clones represent colinear DNA around the FGF1 locus. None of the YAC clones were found to contain the CD 14 and GRL genes, the closest known proximal and distal markers (relative to the centromere) to the FGF1 gene, respectively. This contig is useful for the overlap cloning of the 5q31 region and for reverse genetic strategies for the isolation of disease genes in the region. 46 refs., 7 figs., 5 tabs.

  20. Cloning of the Full-Length Rhesus Cytomegalovirus Genome as an Infectious and Self-Excisable Bacterial Artificial Chromosome for Analysis of Viral Pathogenesis

    OpenAIRE

    Chang, W. L. William; Peter A Barry

    2003-01-01

    Rigorous investigation of many functions encoded by cytomegaloviruses (CMVs) requires analysis in the context of virus-host interactions. To facilitate the construction of rhesus CMV (RhCMV) mutants for in vivo studies, a bacterial artificial chromosome (BAC) containing an enhanced green fluorescent protein (EGFP) cassette was engineered into the intergenic region between unique short 1 (US1) and US2 of the full-length viral genome by Cre/lox-mediated recombination. Infectious virions were re...

  1. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  2. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination. PMID:27349114

  3. Rapid and efficient introduction of a foreign gene into bacterial artificial chromosome-cloned varicella vaccine by Tn7-mediated site-specific transposition

    International Nuclear Information System (INIS)

    Using a rapid and reliable system based on Tn7-mediated site-specific transposition, we have successfully constructed a recombinant Oka varicella vaccine (vOka) expressing the mumps virus (MuV) fusion protein (F). The backbone of the vector was our previously reported vOka-BAC (bacterial artificial chromosome) genome. We inserted the transposon Tn7 attachment sequence, LacZα-mini-attTn7, into the region between ORF12 and ORF13 to generate a vOka-BAC-Tn genome. The MuV-F expressing cassette was transposed into the vOka-BAC genome at the mini-attTn7 transposition site. MuV-F protein was expressed in recombinant virus, rvOka-F infected cells. In addition, the MuV-F protein was cleaved in the rvOka-F infected cells as in MuV-infected cells. The growth of rvOka-F was similar to that of the original recombinant vOka without the F gene. Thus, we show that Tn7-mediated transposition is an efficient method for introducing a foreign gene expression cassette into the vOka-BAC genome as a live virus vector.

  4. Artificial cloning of domestic animals

    OpenAIRE

    Keefer, Carol L.

    2015-01-01

    Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike ...

  5. Artificial cloning of domestic animals.

    Science.gov (United States)

    Keefer, Carol L

    2015-07-21

    Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

  6. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    OpenAIRE

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-01-01

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human g...

  7. Isolation and characterization of bovine herpesvirus 4 (BoHV-4 from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome

    Directory of Open Access Journals (Sweden)

    Cavirani Sandro

    2009-08-01

    Full Text Available Abstract Background Bovine herpesvirus 4 (BoHV-4 is a gammaherpesvirus with a Worldwide distribution in cattle and is often isolated from the uterus of animals with postpartum metritis or pelvic inflammatory disease. Virus strain adaptation to an organ, tissue or cell type is an important issue for the pathogenesis of disease. To explore the mechanistic role of viral strain variation for uterine disease, the present study aimed to develop a tool enabling precise genetic discrimination between strains of BoHV-4 and to easily manipulate the viral genome. Methods A strain of BoHV-4 was isolated from the uterus of a persistently infected cow and designated BoHV-4-U. The authenticity of the isolate was confirmed by RFLP-PCR and sequencing using the TK and IE2 loci as genetic marker regions for the BoHV-4 genome. The isolated genome was cloned as a Bacterial Artificial Chromosome (BAC and manipulated through recombineering technology Results The BoHV-4-U genome was successfully cloned as a BAC, and the stability of the pBAC-BoHV-4-U clone was confirmed over twenty passages, with viral growth similar to the wild type virus. The feasibility of using BoHV-4-U for mutagenesis was demonstrated using the BAC recombineering system. Conclusion The analysis of genome strain variation is a key method for investigating genes associated with disease. A resource for dissection of the interactions between BoHV-4 and host endometrial cells was generated by cloning the genome of BoHV-4 as a BAC.

  8. Construction of human chromosome 21-specific yeast artificial chromosomes.

    Science.gov (United States)

    McCormick, M K; Shero, J H; Cheung, M C; Kan, Y W; Hieter, P A; Antonarakis, S E

    1989-12-01

    Chromosome 21-specific yeast artificial chromosomes (YACs) have been constructed by a method that performs all steps in agarose, allowing size selection by pulsed-field gel electrophoresis and the use of nanogram to microgram quantities of DNA. The DNA sources used were hybrid cell line WAV-17, containing chromosome 21 as the only human chromosome and flow-sorted chromosome 21. The transformation efficiency of ligation products was similar to that obtained in aqueous transformations and yielded YACs with sizes ranging from 100 kilobases (kb) to greater than 1 megabase when polyamines were included in the transformation procedure. Twenty-five YACs containing human DNA have been obtained from a mouse-human hybrid, ranging in size from 200 to greater than 1000 kb, with an average size of 410 kb. Ten of these YACs were localized to subregions of chromosome 21 by hybridization of RNA probes (corresponding to the YAC ends recovered in Escherichia coli) to a panel of somatic cell hybrid DNA. Twenty-one human YACs, ranging in size from 100 to 500 kb, with an average size of 150 kb, were obtained from approximately equal to 50 ng of flow-sorted chromosome 21 DNA. Three were localized to subregions of chromosome 21. YACs will aid the construction of a physical map of human chromosome 21 and the study of disorders associated with chromosome 21 such as Alzheimer disease and Down syndrome.

  9. Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.

    OpenAIRE

    Hermanson, G G; Hoekstra, M F; McElligott, D. L.; Evans, G A

    1991-01-01

    Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker c...

  10. Construction and Characterization of Three Wheat Bacterial Artificial Chromosome Libraries

    Directory of Open Access Journals (Sweden)

    Wenjin Cao

    2014-11-01

    Full Text Available We have constructed three bacterial artificial chromosome (BAC libraries of wheat cultivar Triticum aestivum Wangshuibai, germplasms T. monococcum TA2026 and TA2033. A total of 1,233,792,170,880 and 263,040 clones were picked and arrayed in 384-well plates. On the basis of genome sizes of 16.8 Gb for hexaploid wheat and 5.6 Gb for diploid wheat, the three libraries represented 9.05-, 2.60-, and 3.71-fold coverage of the haploid genomes, respectively. An improved descending pooling system for BAC libraries screening was established. This improved strategy can save 80% of the time and 68% of polymerase chain reaction (PCR with the same successful rate as the universal 6D pooling strategy.

  11. Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species

    Directory of Open Access Journals (Sweden)

    Doležel Jaroslav

    2010-02-01

    Full Text Available Abstract Background The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. Results The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the

  12. Refined human artificial chromosome vectors for gene therapy and animal transgenesis.

    Science.gov (United States)

    Kazuki, Y; Hoshiya, H; Takiguchi, M; Abe, S; Iida, Y; Osaki, M; Katoh, M; Hiratsuka, M; Shirayoshi, Y; Hiramatsu, K; Ueno, E; Kajitani, N; Yoshino, T; Kazuki, K; Ishihara, C; Takehara, S; Tsuji, S; Ejima, F; Toyoda, A; Sakaki, Y; Larionov, V; Kouprina, N; Oshimura, M

    2011-04-01

    Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

  13. Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries

    Energy Technology Data Exchange (ETDEWEB)

    Gingrich, J.C.; Boehrer, D.M.; Garnes, J.A. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-15

    This article discusses the construction and characterization of three human chromosome 2-specific clone libraries. A chromosome 2-specific PAC library was also constructed from a hybrid cell line. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. 47 refs., 3 figs., 1 tab.

  14. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-09-18

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

  15. Cloning of BWS-associated chromosomal breakpoints

    Energy Technology Data Exchange (ETDEWEB)

    Mannens, M.; Hoovers, J.; Redeker, E. [Univ. of Amsterdam (Netherlands)

    1994-09-01

    The Beckwith-Wiedemann syndrome (BWS) is characterized by numerous growth abnormalities and is thought to be subject to {open_quotes}parental imprinting{close_quotes}. There is a striking increased incidence of different types of childhood tumors found in BWS patients of 7.5%. The syndrome is localized to chromosome region 11p15.3-p15.5. A contiguous map of this region of over 10 Mb was constructed and all 25 known genes from this region were localized to this map, including known imprinted genes like IGF2 and H19, or candidate tumor suppressor genes like WEE1, ST5 and rhombotin. In addition, we were able to place the breakpoints of 8 different balanced chromosomal rearrangements, associated with the Beckwith-Wiedemann syndrome, onto this map in two distinct regions that are now known to contain childhood tumor suppressor genes. In one of these BWS clusters (BWSCR1) 5/5 translocation breakpoints could be identified with overlapping cosmids for each breakpoint. A 6.7 kb transcript in all adult tissues tested was identified by several of these cosmids. This transcript was less abundant in fetal tissue. Preliminary results suggest the presence of zinc-finger protein motifs in this gene. This, however, has to be confirmed by sequence analysis. Two breakpoints in the more proximal BWS region (BWSCR2) were associated with clinically distinct BWS phenotypes, of which hemihypertrophy and Wilms` tumor are the most pronounced clinical findings. These breakpoints were found to be overlapped by the same cosmid. In this region, zinc-finger motifs flanking the breakpoints were identified by genomic sequence analysis.

  16. Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yang; ZHANG Xiaojun; Chantel F.SCHEURING; ZHANG Hongbin; LI Fuhua; XIANG Jianhai

    2008-01-01

    Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research.High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I,respectively.The BamH I library consisted of 53760 clones while the Mbo I library consisted of 7680 clones.Approximately 96% of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb,providing a coverage of 5.3 haploid genome equivalents.Similarly,the Mbo I library with an average insert of 145 kb and no insert-empty clones,thus providing a genome coverage of 1.1 haploid genome equivalents.

  17. Cloning of Bovine herpesvirus type 1 and type 5 as infectious bacterial artifical chromosomes

    OpenAIRE

    Ackermann Mathias; Fraefel Cornel; Gabev Evgeni; Tobler Kurt

    2009-01-01

    Abstract Background Bovine herpesviruses type 1 (BoHV1) and type 5 (BoHV5) are two closely related pathogens of cattle. The identity of the two viruses on the amino acid level averages 82%. Despite their high antigenetic similarities the two pathogens induce distinctive clinical signs. BoHV1 causes respiratory and genital tract infections while BoHV5 leads to severe encephalitis in calves. Findings The viral genomes of BoHV1 and BoHV5 were cloned as infectious bacterial artificial chromosomes...

  18. Construction and characterization of the transformation-competent artificial chromosome(TAC)libraries of Leymus multicaulis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.

  19. Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis

    Institute of Scientific and Technical Information of China (English)

    XU YueYu; ZHOU YuLei; SONG LinLin; ZHANG Yan; ZHAO MaoLin

    2008-01-01

    Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants. In order to clone genes highly tolerant to barley yellow dwarf virus (BYDV), Aphids, drought and salt from Leymus multicaulis, the two TAC genomic libraries Ⅰ and Ⅱ were constructed in vector pYLTAC17 and pYLTAC747H/sacB, which contain about 165000 and 236000 recombinant clones sepa-rately. The genome coverage of the two libraries was totally estimated to be about 3-5 haploid genome equivalents, as size selection of genomic DNA fragments was approximately from 9 to 300 kb. Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so, stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTACTM G3 arraying robot after being transferred manually into three 384-well plates. Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates. Nineteen positive clones were detected by using the probe glutahione reductase gene of L. Multicaulis. TAC libraries constructed here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning. Once the target TAC clones were isolated, they could be immediately transferred into plant genomes with the Agrobacterium system.

  20. Positional cloning of disease genes on chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Doggett, N. [Los Alamos National Lab., NM (United States); Bruening, M. [Leiden Univ. (Netherlands); Callen, D. [Adelaide Women`s and Children`s Hospital, North Adelaide, South Australia (Australia); Gardiner, M. [University Coll., London (United Kingdom); Lerner, T. [Massachusetts General Hospital, Boston, MA (United States)

    1996-04-01

    The project seeks to elucidate the molecular basis of an important genetic disease (Batten`s disease) by molecular cloning of the affected gene by utilizing an overlapping clone map of chromosome 16. Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressive loss of vision, seizures, and psychomoter disturbances. The Batten disease gene was genetically mapped to the chromosome region 16p 12.1 in close linkage with the genetic markers D16S299 and D16S298. Exon amplification of a cosmid containing D16S298 yielded a candidate gene that was disrupted by a 1 kb genomic deletion in all patients containing the most common haplotype for the disease. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the gene as the Batten disease gene. The disease gene encodes a novel 438 amino acid membrane binding protein of unknown function.

  1. Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

    Science.gov (United States)

    Juhas, Mario; Ajioka, James W

    2016-06-01

    Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.

  2. Construction and characterization of bacterial artificial chromosomes (BACs) containing herpes simplex virus full-length genomes.

    Science.gov (United States)

    Nagel, Claus-Henning; Pohlmann, Anja; Sodeik, Beate

    2014-01-01

    Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models. PMID:24671676

  3. Final report. Human artificial episomal chromosome (HAEC) for building large genomic libraries

    Energy Technology Data Exchange (ETDEWEB)

    Jean-Michael H. Vos

    1999-12-09

    Collections of human DNA fragments are maintained for research purposes as clones in bacterial host cells. However for unknown reasons, some regions of the human genome appear to be unclonable or unstable in bacteria. Their team has developed a system using episomes (extrachromosomal, autonomously replication DNA) that maintains large DNA fragments in human cells. This human artificial episomal chromosomal (HAEC) system may prove useful for coverage of these especially difficult regions. In the broader biomedical community, the HAEC system also shows promise for use in functional genomics and gene therapy. Recent improvements to the HAEC system and its application to mapping, sequencing, and functionally studying human and mouse DNA are summarized. Mapping and sequencing the human genome and model organisms are only the first steps in determining the function of various genetic units critical for gene regulation, DNA replication, chromatin packaging, chromosomal stability, and chromatid segregation. Such studies will require the ability to transfer and manipulate entire functional units into mammalian cells.

  4. HACking the centromere chromatin code: insights from human artificial chromosomes.

    Science.gov (United States)

    Bergmann, Jan H; Martins, Nuno M C; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations.

  5. Cloning of Bovine herpesvirus type 1 and type 5 as infectious bacterial artifical chromosomes

    Directory of Open Access Journals (Sweden)

    Ackermann Mathias

    2009-10-01

    Full Text Available Abstract Background Bovine herpesviruses type 1 (BoHV1 and type 5 (BoHV5 are two closely related pathogens of cattle. The identity of the two viruses on the amino acid level averages 82%. Despite their high antigenetic similarities the two pathogens induce distinctive clinical signs. BoHV1 causes respiratory and genital tract infections while BoHV5 leads to severe encephalitis in calves. Findings The viral genomes of BoHV1 and BoHV5 were cloned as infectious bacterial artificial chromosomes (BACs. First, recombinant viruses carrying the genetic elements for propagation in bacteria were generated. Second, DNA from these recombinant viruses were transferred into prokaryotic cells. Third, DNA from these bacteria were transferred into eukaryotic cells. Progeny viruses from BAC transfections showed similar kinetics as their corresponding wild types. Conclusion The two viral genomes of BoHV1 and BoHV5 cloned as BACs are accessible to the tools of bacterial genetics. The ability to easily manipulate the viral genomes on a molecular level in future experiments will lead to a better understanding of the difference in pathogenesis induced by these two closely related bovine herpesviruses.

  6. Aquifer parameter identification by the best chromosome clone plus younger generation chromosome prepotency genetic algorithm

    Institute of Scientific and Technical Information of China (English)

    LI Jing-sheng; LI Kai; YAO Lei-hua

    2005-01-01

    This paper developed an improved combinatorial method called the best chromosome clone plus younger generation chromosome prepotency genetic algorithm (BCC-YGCP-GA) to evaluate aquifer parameters. This method is based on a decimal simple genetic algorithm (SGA). A synthetic example for unsteady-state flow in a two-dimensional, inhomogeneous, confined aquifer containing three hydraulically distinct zones, is used to develop data to test the model. The simulation utilizes SGA and BCC-YGCP-GA coupled to the finite element method to identify the mean zonal hydraulic conductivities, and storage coefficients of the three-compartment model. For this geometrically simple model, used as a prototype of more complex systems, the SGA does not reach convergence within 100 generations. Conversely, the convergence rate of the BCC-YGCD-GA model is very fast. The objective function value calculated by BCC-YGCD-GA is reduced to 1/1 000th of the starting value within 100 generations,and the hydraulic conductivity and storage of three zones are within a few percent of the "true" values of the ideal model, highlighting the power of the method for aquifer parameterization.

  7. Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes.

    Science.gov (United States)

    Beier, Sebastian; Himmelbach, Axel; Schmutzer, Thomas; Felder, Marius; Taudien, Stefan; Mayer, Klaus F X; Platzer, Matthias; Stein, Nils; Scholz, Uwe; Mascher, Martin

    2016-07-01

    Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path. PMID:26801048

  8. Mapping and ordered cloning of the human X chromosome

    Energy Technology Data Exchange (ETDEWEB)

    Caskey, C.T.; Nelson, D.L.

    1992-12-01

    Progress is reported on gathering X chromosome specific libraries and integrating those with the library produced in this project. Further studies on understanding Fragile X Syndrome and other hereditary diseases related to the X chromosome are described. (DT)

  9. Human artificial chromosomes for Duchenne muscular dystrophy and beyond: challenges and hopes.

    OpenAIRE

    Tedesco, F. S.

    2015-01-01

    Safe and efficacious vectors able to carry large or several transgenes are of key importance for gene therapy. Human artificial chromosomes can fulfil this essential requirement; moreover, they do not integrate into the host genome. However, drawbacks such as the low efficiency of chromosome transfer and their relatively complex engineering still limit their widespread use. In this article, I summarise the key steps that brought human artificial chromosomes into preclinical research for Duche...

  10. cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/Myeloid Leukemia Factor 2 (MLF2)

    Energy Technology Data Exchange (ETDEWEB)

    Kuefer, M.U.; Valentine, V.; Behm, F.G. [St. Jude Children`s Research Hospital, Memphis, TN (United States)] [and others

    1996-07-15

    A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, and MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements. 19 refs., 2 figs.

  11. Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A bacterial artificial chromosome (BAC) library containing a large genomic DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA inserts is needed. We have developed a BAC library of the restoring line 0-613-2R for isolating the fertility restorer (Rf1) gene and genomic research in cotton (Gossypium hirsutum L.). The BAC library contains 97 825 clones stored in 255 pieces of a 384-well microtiter plate. Random samples of BACs digested with the Notl enzyme indicated that the average insert size is approximately 130 kb, with a range of 80-275 kb,and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 x haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hindlll enzymes. Thus, the stability of a single BAC clone can be sustained at least for 100 generations. Eight simple sequence repeat (SSR) markers flanking the Rf1 gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.

  12. Chromosomal assignment of chicken clone contigs by extending the consensus linkage map

    NARCIS (Netherlands)

    Aerts, J.; Veenendaal, T.; Poel, van der J.J.; Crooijmans, R.P.M.A.; Groenen, M.A.M.

    2005-01-01

    The bacterial artificial clone-based physical map for chicken plays an important role in the integration of the consensus linkage map and the whole-genome shotgun sequence. It also provides a valuable resource for clone selection within applications such as fluorescent in situ hybridization and posi

  13. Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    D' Aiuto, L.; Marzella, R.; Archidiacono, N.; Rocchi, M. (Universita di Bari (Italy)); Antonacci, R. (Instituto Anatomia Umana Normale, Modena (Italy))

    1993-11-01

    The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.

  14. Refined human artificial chromosome vectors for gene therapy and animal transgenesis

    OpenAIRE

    Kazuki, Y.; Hoshiya, H.; Takiguchi, M; Abe, S.; Iida, Y.; Osaki, M.; Katoh, M; Hiratsuka, M; Shirayoshi, Y; HIRAMATSU, K.; Ueno, E; Kajitani, N; Yoshino, T.; Kazuki, K; Ishihara, C.

    2010-01-01

    Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-pro...

  15. Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2013-01-01

    Full Text Available Bacterial artificial chromosome (BAC libraries have been invaluable tools for the genome-wide genetic dissection of complex organisms. Here, we report the construction and characterization of a high-redundancy BAC library from a very valuable pig breed in China, Wuzhishan miniature pig (Sus scrofa, using its blood cells and fibroblasts, respectively. The library contains approximately 153,600 clones ordered in 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 152.3 kb, representing approximately 7.68 genome equivalents of the porcine haploid genome and a 99.93% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. 19 pairs of microsatellite marker primers covering porcine chromosomes were used for screening the BAC library, which showed that each of these markers was positive in the library; the positive clone number was 2 to 9, and the average number was 7.89, which was consistent with 7.68-fold coverage of the porcine genome. And there were no significant differences of genomic BAC library from blood cells and fibroblast cells. Therefore, we identified 19 microsatellite markers that could potentially be used as genetic markers. As a result, this BAC library will serve as a valuable resource for gene identification, physical mapping, and comparative genomics and large-scale genome sequencing in the porcine.

  16. Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome

    Directory of Open Access Journals (Sweden)

    Ting-Ting Wu

    2011-01-01

    Full Text Available Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, γHV-68, also known as MuHV-4 as a bacterial artificial chromosome (BAC. We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open reading frame. The infectious viruses were reconstituted following transfection of the MHV-68 BAC DNA into cells. The MHV-68 BAC-derived virus replicated indistinguishably from the wild-type virus in cultured cells. Excision of the BAC insert was efficiently achieved by coexpressing the Cre recombinase. Although the BAC insertion did not significantly affect acute productive infection in the lung, it severely compromised the ability of MHV-68 to establish splenic latency. Removal of the BAC sequence restored the wild-type level of latency. Site-specific mutagenesis was carried out by RecA-mediated recombination to demonstrate that this infectious BAC clone can be used for genetic studies of MHV-68.

  17. Phage p1-derived artificial chromosomes facilitate heterologous expression of the FK506 gene cluster.

    Directory of Open Access Journals (Sweden)

    Adam C Jones

    Full Text Available We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC library clones containing large natural product gene clusters (≥70 kilobases to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus, a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L(-1 to 5.5 mg L(-1 in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

  18. A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Coen M Adema

    2006-10-01

    Full Text Available To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html, working with the Arizona Genomics Institute (AGI and supported by the National Human Genome Research Institute (NHGRI, produced a high quality bacterial artificial chromosome (BAC library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa consists of 61824 clones (136.3 kb average insert size and provides 9.05 × coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.

  19. Efficient assembly of de novo human artificial chromosomes from large genomic loci

    Directory of Open Access Journals (Sweden)

    Stromberg Gregory

    2005-07-01

    Full Text Available Abstract Background Human Artificial Chromosomes (HACs are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb genomic loci have been reported to be successfully packaged into de novo HACs. Results We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple de novo HACs containing large (100–200 kb genomic loci including therapeutically significant genes for human growth hormone (HGH, polycystic kidney disease (PKD1 and ß-globin. We report significant differences in the ability of different genomic loci to support de novo HAC formation, suggesting possible effects of cis-acting genomic elements. Finally, as a proof of principle, we have observed sustained ß-globin gene expression from HACs incorporating the entire 200 kb ß-globin genomic locus for over 90 days in the absence of selection. Conclusion Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate de novo HAC formation. These genomic loci

  20. Can artificial parthenogenesis sidestep ethical pitfalls in human therapeutic cloning? An historical perspective

    OpenAIRE

    Fangerau, H

    2005-01-01

    The aim of regenerative medicine is to reconstruct tissue that has been lost or pathologically altered. Therapeutic cloning seems to offer a method of achieving this aim; however, the ethical debate surrounding human therapeutic cloning is highly controversial. Artificial parthenogenesis—obtaining embryos from unfertilised eggs—seems to offer a way to sidestep these ethical pitfalls. Jacques Loeb (1859–1924), the founding father of artificial parthogenesis, faced negative public opinion when ...

  1. 454 sequencing of pooled BAC clones on chromosome 3H of barley

    Directory of Open Access Journals (Sweden)

    Yamaji Nami

    2011-05-01

    Full Text Available Abstract Background Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp. Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. Results We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1. Conclusions We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.

  2. Repetitive genome elements in a European corn borer, Ostrinia nubilalis, bacterial artificial chromosome library were indicated by bacterial artificial chromosome end sequencing and development of sequence tag site markers: implications for lepidopteran genomic research.

    Science.gov (United States)

    Coates, Brad S; Sumerford, Douglas V; Hellmich, Richard L; Lewis, Leslie C

    2009-01-01

    The European corn borer, Ostrinia nubilalis, is a serious pest of food, fiber, and biofuel crops in Europe, North America, and Asia and a model system for insect olfaction and speciation. A bacterial artificial chromosome library constructed for O. nubilalis contains 36 864 clones with an estimated average insert size of >or=120 kb and genome coverage of 8.8-fold. Screening OnB1 clones comprising approximately 2.76 genome equivalents determined the physical position of 24 sequence tag site markers, including markers linked to ecologically important and Bacillus thuringiensis toxin resistance traits. OnB1 bacterial artificial chromosome end sequence reads (GenBank dbGSS accessions ET217010 to ET217273) showed homology to annotated genes or expressed sequence tags and identified repetitive genome elements, O. nubilalis miniature subterminal inverted repeat transposable elements (OnMITE01 and OnMITE02), and ezi-like long interspersed nuclear elements. Mobility of OnMITE01 was demonstrated by the presence or absence in O. nubilalis of introns at two different loci. A (GTCT)n tetranucleotide repeat at the 5' ends of OnMITE01 and OnMITE02 are evidence for transposon-mediated movement of lepidopteran microsatellite loci. The number of repetitive elements in lepidopteran genomes will affect genome assembly and marker development. Single-locus sequence tag site markers described here have downstream application for integration within linkage maps and comparative genomic studies. PMID:19132072

  3. Repetitive genome elements in a European corn borer, Ostrinia nubilalis, bacterial artificial chromosome library were indicated by bacterial artificial chromosome end sequencing and development of sequence tag site markers: implications for lepidopteran genomic research.

    Science.gov (United States)

    Coates, Brad S; Sumerford, Douglas V; Hellmich, Richard L; Lewis, Leslie C

    2009-01-01

    The European corn borer, Ostrinia nubilalis, is a serious pest of food, fiber, and biofuel crops in Europe, North America, and Asia and a model system for insect olfaction and speciation. A bacterial artificial chromosome library constructed for O. nubilalis contains 36 864 clones with an estimated average insert size of >or=120 kb and genome coverage of 8.8-fold. Screening OnB1 clones comprising approximately 2.76 genome equivalents determined the physical position of 24 sequence tag site markers, including markers linked to ecologically important and Bacillus thuringiensis toxin resistance traits. OnB1 bacterial artificial chromosome end sequence reads (GenBank dbGSS accessions ET217010 to ET217273) showed homology to annotated genes or expressed sequence tags and identified repetitive genome elements, O. nubilalis miniature subterminal inverted repeat transposable elements (OnMITE01 and OnMITE02), and ezi-like long interspersed nuclear elements. Mobility of OnMITE01 was demonstrated by the presence or absence in O. nubilalis of introns at two different loci. A (GTCT)n tetranucleotide repeat at the 5' ends of OnMITE01 and OnMITE02 are evidence for transposon-mediated movement of lepidopteran microsatellite loci. The number of repetitive elements in lepidopteran genomes will affect genome assembly and marker development. Single-locus sequence tag site markers described here have downstream application for integration within linkage maps and comparative genomic studies.

  4. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

    Science.gov (United States)

    Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

    2014-11-01

    Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

  5. Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses.

    Science.gov (United States)

    Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min

    2015-01-01

    Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase. PMID:26780115

  6. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael;

    2011-01-01

    contigs (184 kb in average) were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes.Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80......BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we...... consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library...

  7. Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genic male-sterile rice 5460S

    Institute of Scientific and Technical Information of China (English)

    邱芳; 金德敏; 伏健民; 张超良; 谢纬武; 王斌; 杨仁崔; 张洪斌

    1999-01-01

    In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.

  8. Construction and characterization of a 10-genome equivalent yeast artificial chromosome library for the laboratory rat, Rattus norvegicus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, L.; Zee, R.Y.L. [Harvard Medical School, Boston, MA (United States); Schalkwyk, L.C. [Max Planck Institute for Molecular Genetics, Berlin (Germany)] [and others

    1997-02-01

    Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20-30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species. 35 refs., 4 figs.

  9. Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Sun, W.; Ferrer-Montiel, A.V.; Schinder, A.F.; Montal, M. (Univ. of California, San Diego, La Jolla (United States)); McPherson, J.P. (Univ. of California, Irvine (United States)); Evans, G.A. (Salk Inst. for Biological Studies, La Jolla, CA (United States))

    1992-02-15

    A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCr analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

  10. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2014-03-01

    Full Text Available Bacterial artificial chromosome (BAC libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12, consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

  11. Bacterial artificial chromosome-derived molecular markers for early bolting in sugar beet.

    Science.gov (United States)

    Gaafar, R M; Hohmann, U; Jung, C

    2005-04-01

    Early bolting in sugar beet (Beta vulgaris L.) is controlled by the dominant gene B. From an incomplete physical map around the B gene, 18 bacterial artificial chromosomes (BACs) were selected for marker development. Three BACs were shotgun-sequenced, and 61 open reading frames (ORFs) were identified. Together with 104 BAC ends from 54 BACs, a total number of 55,464 nucleotides were sequenced. Of these, 37 BAC ends and 12 ORFs were selected for marker development. Thirty-one percent of the sequences were found to be single copy and 24%, low copy. From these sequences, 15 markers from ten different BACs were developed. Ten polymorphisms were determined by simple agarose gel electrophoresis of either restricted or non-restricted PCR products. Another five markers were determined by tetra-primer amplification refractory mutation system-PCR. In order to select candidate BACs for cloning the gene, genetic linkage between seven markers and the bolting gene was calculated using 1,617 plants from an F2 population segregating for early bolting. The recombination values ranged between 0.0033 and 0.0201. In addition, a set of 41 wild and cultivated Beta accessions differing in their early bolting character was genotyped with seven markers. A common haplotype encompassing two marker loci and the b allele was found in all sugar beet varieties, indicating complete linkage disequilibrium between these loci. This suggests that the bolting gene is located in close vicinity to these markers, and the corresponding BACs can be used for cloning the gene.

  12. Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome.

    Science.gov (United States)

    Tobian, J A; Macrina, F L

    1982-10-01

    A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

  13. From amplification to gene in thyroid cancer: A high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X.N.; Gonsky, R.; Korenberg, J.R. [UCLA School of Medicine, Los Angeles, CA (United States). Cedars-Sinai Research Inst.; Knauf, J.A.; Fagin, J.A. [Univ. of Cincinnati, OH (United States). Div. of Endocrinology/Metabolism; Wang, M.; Lai, E.H. [Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Pharmacology; Chissoe, S. [Washington Univ. School of Medicine, St. Louis, MO (United States). Genome Sequencing

    1998-08-01

    Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. The authors now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. They used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3--6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKC{epsilon}), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKC{epsilon} as a previously unmapped candidate gene involved in thyroid tumorigenesis.

  14. Targeted isolation and cloning of 100-kb microbial genomic sequences by Cas9-assisted targeting of chromosome segments.

    Science.gov (United States)

    Jiang, Wenjun; Zhu, Ting F

    2016-05-01

    Cloning of long microbial genomic sequences is an essential tool in synthetic biology and genome engineering. Such long sequences are often difficult to obtain directly by traditional PCR or restriction enzyme digestion, and therefore the cloning of these sequences has remained a technical obstacle in molecular biology. Based on the in vitro application of RNA-guided Cas9 nuclease, the method of Cas9-assisted targeting of chromosome segments (CATCH) cleaves target DNA in vitro from intact bacterial chromosomes embedded in agarose plugs, which can be subsequently ligated with cloning vector through Gibson assembly. Here we describe an optimized protocol of CATCH cloning for the targeted cloning of long genomic sequences of up to 100 kb from microorganisms. The protocol uses standard laboratory equipment and takes ∼8 h of bench time over several days, and it may potentially simplify and accelerate efforts to isolate and clone large gene clusters from microorganisms. PMID:27101517

  15. Efeito da desrama artificial na qualidade da madeira de clones de eucalipto em sistema agrossilvipastoril

    OpenAIRE

    Vale Rodrigo Silva do; Macedo Renato Luiz Grisi; Venturin Nelson; Mori Fábio Akira; Morais Augusto Ramalho de

    2002-01-01

    O objetivo do presente trabalho foi avaliar o efeito da desrama artificial na qualidade da madeira de clones de eucalipto, em sistema agrossilvipastoril, na fazenda Riacho, pertencente à Companhia Mineira de Metais, município de Paracatu-MG, Brasil. O experimento foi instalado em dezembro de 1994, com o plantio das mudas de três clones de híbridos naturais de Eucalyptus camaldulensis Dehnh. (código 06) e de Eucalyptus urophylla S.T. Blake (código 13 e 44), no espaçamento de 10 x 4 m. O deline...

  16. Cloning, chromosome localization and features of a novel human gene, MATH2

    Indian Academy of Sciences (India)

    Lingchen Guo; Min Jiang; Yushu Ma; Haipeng Cheng; Xiaohua Ni; Yangsheng Jin; Yi Xie; Yumin Mao

    2002-04-01

    We report cloning and some features of a novel human gene, MATH2, which encodes a protein of 337 amino acid residues with a basic helix–loop–helix domain and exhibits 98% similarity to mouse Math2. Results of Northern blot analysis revealed two transcripts of the MATH2 gene of 1.7 kb and 2.4 kb in human brain. We localized MATH2 to chromosome 7 at 7p14–15 by matching with the Human Genome Sequence Database. Human MATH2 and mouse Math2 may have the same functions in the nervous system.

  17. Construction of a nurse shark (Ginglymostoma cirratum bacterial artificial chromosome (BAC library and a preliminary genome survey

    Directory of Open Access Journals (Sweden)

    Inoko Hidetoshi

    2006-05-01

    Full Text Available Abstract Background Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. Aims In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC library for the nurse shark, Ginglymostoma cirratum. Results The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 × 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6–28 primary positive clones per probe of which 50–90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. Conclusion We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

  18. Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Mitra Partha P

    2009-08-01

    Full Text Available Abstract Background Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host. Results As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5–7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology. Conclusion The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds

  19. The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

    Science.gov (United States)

    Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

    1992-07-01

    We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.

  20. Cloning, sequencing, and analysis of inv8 chromosome breakpoints associated with recombinant 8 syndrome.

    Science.gov (United States)

    Graw, S L; Sample, T; Bleskan, J; Sujansky, E; Patterson, D

    2000-03-01

    Rec8 syndrome (also known as "recombinant 8 syndrome" and "San Luis Valley syndrome") is a chromosomal disorder found in individuals of Hispanic descent with ancestry from the San Luis Valley of southern Colorado and northern New Mexico. Affected individuals typically have mental retardation, congenital heart defects, seizures, a characteristic facial appearance, and other manifestations. The recombinant chromosome is rec(8)dup(8q)inv(8)(p23.1q22.1), and is derived from a parental pericentric inversion, inv(8)(p23.1q22.1). Here we report on the cloning, sequencing, and characterization of the 8p23.1 and 8q22 breakpoints from the inversion 8 chromosome associated with Rec8 syndrome. Analysis of the breakpoint regions indicates that they are highly repetitive. Of 6 kb surrounding the 8p23.1 breakpoint, 75% consists of repetitive gene family members-including Alu, LINE, and LTR elements-and the inversion took place in a small single-copy region flanked by repetitive elements. Analysis of 3.7 kb surrounding the 8q22 breakpoint region reveals that it is 99% repetitive and contains multiple LTR elements, and that the 8q inversion site is within one of the LTR elements.

  1. Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

    Institute of Scientific and Technical Information of China (English)

    HE Cong-fen; MA You-zhi; XIN Zhi-yong; XU Qiong-fang; LI Lian-cheng

    2004-01-01

    The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) was identified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes were microisolated and collected. After two rounds of PCR amplification, the PCR products were ranged from 150 - 3 000 bp,with predominant fragments at about 200 - 2 000 bp. Using Ag.intermediumgenomic DNA as a probe, Southern blotting analysis confirmed the products originated from Ag. intermediumgenome. The products were purified, ligated to pUC18 and then transformed into competence E.coli DH5α to produce a 2Ai-2 chromosome DNA library. The microcloning experiments produced approximately 5×105 clones, the size range of the cloned inserts was 200- 1 500 bp, with an average of 580bp. Using Ag. intermediumgenomic DNA as a probe, dot blotting results showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences in the library. Four Ag. intermedium clones were screened from the library by RFLP, and three clones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitive sequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitive DNA sequence.

  2. Molecular cloning and chromosomal localization of the ADH7 gene encoding human class IV ({sigma}) ADH

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Hirokazu; Baraona, E.; Lieber, C.S. [Mount Sinai School of Medicine, Bronx, NY (United States)

    1996-01-15

    The ADH7 gene encoding human Class IV ({sigma}) alcohol dehydrogenase (ADH) was cloned from a Caucasian genomic DNA library and characterized. It has nine exons and eight introns that span about 22 kb, and its intron insertion is identical to that of the other ADH genes (ADH1 to ADH5). The nucleotide sequences of the exons encoding 374 amino acids are identical to the previously reported cDNA sequence of {sigma} ADH. Fluorescence in situ hybridization analysis showed that ADH7 is located on human chromosome 4q23-q24, close to the ADH cluster locus (4q21-q25). These data are consistent with the view that Class IV ADH is a member of the ADH family and is phylogenetically close to the other ADHs. 15 refs., 2 figs., 1 tab.

  3. STAND-LEVEL PROGNOSIS OF EUCALYPTUS CLONES USING ARTIFICIAL NEURAL NETWORKS

    Directory of Open Access Journals (Sweden)

    Mayra Luiza Marques da Silva Binoti

    2015-03-01

    Full Text Available The objective of this study was to train, implement and evaluate the efficiency of artificial neural networks (ANN to perform production prognosis of even-aged stands of eucalyptus clones. The data used were from plantations located in southern Bahia, totaling about 2,000 acres of forest. Numeric variables, such as age, basal area, volume and categorical variables, such as soil class texture, spacing, land relief, project and clone were used. The data were randomly divided into two groups: training (80% and generalization (20%. Three types of networks were trained: perceptron, multilayer perceptron networks and radial basis function. The RNA that showed the best performance in training and generalization were selected to perform the prognosis with data from the first forest inventory. We conclude that the RNA had satisfactory results, showing the potential and applicability of the technique in solving measurement and forest management problems.

  4. A yeast artificial chromosome contig of the critical region for cri-du-chat syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Goodart, S.A.; Rojas, K.; Overhauser, J. [Thomas Jefferson Univ., Philadelphia, PA (United States)] [and others

    1994-11-01

    Cri-du-chat is a chromosomal deletion syndrome characterized by partial deletion of the short arm of chromosome 5. The clinical symptoms include growth and mental retardation, microcephaly, hypertelorism, epicanthal folds, hyptonia, and a high-pitched monochromatic cry that is usually considered diagnostic for the syndrome. Recently, a correlation between clinical features and the extent of the chromosome 5 deletions has identified two regions of the short arm that appear to be critical for the abnormal development manifested in this syndrome. Loss of a small region in 5p15.2 correlates with all of the clinical features of cri-du-chat with the exception of the cat-like cry, which maps to 5p15.3. Here the authors report the construction of a YAC contig that spans the chromosomal region in 5p15.2 that plays a major role in the etiology of the cri-du-chat syndrome. YACs that span the 2-Mb cri-du-chat critical region have been identified and characterized. This YAC contig lays the groundwork for the construction of a transcriptional map of this region and the eventual identification of genes involved in the clinical features associated with the cri-du-chat syndrome. It also provides a new diagnostic tool for cri-du-chat in the shape of a YAC clone that may span the entire critical region. 24 refs., 4 figs., 2 tabs.

  5. Molecular cloning, chromosomal mapping, and characterization of the mouse UDP-galactose: Ceramide galactosyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Coetzee, T.; Fujita, N.; Marcus, J. [Univ. of North Carolina, Chapel Hill, NC (United States)] [and others

    1996-07-01

    UDP-galactose:ceramide galactosyltransferase (CGT) (EC 2.11.62) catalyzes the final step in the synthesis of galactocerebroside, a glycosphingolipid characteristically abundant in myelin. In this report, we describe the isolation of genomic clones spanning the mouse CGT gene. The mouse CGT gene consists of six exons that span a minimum of 70 kb of DNA and that encode a 541 amino acid translation product with extensive sequence similarity to the rat CGT enzyme and to UDP-glucuronosyltransferases (UGT). The 5{prime}-untranslated region of the mouse CGT gene is encoded by a separate exon located approximately 25 kb upstream of the first protein-encoding exon. Furthermore, the genomic organization of the five encoding region exons of the mouse CGT gene resembles that of the human UGT1 and rat UGT2B1 genes. Finally, analysis of somatic cell hybrids by PCR and fluorescence in situ hybridization to metaphase chromosomes has localized the mouse CGT gene to chromosome 3, bands E3-F1. 26 refs., 5 figs., 1 tab.

  6. Screening of YAC clones and building a map of the chromosome 13 region often deleted during chronic B-cell lymphocytic leucosis

    NARCIS (Netherlands)

    Brodyanskii, VM; Sulimova, GE; Udina, IG; Aitova, SS; Shaikhaev, GO; Sharikova, OA; Zakharev, VM; Fedorova, LI; Zelenin, AV; Eikhorn, S; Baush, C; Laland, M; Ross, M; Yankovskii, NK

    1995-01-01

    Pools of YAC clones from the ICRF library were analyzed by PCR using PBKpt, MGG15, and D13S25 markers that flank the chromosome 13 region often deleted during chronic lymphocytic leukemia. Ten clones were found and described. Nine mega-YAC clones from the CEPH library flanking the region of interest

  7. A yeast artificial chromosome (YAC) contig encompassing the critical region of the X-linked lymphoproliferative disease (XLP) locus.

    Science.gov (United States)

    Lanyi, A; Li, B; Li, S; Talmadge, C B; Brichacek, B; Davis, J R; Kozel, B A; Trask, B; van den Engh, G; Uzvolgyi, E; Stanbridge, E J; Nelson, D L; Chinault, C; Heslop, H; Gross, T G; Seemayer, T A; Klein, G; Purtilo, D T; Sumegi, J

    1997-01-01

    X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein-Barr virus (EBV) infection. Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma. Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence. From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated. Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients. Three more families with interstitial deletions were found. Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescence in situ hybridization and which consists of 15 overlapping YAC clones, has been constructed. A detailed restriction enzyme map of the region has been constructed. YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction

  8. GenMapDB: a database of mapped human BAC clones

    OpenAIRE

    Morley, Michael; Arcaro, Melissa; Burdick, Joshua; Yonescu, Raluca; Reid, Thomas; Kirsch, Ilan R.; Cheung, Vivian G.

    2001-01-01

    GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9–22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequen...

  9. Cloning, Structural Organization and Chromosomal Mapping of Rat Costimulatory Molecule 4-1BBL

    Institute of Scientific and Technical Information of China (English)

    Qiu-Ming DONG; Xue-Guang ZHANG; Li-Jie MA; Guang-Bo ZHANG; Ya-Fang WU; Jia-Yao SHEN; Ying CHEN; Yong-Jing CHEN; Xiang-Ke PU; Sai-Yu HANG

    2005-01-01

    4-1BBL (TNFSF9) is a member of the tumor necrosis factor (TNF) ligand superfamily, which is expressed on some activated antigen presenting cells and B cells. We isolated a rat cDNA clone encoding the rat homologue of the human 4-1BBL (GenBank accession No. AY259541). The deduced rat 4-1BBL protein, consisting of 308 amino acids with a molecular weight of 33,469 Da, was a typical type Ⅱ transmembrane glycoprotein, the same as human and murine 4-1BBL. "SDAA" in the cytoplasmic domain of rat 4-1BBL was deduced to act as the phosphorylation site for casein kinase I ("SXXS" motif), which is present in the cytoplasmic domains of human and murine 4-1BBL, and all other TNF ligand family members known to utilize reverse signaling. The two introns of 4-1BBL were also cloned (GenBank accession No.AY332409). Rat 4-1BBL is much more homologous with murine 4-1BBL than with human 4-1BBL, in both nucleotide and amino acid sequences. Rat 4-1BBL was expressed in all tested tissues: brain, lung, colon, liver,thymus, testicle, kidney, adrenal, stomach, spleen and heart. The chromosomal location of rat 4-1BBL was first identified by bioinformatics, then by fluorescence in situ hybridization at 9q11 (GenBank accession UniGene No. Rn.46783). Rat, murine and human 4-1BBL genes are evolved from a common gene. The identification and characterization of the rat counterpart of human 4-1BBL will facilitate studies of the biological function of this molecule.

  10. Cloning, tissue expression pattern, and chromosome localization of human protein kinase Bγ gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Protein kinase B (PKB) is a member of the second messenger-regulated subfamily of protein kinases, and plays a key role in cell-cycle regulation, glucose uptake and promotion of cell differentiation. Evidence shows that PKB undergoes activation in some human tumors and is involved in Ras pathway, which implies that PKB can trigger a pathway to induce oncogenic transformation. A nucleotide sequence of mouse Pkb? was used as a probe to screen homolog in a human liver cDNA library. A fragment of 1998 bp containing a 1440 bp ORF encoding 479 amino acid residues was obtained. Then the 3'-terminal of this fragment was extended to 2788 bp by 'electronic walking' screening, and the extended fragment was confirmed by PCR amplification. The protein deduced by the gene had a high identity of 83% and 78% to the human PKBγ and γ, respectively, and was designated as human PKB?. Northern hybridization detected two equally expressed transcripts of 8.5 and 6.5 kb in length in all 16 human tissues tested, with the highest expression level in brain, and lower levels with variation in the other tissues. By RH mapping, the PKBγ was placed on chromosome 1q43, between markers D1S304 and D1S2693. It is a valuable clue for cloning the candidate genes related to prostate cancer; Arrhythmogenic Right Ventricular Dysplasia (ARVD); Chediak-Higashi, NK cell Deficiency (CHS); and Hypoparathyrodism with Short Stature, Mental Retardation and Seizures which have already been mapped in this chromosomal region.

  11. EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Niels Bjerg; Strucko, Tomas; Kildegaard, Kanchana Rueksomtawin;

    2014-01-01

    Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression...... in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out....

  12. 白眉长臂猿基因组BAC文库的构建%Construction of Genome Bacterial Artificial Chromosome Library of Hylobates Hoolock

    Institute of Scientific and Technical Information of China (English)

    王起明; 孙烨超; 厉申捷; 叶建平

    2015-01-01

    High quality genomic DNA of Hylobates hoolock was obtained by gentle physical homogenization. The DNA was partially digested with EcoRⅠand EcoRⅠmethylase, and cloned to pCC1BAC vector. The positive clones were stored in 384-well plates. The constructed BAC library consists of 85800 clones. DNA from randomly selected 250 BAC clones was restricted with Not I restriction enzyme and fragments were separated by pulsed field gel electrophoresis. The result shows that the average insert size is estimated as approximately 110 kb, and the ratio of non-recombinant clones is 10. 0%. If the genome size of Hylobates hoolock is 3 ×106 kilo-base, the library could cover 3 times the number of genome.%通过温和的物理方法获得白眉长臂猿高质量的基因组DNA,EcoRⅠ和EcoRⅠ甲基化酶部分酶切后经回收、连接、转化、阳性克隆的保存,构建了含有85800个克隆的全基因组BAC( Bacterial artificial chromosome)文库.随机选取250个BAC克隆进行Not I酶切及脉冲场电泳分析,结果表明该文库的平均插入片段大小为110 kb,非重组克隆(无插入片段)的比率为10.0%.假定白眉长臂猿的基因组大小为3×106 kb,根据文库的平均插入片段大小,则该文库具有3倍的基因组覆盖率.

  13. The Selection and Use of Sorghum (Sorghum propinquum Bacterial Artificial Chromosomes as Cytogenetic FISH Probes for Maize (Zea mays L.

    Directory of Open Access Journals (Sweden)

    Debbie M. Figueroa

    2011-01-01

    Full Text Available The integration of genetic and physical maps of maize is progressing rapidly, but the cytogenetic maps lag behind, with the exception of the pachytene fluorescence in situ hybridization (FISH maps of maize chromosome 9. We sought to produce integrated FISH maps of other maize chromosomes using Core Bin Marker loci. Because these 1 Kb restriction fragment length polymorphism (RFLP probes are below the FISH detection limit, we used BACs from sorghum, a small-genome relative of maize, as surrogate clones for FISH mapping. We sequenced 151 maize RFLP probes and compared in silico BAC selection methods to that of library filter hybridization and found the latter to be the best. BAC library screening, clone verification, and single-clone selection criteria are presented along with an example of transgenomic BAC FISH mapping. This strategy has been used to facilitate the integration of RFLP and FISH maps in other large-genome species.

  14. The selection and use of sorghum (Sorghum propinquum) bacterial artificial chromosomes as cytogenetic FISH probes for maize (Zea mays L.).

    Science.gov (United States)

    Figueroa, Debbie M; Davis, James D; Strobel, Cornelia; Conejo, Maria S; Beckham, Katherine D; Ring, Brian C; Bass, Hank W

    2011-01-01

    The integration of genetic and physical maps of maize is progressing rapidly, but the cytogenetic maps lag behind, with the exception of the pachytene fluorescence in situ hybridization (FISH) maps of maize chromosome 9. We sought to produce integrated FISH maps of other maize chromosomes using Core Bin Marker loci. Because these 1 Kb restriction fragment length polymorphism (RFLP) probes are below the FISH detection limit, we used BACs from sorghum, a small-genome relative of maize, as surrogate clones for FISH mapping. We sequenced 151 maize RFLP probes and compared in silico BAC selection methods to that of library filter hybridization and found the latter to be the best. BAC library screening, clone verification, and single-clone selection criteria are presented along with an example of transgenomic BAC FISH mapping. This strategy has been used to facilitate the integration of RFLP and FISH maps in other large-genome species. PMID:21234422

  15. Diagnosis and Prognostication of Ductal Adenocarcinomas of the Pancreas Based on Genome-Wide DNA Methylation Profiling by Bacterial Artificial Chromosome Array-Based Methylated CpG Island Amplification

    Directory of Open Access Journals (Sweden)

    Masahiro Gotoh

    2011-01-01

    Full Text Available To establish diagnostic criteria for ductal adenocarcinomas of the pancreas (PCs, bacterial artificial chromosome (BAC array-based methylated CpG island amplification was performed using 139 tissue samples. Twelve BAC clones, for which DNA methylation status was able to discriminate cancerous tissue (T from noncancerous pancreatic tissue in the learning cohort with a specificity of 100%, were identified. Using criteria that combined the 12 BAC clones, T-samples were diagnosed as cancers with 100% sensitivity and specificity in both the learning and validation cohorts. DNA methylation status on 11 of the BAC clones, which was able to discriminate patients showing early relapse from those with no relapse in the learning cohort with 100% specificity, was correlated with the recurrence-free and overall survival rates in the validation cohort and was an independent prognostic factor by multivariate analysis. Genome-wide DNA methylation profiling may provide optimal diagnostic markers and prognostic indicators for patients with PCs.

  16. A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish.

    Science.gov (United States)

    Fuentes, Fernando; Reynolds, Eric; Lewellis, Stephen W; Venkiteswaran, Gayatri; Knaut, Holger

    2016-01-01

    Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels. PMID:26818072

  17. DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

    International Nuclear Information System (INIS)

    Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2

  18. Plant artificial chromosome technology and its potential application in genetic engineering.

    Science.gov (United States)

    Yu, Weichang; Yau, Yuan-Yeu; Birchler, James A

    2016-05-01

    Genetic engineering with just a few genes has changed agriculture in the last 20 years. The most frequently used transgenes are the herbicide resistance genes for efficient weed control and the Bt toxin genes for insect resistance. The adoption of the first-generation genetically engineered crops has been very successful in improving farming practices, reducing the application of pesticides that are harmful to both human health and the environment, and producing more profit for farmers. However, there is more potential for genetic engineering to be realized by technical advances. The recent development of plant artificial chromosome technology provides a super vector platform, which allows the management of a large number of genes for the next generation of genetic engineering. With the development of other tools such as gene assembly, genome editing, gene targeting and chromosome delivery systems, it should become possible to engineer crops with multiple genes to produce more agricultural products with less input of natural resources to meet future demands. PMID:26369910

  19. Mapping and ordered cloning of the human X chromosome. Progress report, September 1991--November 1992

    Energy Technology Data Exchange (ETDEWEB)

    Caskey, C.T.; Nelson, D.L.

    1992-12-01

    Progress is reported on gathering X chromosome specific libraries and integrating those with the library produced in this project. Further studies on understanding Fragile X Syndrome and other hereditary diseases related to the X chromosome are described. (DT)

  20. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  1. Chromosome

    Science.gov (United States)

    Chromosomes are structures found in the center (nucleus) of cells that carry long pieces of DNA. DNA ... is the building block of the human body. Chromosomes also contain proteins that help DNA exist in ...

  2. Integration of the cytogenetic, genetic, and physical maps of the human genome by FISH mapping of CEPH YAC clones

    Energy Technology Data Exchange (ETDEWEB)

    Bray-Ward, P.; Menninger, J.; Lieman, J. [Yale Univ. School of Medicine, New Haven, CT (United States)] [and others

    1996-02-15

    This article discusses the genetic mapping of over 950 yeast artificial chromosome (YAC) clones on human chromosomes. This integration of the cytogenetic, genetic and physical maps of the human genome was accomplished using fluorescence in situ hybridization (FISH) mapping and the CEPH library of YAC clones. 27 refs., 2 figs., 1 tab.

  3. Diversity of staphylococcal cassette chromosome mec elements in predominant methicillin-resistant Staphylococcus aureus clones in a small geographic area.

    Science.gov (United States)

    Basset, Patrick; Senn, Laurence; Vogel, Valérie; Zanetti, Giorgio; Blanc, Dominique S

    2010-11-01

    Recent population genetic studies suggest that staphylococcal cassette chromosome mec (SCCmec) was acquired much more frequently than previously thought. In the present study, we aimed to investigate the diversity of SCCmec elements in a local methicillin-resistant Staphylococcus aureus (MRSA) population. Each MRSA isolate (one per patient) recovered in the Vaud canton of Switzerland from January 2005 to December 2008 was analyzed by the double-locus sequence typing (DLST) method and SCCmec typing. DLST analysis indicated that 1,884/2,036 isolates (92.5%) belong to four predominant clones. As expected from the local spread of a clone, most isolates within clones harbored an identical SCCmec type. However, three to seven SCCmec types have been recovered in every predominant DLST clone, suggesting that some of these elements might have been acquired locally. This pattern could also be explained by distinct importations of related isolates into the study region. The addition of a third highly variable locus to further increase the discriminatory power of typing as well as epidemiological data suggested that most ambiguous situations were explained by the second hypothesis. In conclusion, our study showed that even if the acquisition of new SCCmec elements at a local level likely occurs, it does not explain all the diversity observed in the study region. PMID:20713672

  4. Association Between Pachytene Chromosomes and Linkage Groups in Carrot

    Science.gov (United States)

    The genome of carrot (Daucus carota L.) consists of ~ 480 Mb/1C organized in 9 chromosome pairs. The importance of carrots in human nutrition is triggering the development of genomic resources, including carrot linkage maps, a bacterial artificial chromosome (BAC) clone library and BAC end sequence...

  5. Cloning

    Science.gov (United States)

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  6. Development of a Safeguard System Using an Episomal Mammalian Artificial Chromosome for Gene and Cell Therapy

    Science.gov (United States)

    Uno, Narumi; Uno, Katsuhiro; Komoto, Shinya; Suzuki, Teruhiko; Hiratsuka, Masaharu; Osaki, Mitsuhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo

    2015-01-01

    The development of a safeguard system to remove tumorigenic cells would allow safer clinical applications of stem cells for the treatment of patients with an intractable disease including genetic disorders. Such safeguard systems should not disrupt the host genome and should have long-term stability. Here, we attempted to develop a tumor-suppressing mammalian artificial chromosome containing a safeguard system that uses the immune rejection system against allogeneic tissue from the host. For proof-of-concept of the safeguard system, B16F10 mouse melanoma cells expressing the introduced H2-K(d) major histocompatibility complex (MHC class I)-allogenic haplotype were transplanted into recipient C57BL/6J mice expressing MHC H2-K(b). Subcutaneous implantation of B16F10 cells into C57BL/6J mice resulted in high tumorigenicity. The volume of tumors derived from B16F10 cells expressing allogenic MHC H2-K(d) was decreased significantly (P < 0.01). Suppression of MHC H2-K(d)-expressing tumors in C57BL/6J mice was enhanced by immunization with MHC H2-K(d)-expressing splenocytes (P < 0.01). These results suggest that the safeguard system is capable of suppressing tumor formation by the transplanted cells. PMID:26670279

  7. A bacterial artificial chromosome transgenic mouse model for visualization of neurite growth.

    Science.gov (United States)

    Tao, Tao; Chen, Chen; Sun, Jie; Peng, YaJing; Zhu, MinSheng

    2015-04-01

    Class III β-tubulin (Tubb3) is a component of the microtubules in neurons and contributes to microtubule dynamics that are required for axon outgrowth and guidance during neuronal development. We here report a novel bacterial artificial chromosome (BAC) transgenic mouse line that expresses Class III β-tubulin fused to mCherry, an improved monomeric red fluorescent protein, for the visualization of microtubules during neuronal development. A BAC containing Tubb3 gene was modified by insertion of mCherry complementary DNA downstream of Tubb3 coding sequence via homologous recombination. mCherry fusion protein was expressed in the nervous system and testis of the transgenic animal, and the fluorescent signal was observed in the neurons that located in the olfactory bulb, cerebral cortex, hippocampal formation, cerebellum, as well as the retina. Besides, Tubb3-mCherry fusion protein mainly distributed in neurites and colocalized with endogenous Class III β-tubulin. The fusion protein labels Purkinje cell dendrites during cerebellar circuit formation. Therefore, this transgenic line might be a novel tool for scientific community to study neuronal development both in vitro and in vivo.

  8. 棉花细菌人工染色体文库构建方法探讨%Studies on Construction Method of Cotton Bacterial Artificial Chromosome Library

    Institute of Scientific and Technical Information of China (English)

    高海燕; 王省芬; 刘方; 彭仁海; 张艳; 马峙英; 王坤波

    2013-01-01

    细菌人工染色体(Bacterial artificial chromosome,BAC)文库是开展基因组测序、基因图位克隆、分子标记、物理作图等研究的重要基因组资源.本文在构建了二倍体野生棉阿非利加棉(Gossypium herbaceum var.africanum)BAC文库的基础上,就棉花细菌人工染色体基因组文库构建过程中高分子量基因组DNA的提取、部分酶切片段选择、DNA的回收、连接转化以及BAC文库的保存等过程中一些细节和注意事项进行了比较详细的分析比较,希望能为棉花BAC文库的构建提供一些可供借鉴的经验.%Bacterial artificial chromosome (BAC) library is an important genome resources to such research as genome sequencing, map-based cloning, molecular markers, and physical mapping. On the base of the construction of BAC library for Gossypi-um herbaceum var. africanum, this paper presents an exhaustive analysis on details and notices of the BAC library construction process. It includes extraction of high molecular weight (HMW) nuclear DNA, determination of the optimized enzyme for partial digestion of HMW DNA, two rounds of size fractionation, recovery of large fragments DNA, ligation and transformation of large fragments of DNA and storage of BAC library. Thus being able to supply an experience for constructing high efficiency cotton BAC library.

  9. Highly Efficient Modification of Bacterial Artificial Chromosomes (BACs) Using Novel Shuttle Vectors Containing the R6Kγ Origin of Replication

    OpenAIRE

    Gong, Shiaoching; Yang, Xiangdong William; Li, Chenjian; Heintz, Nathaniel

    2002-01-01

    Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the ...

  10. Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse

    OpenAIRE

    Choi, Inho; Chung, Hee Kyoung; Ramu, Swapnika; Lee, Ha Neul; Kim, Kyu Eui; Lee, Sunju; Yoo, Jaehyuk; Choi, Dongwon; Lee, Yong Suk; Aguilar, Berenice; Hong, Young-Kwon

    2011-01-01

    Although the blood vessel-specific fluorescent transgenic mouse has been an excellent tool to study vasculogenesis and angiogenesis, a lymphatic-specific fluorescent mouse model has not been established to date. Here we report a transgenic animal model that expresses the green fluorescent protein under the promoter of Prox1, a master control gene in lymphatic development. Generated using an approximately 200-kb-long bacterial artificial chromosome harboring the entire Prox1 gene, this Prox1-g...

  11. CHARACTERIZATION AND CHROMOSOMAL ASSIGNMENT OF YEAST ARTIFICIAL CHROMOSOMES CONTAINING HUMAN 3P13-P21-SPECIFIC SEQUENCE-TAGGED SITES

    NARCIS (Netherlands)

    MICHAELIS, SC; BARDENHEUER, W; LUX, A; SCHRAMM, A; GOCKEL, A; SIEBERT, R; WILLERS, C; SCHMIDTKE, K; TODT, B; VANDERHOUT, AH; BUYS, CHCM; HEPPELLPARTON, AC; RABBITTS, PH; UNGAR, S; SMITH, D; LEPASLIER, D; COHEN, D; OPALKA, B; SCHUTTE, J

    1995-01-01

    Human chromosomal region 3p12-p23 is proposed to harbor at least three tumor suppressor genes involved in the development of lung cancer, renal cell carcinoma, and other neoplasias. In order to identify one of these genes we defined sequence tagged sites (STSs) specific for 3p13-p24.2 by analyzing a

  12. Identification of 2nd chromosome region translocated onto the W chromosome by RFLP with EST-cDNA clones in the Gensei-kouken strains of the mulberry silkworm, Bombyx mori L

    Directory of Open Access Journals (Sweden)

    Sivaramakurup Sreekumar

    2010-01-01

    Full Text Available In silkworms, sex-limited strains are either obtained spontaneously or induced by X-rays or gamma rays. When a fragment of an autosome carrying a dominant allele of those genes responsible for certain characters is translocated onto a W chromosome, the female of the successive generations will express these phenotypic characters and sex discrimination can be facilitated. Gensei-kouken strains are sex-limited strains of silkworms developed by irradiating the pupae with gamma rays, by which a portion of the second chromosome is translocated onto the W chromosome. In these improved strains, the females are yellow-blooded and spin yellow cocoons. By using the EST-cDNA clones mapped on the Z chromosome, we identified the sex according to the polymorphic banding pattern or intensity of the signals. Furthermore, by using the clones on the second chromosome, the region of the second chromosome translocated onto the W chromosome was also defined. In both the A95 and A 96 strains selected for the present study, only the mid-portion of the second chromosome was translocated. The differences in length of the fragments translocated in these strains are discussed.

  13. Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

    Institute of Scientific and Technical Information of China (English)

    XU; Junwang(徐军望); FENG; Dejiang(冯德江); LI; Xugang(李旭刚); CHANG; Tuanjie(常团结); ZHU; Zhen(朱祯)

    2002-01-01

    The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.

  14. Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors.

    OpenAIRE

    Tao, Q.; Zhang, H. B.

    1998-01-01

    Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) systems were previously developed for cloning of very large eukaryotic DNA fragments in bacteria. We report the feasibility of cloning very large fragments of eukaryotic DNA in bacteria using conventional plasmid-based vectors. One conventional plasmid vector (pGEM11), one conventional binary plasmid vector (pSLJ1711) and one conventional binary cosmid vector (pCLD04541) were investigated using the widely used BA...

  15. Dysregulation of gene expression in the artificial human trisomy cells of chromosome 8 associated with transformed cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Hisakatsu Nawata

    Full Text Available A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression.

  16. Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

    OpenAIRE

    Lee, R T; Peterson, C L; Calman, A F; Herskowitz, I.; O'Donnell, J J

    1992-01-01

    A human cDNA encoding a galactokinase (EC 2.7.1.6) was isolated by complementation of a galactokinase-deficient (gal1-) strain of Saccharomyces cerevisiae. This cDNA encodes a predicted protein of 458 amino acids with 29% identity to galactokinase of Saccharomyces carlsbergensis. Previous studies have mapped a human galactokinase gene (GK1) to chromosome 17q23-25, closely linked to thymidine kinase. The galactokinase gene that we have isolated (GK2) is located on chromosome 15. The relationsh...

  17. Aberration distribution and chromosomally marked clones in x-irradiated skin

    International Nuclear Information System (INIS)

    Samples of clinically normal human skin were removed from therapeutically X-irradiated areas at intervals of time ranging from one hour to 60 years after completion of radiation treatment. Primary cultures of untransformed fibroblasts from these samples were analysed for surviving chromosomal structural changes using ASG banding techniques. Aberrations of four basic types, reciprocal translocations, terminal deletions, pericentric inversions and paracentric inversions (the last very rare) were found in all samples. Evidence indicates that many of these are secondary aberrations derived from primary chromatid types. Presumptive break points for all aberrations were mapped, and various tests applied to investigate their within-chromosome distributions (the data are unsuitable for valid between-chromosome analysis). For translocations, the within-arm distributions are non-random, principally as the result of a very significant deficiency of break points in terminal segments. Tests for the intrachromosomal changes (pericentric inversions and deletions) are simpler, and in neither case were there significant departure from randomness Two lines of evidence are present in the data for division and migration of chromosomally abnormal cells in vivo: (a) the presence of identical aberrations in cells from different parts of the biopsy; (b) the presence of cells with sequential changes, indicating cell division between the dose fractions of the therapeutic regime. (author)

  18. Narrowing the genetic interval and yeast artificial chromosome map in the branchio-oto-renal region on chromosome 8q

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Shrawan; Kimberling, W.J.; Pinnt, J. [Boys Town National Research Hospital, Omaha, NE (United States)] [and others

    1996-01-01

    Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial abnormality, hearing loss, and renal anomalies. Recently, the disease gene has been localized to chromosome 8q. Here, we report genetic studies that further refine the disease gene region to a smaller interval and identify several YACs from the critical region. We studied two large, clinically well-characterized BOR families with a set of 13 polymorphic markers spanning the D8S165-D8S275 interval from the chromosome 8q region. Based on multipoint analysis, the highest likelihood for the location of the BOR gene is between markers D8S543 and D8S530, a distance of about 2 cM. YACs that map in the BOR critical region have been identified and characterized by fluorescence in situ hybridization and pulsed-field gel electrophoresis. A YAC contig, based on the STS content map, that covers a minimum of 4 Mb of human DNA in the critical region of BOR is assembled. This lays the groundwork for the construction of a transcriptional map of this region and the eventual identification of genes involved in BOR syndrome. 40 refs., 4 figs., 1 tab.

  19. Telomere length homeostasis and telomere position effect on a linear human artificial chromosome are dictated by the genetic background.

    Science.gov (United States)

    Weuts, An; Voet, Thierry; Verbeeck, Jelle; Lambrechts, Nathalie; Wirix, Evelyne; Schoonjans, Luc; Danloy, Sophie; Marynen, Peter; Froyen, Guy

    2012-12-01

    Telomere position effect (TPE) is the influence of telomeres on subtelomeric epigenetic marks and gene expression. Previous studies suggested that TPE depends on genetic background. As these analyses were performed on different chromosomes, cell types and species, it remains unclear whether TPE represents a chromosome-rather than genetic background-specific regulation. We describe the development of a Linear Human Artificial Chromosome (L-HAC) as a new tool for telomere studies. The L-HAC was generated through the Cre-loxP-mediated addition of telomere ends to an existing circular HAC (C-HAC). As it can be transferred to genetically distinct cell lines and animal models the L-HAC enables the study of TPE in an unprecedented manner. The HAC was relocated to four telomerase-positive cell lines via microcell-mediated chromosome transfer and subsequently to mice via blastocyst injection of L-HAC(+)-ES-cells. We could show consistent genetic background-dependent adaptation of telomere length and telomere-associated de novo subtelomeric DNA methylation in mouse ES-R1 cells as well as in mice. Expression of the subtelomeric neomycin gene was inversely correlated with telomere length and subtelomeric methylation. We thus provide a new tool for functional telomere studies and provide strong evidence that telomere length, subtelomeric chromatin marks and expression of subtelomeric genes are genetic background dependent.

  20. Artificially introduced aneuploid chromosomes assume a conserved position in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Kundan Sengupta

    Full Text Available BACKGROUND: Chromosomal aneuploidy is a defining feature of carcinomas. For instance, in colon cancer, an additional copy of Chromosome 7 is not only observed in early pre-malignant polyps, but is faithfully maintained throughout progression to metastasis. These copy number changes show a positive correlation with average transcript levels of resident genes. An independent line of research has also established that specific chromosomes occupy a well conserved 3D position within the interphase nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated whether cancer-specific aneuploid chromosomes assume a 3D-position similar to that of its endogenous homologues, which would suggest a possible correlation with transcriptional activity. Using 3D-FISH and confocal laser scanning microscopy, we show that Chromosomes 7, 18, or 19 introduced via microcell-mediated chromosome transfer into the parental diploid colon cancer cell line DLD-1 maintain their conserved position in the interphase nucleus. CONCLUSIONS: Our data is therefore consistent with the model that each chromosome has an associated zip code (possibly gene density that determines its nuclear localization. Whether the nuclear localization determines or is determined by the transcriptional activity of resident genes has yet to be ascertained.

  1. Cloning, characterization and chromosomal location of a satellite DNA from the Pacific oyster, Crassostrea gigas

    Digital Repository Service at National Institute of Oceanography (India)

    Clabby, C.; Goswami, U.; Flavin, F.; Wilkins, N.P.; Houghton, J.A.; Powell, R.

    regions on two homologous chromosome pairs. ACKNOWLEDGEMENTS C.C. and U.G. contributed equally to this work and are considered co-first authors. This work was supported in part by the European Union FAR Programme. C.C. was supported by both FORBAIRT... and UCG postgrad- uate fellowships. U.G. is grateful to the European Commission and the Department of Science and Technology (Government of India, New Delhi), for the award of a Marie Curie fellowship and to the National Institute of Oceanography...

  2. Porcine gamma-synuclein: molecular cloning, expression analysis, chromosomal localization and functional expression

    DEFF Research Database (Denmark)

    Frandsen, Pernille Munk; Madsen, Lone Bruhn; Bendixen, Christian;

    2009-01-01

    and pituitary gland. Expression analysis also showed that porcine SNCG transcripts could be detected in different brain regions during early stages of embryo development. The porcine SNCG orthologue was mapped to chromosome 14q25-q29. The distribution of recombinant porcine γ-synuclein was studied in three......The γ-synuclein protein is involved in breast carcinogenesis and has also been implicated in other forms of cancer and in ocular diseases. Furthermore, γ-synuclein is believed to have a role in certain neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. This work...... different transfected cell lines and the protein was found to be predominantly localized in the cytoplasm...

  3. Evolution of Chromosome 6 of Solanum Species Revealed by Comparative Fluorescence in Situ Hybridization Mapping

    Science.gov (United States)

    Comparative genome mapping is an important tool in evolutionary research. Here we demonstrate a comparative fluorescent in situ hybridization (FISH) mapping strategy. A set of 13 bacterial artificial chromosome (BAC) clones derived from potato chromosome 6 was used for FISH mapping in seven differen...

  4. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

  5. Construction of a bacterial artificial chromosome library of S-type CMS maize mitochondria

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In order to isolate mitochondrial genes easily, we have developed a new method to construct S-type CMS maize mitochondrial gene library by means of embedding mitochondria and enzymatic digesting mitochondria in situ, preparing mtDNA by electrophoresis, digesting LMP agarose with β-agarase, using BAC vector and electroporation. About 2 500 white clones of Mo17 CMS-J mitochondrial gene library were obtained with the average size of 18.24 kb, ranging from 5 to 40 kb, 63.6% inserts came from mitochondrial genome and represented 48 ′ mitochondrial genome equivalents. All the probes had detected the positive clones in the gene library. It is helpful to elucidating the maize mitochondrial genome structure and mechanism of S-type CMS, and may give some valuable reference to the construction of other plant mitochondrial genome library.

  6. Construction of a full bacterial artificial chromosome (BAC) library of Oryza sativa genome

    Institute of Scientific and Technical Information of China (English)

    TAOQUANZHOU; HAIYINGZHAO; 等

    1994-01-01

    We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 106 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.

  7. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3

    Energy Technology Data Exchange (ETDEWEB)

    Haiming Chen; Lalioti, M.D.; Perrin, G.; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)] [and others

    1996-07-01

    In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to 2 genes from other species) and to a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3{prime} UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown. 56 refs., 8 figs., 1 tab.

  8. The construction of a yeast artificial chromosome (YAC) contig in the vicinity of the Usher syndrome type IIa (USH2A) gene in 1q41

    Energy Technology Data Exchange (ETDEWEB)

    Sumegi, Janos; Wang, Ji-Yi; Zhen, Dong-Kai [Univ. of Nebraska Medical Center, Omaha, NE (United States)] [and others

    1996-07-01

    The gene for Usher syndrome type II (USH2A), and autosomal recessive syndromic deafness, has been mapped to a region of 1q41 flanked proximally by D1S217 and distally by D1S439. Using sequence-tagged sites (STSs) within the region, a total of 21 yeast artificial chromosome (YAC) clones were isolated and ordered into a single contig that spans approximately 11.0 Mb. The order of microsatellite and STS markers in this region was established as D1S505-D1S425-DXS217-D1S556-D1S237-D1S474-EB1-KB6-AFM144XF2-KB1-KB4-D1S229-D1S490-D1S227-TGF{beta}2-D1S439. Analysis of newly positioned polymorphic markers in recombinant individuals in two Usher syndrome type IIa families has enabled us to identify DXS474 and AFM144XF2 as two flanking markers for the Usher type IIa locus. The physical distance between the two markers is 1.0 Mb. This region is covered by eight YACs from the CEPH library: 945f7, 867g9, 762a6, 919h3, 794b8, 785h4, 848b9, and 841g2. A long range physical map of the Usher type IIa critical region, using MluI, BssHII, NotI, EagI, and SacII, has been developed. 41 refs., 5 figs.

  9. Genome-wide copy number profiling on high-density bacterial artificial chromosomes, single-nucleotide polymorphisms, and oligonucleotide microarrays: a platform comparison based on statistical power analysis.

    NARCIS (Netherlands)

    Hehir-Kwa, J.Y.; Egmont-Peterson, M.; Janssen, I.M.; Smeets, D.F.C.M.; Geurts van Kessel, A.H.M.; Veltman, J.A.

    2007-01-01

    Recently, comparative genomic hybridization onto bacterial artificial chromosome (BAC) arrays (array-based comparative genomic hybridization) has proved to be successful for the detection of submicroscopic DNA copy-number variations in health and disease. Technological improvements to achieve a high

  10. Detailed comparison between the wheat chromosome group 7 short arms and the rice chromosome arms 6S and 8L with special reference to genes involved in starch biosynthesis

    DEFF Research Database (Denmark)

    Li, Zhongyi; Huang, Bingyan; Rampling, Lynette;

    2004-01-01

    Rice bacterial artificial chromosome (BAC) clones have been identified that contain sequences orthologous to each EST localized to wheat chromosome 7AS deletion stocks by Southern blot hybridization. This information has been used to relate the DNA sequence included in each wheat deletion stock t...

  11. Physical Map and Organization of Chromosome 7 in the Rice Blast Fungus, Magnaporthe grisea

    OpenAIRE

    Zhu, Heng; Blackmon, Barbara P.; Sasinowski, Maciek; Dean, Ralph A.

    1999-01-01

    The rice blast fungus Magnaporthe grisea is a highly destructive plant pathogen and one of the most important for studying various aspects of host-plant interactions. It has been widely adopted as a model organism because it is ideally suited for genetic and biological studies. To facilitate map-based cloning, chromosome walking, and genome organization studies of M. grisea, a complete physical map of chromosome 7 was constructed using a large-insert (130 kb) bacterial artificial chromosome (...

  12. Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters

    OpenAIRE

    Jiang, Wenjun; Zhao, Xuejin; Gabrieli, Tslil; Lou, Chunbo; Ebenstein, Yuval; Zhu, Ting F.

    2015-01-01

    The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is ex...

  13. Cloning, tissue expression pattern characterization and chromosome localization of human peptide methionine sulfoxide reductase cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Oxidation and reduction of some amino acids are one of the molecular mechanisms for regulating the function of proteins. The oxidation of methionine (Met) to methionine sulfoxide (Met(O)) results in decreasing or loss of the biological activity of related proteins. It was found that peptide methionine sulfoxide reductase (msrA) can reduce Met(O) to Met and therefore restored the biological function of the oxidized proteins. To reveal the methionine oxidation-reduction mechanism in human body, in this study, the cDNA sequence of bovine msrA was used as an information-probe to screen the human EST database. Based on a contig assembled from homologous ESTs, a 1 256-bp human MSRA cDNA was cloned from several human cDNA libraries. The cDNA contains an open reading frame (ORF) of 705 bp in length, which encodes 235 amino acid residues. Homology comparison revealed that human MSRA shares 88% and 61% identities with bovine and Escherichia coli msrA protein respectively. Expression pattern analysis revealed a single 1.6-kb transcript of human MSRA in most human tissues and with highest expression in kidney. By radiation hybrid panel mapping, the gene was localized to human chromosome 8p22-23 between markers D8S518 and D8S550. There are 2 human inherited diseases Keratolytic Winter Erythema and Microcephaly related genes in this region, it is inferred that human MSRA might be the candidate of the two diseases.

  14. Towards the cloning of imprinted genes in the Prader-Willi/Angelman region of chromosome 15q11-q13

    Energy Technology Data Exchange (ETDEWEB)

    Nakao, M.; Sutcliffe, J.S.; Beaudet, A.L. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct clinical phenotypes resulting from paternal and maternal deficiencies respectively in human chromosome 15q11-q13. The data suggest the presence of oppositely imprinted genes in the region, and the gene for small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) has been identified as a candidate gene for PWS. Previous strategies for positional cloning identified a number of transcripts from the PWS/AS region, and two of them, PAR-5 (D15S226E) and PAR-1 (D15S227E), are paternally expressed in cultured human cells from patients deleted for 15q11-q13 as is SNRPN. Cosmid contig maps have been developed from the following YACs (contained loci in parentheses): 307A12 (D15S13), 457B4 (SNRPN), 132D4 (D15S10), A229A2, and 378A12 (D15S113), to facilitate molecular studies of PWS and AS. Exon trapping has been employed to isolate putative exons from these overlapping cosmids. Two trapped fragments from the D15S113 region and one fragment from the SNRPN region has been isolated. Sequence information is available for all of the fragments. In addition to imprinting analysis in cultured human cells, we have developed a method to detect imprinting in mouse and human using a GC-clamped denaturing gradient gel electrophoresis strategy, in combination with reverse transcription-polymerase chain reaction. The imprinting analyses of putative exons are in progress to investigate their possible candidacy for involvement in PWS or AS phenotypes.

  15. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D;

    1990-01-01

    of the Arg-Gly-Asp (RGD) cell attachment site. Chromosomal mapping of the osteopontin gene (OPN) using human-rodent cell hybrids demonstrated a location on chromosome 4 in the human genome. In situ hybridization of metaphase chromosomes using radiolabeled OP1a as a probe indicated that the gene is located...

  16. Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences

    Directory of Open Access Journals (Sweden)

    B. Karsten Tischer

    2012-01-01

    Full Text Available Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

  17. Construction of BAC Libraries from Flow-Sorted Chromosomes.

    Science.gov (United States)

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

    Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes. PMID:27511172

  18. ADVANCES IN REPRODUCTIVE TECHNOLOGIES IN CATTLE: FROM ARTIFICIAL INSEMINATION TO CLONING

    Directory of Open Access Journals (Sweden)

    M Bertolini

    2009-01-01

    Full Text Available El afán por controlar los procesos reproductivos en animales ha llevado a una gran ganancia en conocimiento, impulsando el desarrollo de cuatro tecnologías reproductivas asistidas (ARTS para animales y humanos. El uso de ARTS ha sido de gran importancia en la producción ganadera. En términos generales, las tres principales generaciones de ART, incluyendo 1 inseminación artificial (AI y congelación de gametos y embriones, 2 superovulación y transferencia de embriones (MOET y 3 procedimientos de fertilización in Vitro, han madurado en aplicaciones comerciales exitosas, facilitando el incremento en la producción a través de la genética, reducción del intervalo generacional, control de enfermedades, y reducción de costos de producción. La cuarta generación de ARTS incluye procesos que aún son muy experimentales, como transferencia de núcleos (NT de células somáticas, transgénesis, y biología de células madre. Estas tecnologías se intercalan las unas con las otras y con las herramientas moleculares actuales, dependen completamente de las generaciones de tecnologías previas. Sin embargo, hay muchos retos reproductivos que no permiten alcanzar el potencial reproductivo máximo, afectando la productividad y la rentabilidad. Es claro que la aplicación de tales tecnologías como actividades lucrativas se mantendrán cuestionadas si no se asocian a otros componentes de la producción pecuaria, como la salud animal, nutrición, y prácticas de manejo adecuadas.

  19. Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

    DEFF Research Database (Denmark)

    Nilsson, Dan; Hove-Jensen, Bjarne

    1987-01-01

    The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting...... in a DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up...... to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA....

  20. Red-Mediated Transposition and Final Release of the Mini-F Vector of a Cloned Infectious Herpesvirus Genome

    OpenAIRE

    Wussow, Felix; Fickenscher, Helmut; Tischer, B Karsten

    2009-01-01

    Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via...

  1. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    Directory of Open Access Journals (Sweden)

    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  2. Gata3 Hypomorphic Mutant Mice Rescued with a Yeast Artificial Chromosome Transgene Suffer a Glomerular Mesangial Cell Defect.

    Science.gov (United States)

    Moriguchi, Takashi; Yu, Lei; Otsuki, Akihito; Ainoya, Keiko; Lim, Kim-Chew; Yamamoto, Masayuki; Engel, James Douglas

    2016-09-01

    GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generated Gata3 hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb Gata3 yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb Gata3 YAC transgene recapitulated Gata3 expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease. PMID:27296697

  3. Fluorescent in-situ hybridization of cattle and sheep chromosomes with cloned human fragile-X DNA

    DEFF Research Database (Denmark)

    Ali, Ahmd; Thomsen, Preben Dybdahl; Babar, M.E.

    2009-01-01

    /CGG(n) trinucleotide repeat sequence was carried out to determine homology between human and bovine fragile-X. The hybridisation results showed only a weak signal on a human chromosome that was not an X with all three fragile site probes. No signals were detected in sheep chromosomes. The signal of all three human...... fragile-X probes on cattle chromosomes was however, medium-prominent sub-centromeric signal on two homologues. BrdU administration in 12 h before harvesting identified these homologues to be chromosome number 5. In addition retrospective slides of cattle and sheep chromosomes used for fragile site studies...... showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X....

  4. Complex Role of the Mitochondrial Targeting Signal in the Function of Steroidogenic Acute Regulatory Protein Revealed by Bacterial Artificial Chromosome Transgenesis in Vivo

    OpenAIRE

    Sasaki, Goro; Ishii, Tomohiro; Jeyasuria, Pancharatnam; Jo, Youngah; Bahat, Assaf; Orly, Joseph; Hasegawa, Tomonobu; Parker, Keith L.

    2008-01-01

    The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green...

  5. Autoexcision of Bacterial Artificial Chromosome Facilitated by Terminal Repeat-Mediated Homologous Recombination: a Novel Approach for Generating Traceless Genetic Mutants of Herpesviruses ▿

    OpenAIRE

    Zhou, Fuchun; Li, Qiuhua; Wong, Scott W.; Gao, Shou-jiang

    2010-01-01

    Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe t...

  6. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    International Nuclear Information System (INIS)

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16+ natural killer cells and CD3+, CD16- T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

  7. 大白菜细菌人工染色体文库的构建及鉴定%Construction and Characterization of a Bacterial Artificial Chromosome Library from Chinese Cabbage

    Institute of Scientific and Technical Information of China (English)

    冯大领; 石学萍; 杨煜; 王彦华; 轩淑欣; 赵建军; 申书兴

    2011-01-01

    以我国优良的大白菜自交系'85-1'为材料,利用 pIndigoBAC-5 为载体,通过对高分子量DNA 的制备、大片段 DNA 的选择、连接转化条件等几个方面的优化,构建了大白菜细菌人工染色体文库.该文库由 57 600个克隆组成,平均大小为98.4 kb,空载率为1.5%;覆盖大白菜基因组 10.3 倍;挑取 6 个克隆培养5 d 后,经HindⅢ完全酶切检测,其指纹图谱稳定一致.大白菜细菌人工染色体文库的构建为重要功能基因的克隆和定位及比较基因组研究奠定了基础.%A bacterial artificial chromosome library of Brassica campestris L. ssp. pekinensis ( Lour.)Olsson (Chinese cabbage) was constructed from inbred line‘ 85-1’ with the vector pIndigoBAC-5. The key processes of the construction, such as preparation of high molecular weight DNA, selection of digested fragments, condition of ligation and transformation, were studied. The library consists of 57 600 clones in which the average insert size is about 98.4 kb and the empty clones are about 1.5%. The library represents an equivalent of 10.3 fold size of Chinese cabbage genome. Six clones randomly picked from this library show no HindⅢ fingerprint changes after 5 days' successive culture, which indicates that the clones in the library are stable. The library will lay the foundation for gene clone, location and comparative genomics research of Brassica.

  8. Uroporphyrinogen-III synthase: Molecular cloning, nucleotide sequence, expression of a mouse full-length cDNA, and its localization on mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Xu, W.; Desnick, R.J. [Mount Sinai School of Medicine, New York, NY (United States); Kozak, C.A. [National Institute of Health, Bethesda, MD (United States)

    1995-04-10

    Uroporphyrinogen-III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 X 10{sup 6} recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5{prime} and 3{prime} untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli. In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy. 38 refs., 1 tab.

  9. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    Directory of Open Access Journals (Sweden)

    Shigeyuki Yamaguchi

    Full Text Available The production of cells capable of expressing gene(s of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  10. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice.

    Science.gov (United States)

    Cubells, Joseph F; Schroeder, Jason P; Barrie, Elizabeth S; Manvich, Daniel F; Sadee, Wolfgang; Berg, Tiina; Mercer, Kristina; Stowe, Taylor A; Liles, L Cameron; Squires, Katherine E; Mezher, Andrew; Curtin, Patrick; Perdomo, Dannie L; Szot, Patricia; Weinshenker, David

    2016-01-01

    Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency

  11. Cloning and chromosomal localization of a human kidney cDNA involved in cystine, dibasic, and neutral amino acid transport.

    OpenAIRE

    Lee, W S; Wells, R G; Sabbag, R V; Mohandas, T K; Hediger, M A

    1993-01-01

    We have recently cloned, sequenced, and characterized a rat kidney cDNA (D2) that stimulates cystine as well as dibasic and neutral amino acid transport. In order to evaluate the role of this protein in human inherited diseases such as cystinuria, we have isolated a human D2 clone (D2H) by low stringency screening of a human kidney cDNA library using the radiolabeled D2 insert as a probe. The D2H cDNA is 2284 nucleotides long and encodes a 663 amino acid protein that is 80% identical to the r...

  12. Met-ase: Cloning and distinct chromosomal location of a serine protease preferentially expressed in human natural killer cells

    Energy Technology Data Exchange (ETDEWEB)

    Smyth, M.J.; Trapani, J.A. (Austin Research Institute, Austin Hospital, Victoria (Australia)); Sayers, T.J.; Wiltrout, T. (NCI-Frederick Cancer Research and Development Center, Frederick, MD (United States)); Powers, J.C. (Georgia Institute of Technology, Atlanta, GA (United States))

    1993-12-01

    A cDNA clone encoding a human NK serine protease was obtained by screening a [lambda]-gt10 library from the Lopez NK leukemia with the rat natural killer Met-ase (RNK-Met-1) cDNA clone. In Northern blot analysis human Met-ase (Hu-Met-1) cDNA hybridized with a 0.9-kb mRNA in two human NK leukemia cell lines, unstimulated human PBMC, and untreated purified CD3[sup [minus

  13. (Developing a physical map of human chromosome 22)

    Energy Technology Data Exchange (ETDEWEB)

    Simon, M.I.

    1991-01-01

    We have developed bacterial F-factor based systems for cloning large fragments of human DNA in E. coli. In addition to large size, these systems are capable of maintaining human DNA with a high degree of stability. The cosmid size clones are called Fosmids and the clones containing larger inserts (100--200 kb) are called bacterial artificial chromosomes (BACs). The ultimate test of the effectiveness of cloning and mapping technology is the degree to which it can be efficiently applied to solve complex mapping problems. We, therefore, plan to use the large fragment cloning procedure as well as a variety of other approaches to generate a complete map of overlapping clones corresponding to human chromosome 22. We have thus far prepared two human chromosome 22 specific Fosmid libraries and we are in the process of constructing a chromosome 22 specific BAC library composed of fragments larger than 100 kb. We will further optimize the technology so that libraries of fragments larger than 200 kb can be readily prepared.

  14. [Developing a physical map of human chromosome 22]. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Simon, M.I.

    1991-12-31

    We have developed bacterial F-factor based systems for cloning large fragments of human DNA in E. coli. In addition to large size, these systems are capable of maintaining human DNA with a high degree of stability. The cosmid size clones are called Fosmids and the clones containing larger inserts (100--200 kb) are called bacterial artificial chromosomes (BACs). The ultimate test of the effectiveness of cloning and mapping technology is the degree to which it can be efficiently applied to solve complex mapping problems. We, therefore, plan to use the large fragment cloning procedure as well as a variety of other approaches to generate a complete map of overlapping clones corresponding to human chromosome 22. We have thus far prepared two human chromosome 22 specific Fosmid libraries and we are in the process of constructing a chromosome 22 specific BAC library composed of fragments larger than 100 kb. We will further optimize the technology so that libraries of fragments larger than 200 kb can be readily prepared.

  15. Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

    OpenAIRE

    Noskov, V. N.; Koriabine, M.; Solomon, G.; Randolph, M; Barrett, J C; Leem, S.-H.; Stubbs, L; Kouprina, N; Larionov, V.

    2001-01-01

    The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) contain...

  16. Protective efficacy of a recombinant BAC clone of Marek's disease virus containing REV-LTR

    Science.gov (United States)

    Insertion of reticuloendotheliosis virus (REV) long-terminal repeat (LTR) into a bacterial artificial chromosome (BAC) clone of a very virulent strain of Marek’s disease (MD) virus (MDV), Md5 (Kim et al, 2011) rendered the resultant recombinant virus termed rMd5 REV-LTR BAC fully attenuated at passa...

  17. First Birth after Sperm Selection through Discontinuous Gradient Centrifugation and Artificial Insemination from a Chromosomal Translocation Carrier

    OpenAIRE

    Alexandre Rouen; Capucine Hyon; Richard Balet; Nicole Joyé; Nino Guy Cassuto; Jean-Pierre Siffroi

    2014-01-01

    Introduction. Balanced chromosomal carriers, though usually healthy, are confronted with recurrent spontaneous abortions and malformations in the offspring. Those are related to the transmission of an abnormal, chromosomally unbalanced genotype. We evidenced that the proportion of unbalanced spermatozoa can be significantly decreased through a sperm preparation process called discontinuous gradient centrifugation (DGC). We therefore started offering intrauterine inseminations with this proced...

  18. Some observations on the pathogenicity of a molecular clone of a very virulent strain of Marek’s disease virus containing an insert of long terminal repeat of reticuloendotheliosis virus

    Science.gov (United States)

    We recently artificially inserted REV LTR into a bacterial artificial chromosome (BAC) clone of a very virulent strain of Marek’s disease (MD) virus (MDV), Md5; the virus was designated rMd5-RM1-LTR (Kim et al., 2011). In the present study, susceptible chickens of ADOL line 15I5 X 71 with and witho...

  19. Cloning and characterization of a novel gene (C17orf25) from the deletion region on chromosome 17p13.3 in hepatocelular carcinoma

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using a combination of hybridization of PAC to a cDNA library and RACE technique, we isolated a novel cDNA, designated as C17orf25 (Chromosome 17 open reading frame 25, previously named it HC71A), from the deletion region on chromosome 17p13.3. The cDNA encodes a protein of 313 amino acids with a calculated molecular mass of 34.8 kDa. C17orf25 is divided into 10 exons and 9 introns, spanning 23 kb of genomic DNA. Northern blot analysis showed that the mRNA expression of C17orf25 was decreased in hepatocellular carcinoma samples as compared to adjacent noncancerous liver tissues from the same patients. The transfection of C17orf25 into the hepatocellular carcinoma cell SMMC7721 and overexpression could inhibit the cell growth. The above results indicate that C17orf25 is a novel human gene, and the cloning and preliminary characterization of C17orf25 is a prerequisite for further functional analysis of this novel gene in human hepatocellular carcinoma.

  20. Fusion primer and nested integrated PCR (FPNI-PCR: a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

    Directory of Open Access Journals (Sweden)

    Wang Zhen

    2011-11-01

    Full Text Available Abstract Background The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Results Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs. These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. Conclusions The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures.

  1. Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X.

    Science.gov (United States)

    Uchida, K; Tsuchida, J; Tanaka, H; Koga, M; Nishina, Y; Nozaki, M; Yoshinaga, K; Toshimori, K; Matsumiya, K; Okuyama, A; Nishimune, Y

    2000-10-01

    We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation. PMID:10993819

  2. From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

    Science.gov (United States)

    Kouprina, Natalay; Lee, Nicholas C O; Kononenko, Artem V; Samoshkin, Alexander; Larionov, Vladimir

    2015-01-01

    Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed. PMID:25239739

  3. Comparative mapping on the mouse and human X chromosomes of a human cDNA clone encoding the vasopressin renal-type receptor (AVP2R)

    Energy Technology Data Exchange (ETDEWEB)

    Faust, C.J.; Gonzales, J.C.; Seibold, A.; Birnbaumer, M.; Herman, G.E. (Baylor College of Medicine, Houston, TX (United States))

    1993-02-01

    Mutation in the gene for the human renal-type vasopressin receptor (V2R) have recently been identified in patients with nephrogenic diabetes insipidus (NDI). Both V2R and NDI have been independently mapped to Xq28. Using a combination of genetic and physical mapping, we have localized the murine V2r locus to within 100 kb of L1Cam on the mouse X chromosome in a region syntenic with human Xq28. Based on conserved gene order of mouse and human loci in this region, physical mapping using DNA derived form human lymphoblasts has established that the corresponding human loci V2R and L1CAM are linked within 210 kb. The efficiency and precision of genetic mapping of V2r and other loci in the mouse suggest that it might be easier to map additional human genes in the mouse first and infer the corresponding human location. More precise physical mapping in man could then be performed using pulsed-field gel electrophoresis and/or yeast artificial chromosomes. 16 refs., 1 fig. 1 tab.

  4. The use of chromosome-based vectors for animal transgenesis.

    Science.gov (United States)

    Kuroiwa, Y; Yoshida, H; Ohshima, T; Shinohara, T; Ohguma, A; Kazuki, Y; Oshimura, M; Ishida, I; Tomizuka, K

    2002-06-01

    This article summarizes our efforts to use chromosome-based vectors for animal transgenesis, which may have a benefit for overcoming the size constraints of cloned transgenes in conventional techniques. Since the initial trial for introducing naturally occurring human chromosome fragments (hCFs) with large and complex immunogulobulin (Ig) loci into mice we have obtained several lines of trans-chromosomic (Tc) mice with transmittable hCFs. As expected the normal tissue-specific expression of introduced human genes was reproduced in them by inclusion of essential remote regulatory elements. Recent development of 'chromosome cloning' technique that enable construction of human artificial chromosomes (HACs) containing a defined chromosomal region should prevent the introduction of additional genes other than genes of interest and thus enhance the utility of chromosome vector system. Using this technique a panel of HACs harboring inserts ranging in size from 1.5 to 10 Mb from three human chromosomes (hChr2, 7, 22) has been constructed. Tc animals containing the HACs may be valuable not only as a powerful tool for functional genomics but also as an in vivo model to study therapeutic gene delivery by HACs.

  5. Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon

    Directory of Open Access Journals (Sweden)

    Lawrence Mark L

    2010-07-01

    Full Text Available Abstract Background Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum. Results We found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies. Conclusions This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.

  6. The Mouse Clock Locus: Sequence and Comparative Analysis of 204 Kb from Mouse Chromosome 5

    OpenAIRE

    Wilsbacher, Lisa D.; Sangoram, Ashvin M.; Antoch, Marina P.; Takahashi, Joseph S.

    2000-01-01

    The Clock gene encodes a basic helix-loop-helix (bHLH)–PAS transcription factor that regulates circadian rhythms in mice. We previously cloned Clock in mouse and human using a battery of behavioral and molecular techniques, including shotgun sequencing of two bacterial artificial chromosome (BAC) clones. Here we report the finished sequence of a 204-kb region from mouse chromosome 5. This region contains the complete loci for the Clock and Tpardl (pFT27) genes, as well as the 3′ partial locus...

  7. Toward a Molecular Cytogenetic Map for Cultivated Sunflower (Helianthus annuus L.) by Landed BAC/BIBAC Clones

    OpenAIRE

    Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

    2013-01-01

    Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivate...

  8. Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones

    Institute of Scientific and Technical Information of China (English)

    Yuan Guan; Qi Chen; Junsong Pan; Zheng Li; Huanle He; Aizhong Wu; Rentao Song; Run Cai

    2008-01-01

    A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each link-age group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen mark-ers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.

  9. Response to artificial inoculation with Phytophthora infestans of potato clones insensitive to culture filtrate of the fungus

    International Nuclear Information System (INIS)

    Induction of general resistance to Phytophthora infestans (Mont.) de Bary by in vitro culture has been reported for cvs. 'Bintje' and 'Kennebec'. In both cases, sporangia suspension of the fungus were used as selective agent. As reported for many host-pathogen interactions, culture filtrate or purified toxins may also contribute to the selection of resistant genotypes. Challenge of mutagenized buds of cv. 'Spunta' with culture filtrate of P. infestans allowed selection of insensitive clones. To verify the possible correlation between culture filtrate insensitivity and disease resistance, different inoculation technique were applied to these clones. Considering that general resistance to late blight is a complex trait, different tests were performed to evaluate the behaviour during various stages of pathogenesis, such as infection, invasion, mycelial growth and sporulation. 7 refs, 1 tab

  10. Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

    Institute of Scientific and Technical Information of China (English)

    吴清江; 叶玉珍; 董新红

    2003-01-01

    A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.

  11. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    DEFF Research Database (Denmark)

    Wewer, U M; Gerecke, D R; Durkin, M E;

    1994-01-01

    Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... beta 2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human beta 2 chain is predicted to have all of the seven structural domains typical of the beta chains of laminin, including the short cysteine-rich alpha region. The amino acid sequence of human beta 2...... chain showed 86.1% sequence identity to the rat beta 2 chain, 50.0% to the human beta 1 chain, and 36.3% to the human beta 3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the beta 2 chain of laminin purified from human...

  12. Molecular cloning, expression analysis and chromosomal mapping of salt-responsive cDNAs in rice ( Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    李子银; 张劲松; 陈受宜

    1999-01-01

    By using differential display PCR (DD-PCR) technique, two salt-inducible and one salt-repressed cDNA fragments were isolated from rice. The three eDNA fragments were characterized respectively as partial sequence of rice S-adenosylmethionine deearboxylase (SAMDC) gene, a new member of translation elongation factor 1A gene (named REF1A ), and a novel gene whose function is unknown (named SRG1). The full-length cDNA of SAMDC gene (named SAMDC1) was further isolated by RT-PCR approach and the deduced polypeptide was found to be homologous to SAMDC proteins of other plants, yeast and human. Northern hybridization revealed that expression of SAMDC1 and REF1A was induced, while SRG1 was dramatically repressed, by salinity stress. Southern blot analysis demonstrated that SAMDC1 and SRG1 were present as a single copy gene in rice genome, whereas rice REF1A gene was organized as a gene family. The REF1A, SAMDC1, and SRG1 genes were located on chromosome 3, 4, and 6 respectively by RFLP mapping approach using ZYQ

  13. Isolation and properties of Drosophila melanogaster ferritin--molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome.

    Science.gov (United States)

    Charlesworth, A; Georgieva, T; Gospodov, I; Law, J H; Dunkov, B C; Ralcheva, N; Barillas-Mury, C; Ralchev, K; Kafatos, F C

    1997-07-15

    Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed. PMID:9266686

  14. Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1.

    OpenAIRE

    Valvano, M A; Crosa, J H

    1989-01-01

    We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material whi...

  15. First Birth after Sperm Selection through Discontinuous Gradient Centrifugation and Artificial Insemination from a Chromosomal Translocation Carrier

    Directory of Open Access Journals (Sweden)

    Alexandre Rouen

    2014-01-01

    Full Text Available Introduction. Balanced chromosomal carriers, though usually healthy, are confronted with recurrent spontaneous abortions and malformations in the offspring. Those are related to the transmission of an abnormal, chromosomally unbalanced genotype. We evidenced that the proportion of unbalanced spermatozoa can be significantly decreased through a sperm preparation process called discontinuous gradient centrifugation (DGC. We therefore started offering intrauterine inseminations with this procedure to couples with a male translocation carriers. Case Presentation. We report the case of a 37-year-old man carrying a t(3;10(q25;p13 reciprocal translocation. He and his partner had had trouble conceiving for ten years and had four spontaneous abortions. DGC in this patient decreased the proportion of unbalanced spermatozoa from 63.6% to 52.3%. They were therefore offered intrauterine insemination with DGC, which eventually led to the birth of a healthy female child carrying the paternal translocation. Conclusion. We showed that translocation carriers could be offered intrauterine inseminations with DGC. Before this, the only two options were natural conception with prenatal diagnosis and termination of chromosomally unbalanced fetuses or preimplantation genetic diagnosis, which is a much heavier and costly procedure. We are currently offering this option through a multicentric program in France, and this is the first birth originating from it.

  16. First Birth after Sperm Selection through Discontinuous Gradient Centrifugation and Artificial Insemination from a Chromosomal Translocation Carrier.

    Science.gov (United States)

    Rouen, Alexandre; Hyon, Capucine; Balet, Richard; Joyé, Nicole; Cassuto, Nino Guy; Siffroi, Jean-Pierre

    2014-01-01

    Introduction. Balanced chromosomal carriers, though usually healthy, are confronted with recurrent spontaneous abortions and malformations in the offspring. Those are related to the transmission of an abnormal, chromosomally unbalanced genotype. We evidenced that the proportion of unbalanced spermatozoa can be significantly decreased through a sperm preparation process called discontinuous gradient centrifugation (DGC). We therefore started offering intrauterine inseminations with this procedure to couples with a male translocation carriers. Case Presentation. We report the case of a 37-year-old man carrying a t(3;10)(q25;p13) reciprocal translocation. He and his partner had had trouble conceiving for ten years and had four spontaneous abortions. DGC in this patient decreased the proportion of unbalanced spermatozoa from 63.6% to 52.3%. They were therefore offered intrauterine insemination with DGC, which eventually led to the birth of a healthy female child carrying the paternal translocation. Conclusion. We showed that translocation carriers could be offered intrauterine inseminations with DGC. Before this, the only two options were natural conception with prenatal diagnosis and termination of chromosomally unbalanced fetuses or preimplantation genetic diagnosis, which is a much heavier and costly procedure. We are currently offering this option through a multicentric program in France, and this is the first birth originating from it. PMID:24587925

  17. Application of high molecular weight DNA cloning in legume nodulation gene analysis

    International Nuclear Information System (INIS)

    High molecular weight (HMW) DNA was isolated from Glycine max (soybean) and the model legume Lotus japonicus for the purpose of legume genome analysis. The primary objectives were the gene regions that control nodulation, early plant-microbe interaction and cell division responses. HMW DNA was separated by pulse field gel electrophoresis (CHEF-PFGE) and analyzed with closely linked restriction fragment length polymorphism (RFLP) markers co-hybridized with clones, permitting estimation of the regional physical distances as they relate to recombination frequency. In the distal region of molecular linkage group H containing one of the genes controlling nodule number autoregulation and symbiotic nitrate tolerance (i.e. the nts gene), 1 cM was equivalent to less than 500 kb. Partially digested EcoRI soybean and L. japonicus HMW DNA were cloned into pYAC4. Stable yeast artificial chromosomes (YACs) carrying up to 960 kb DNA were generated. The average insert size was 200 kb. Hybridization with total genomic soybean DNA revealed YACs with different amounts of repeated DNA sequences. Mapping of the end clones demonstrated whether the YACs were chimeric. YACs of different complexity were used for chromosome identification using degenerate primer polymerase chain reaction and fluorescent in situ hybridization. This approach is a fast alternative to testing for YAC chimerism. Single arbitrary and structured mini-hairpin primers were used to amplify and DNA fingerprint the YACs, providing a means of identifying the additional markers needed for contig construction. HMW DNA was cloned into the F plasmid bacterial artificial chromosome (BAC) vector. The YACs and BACs were also constructed with DNA from the small genome/highly transformable legume L. japonicus. Mapping of the YAC and BAC clones with molecular markers will help to ascertain the degree of chimerism and stability in the different cloning systems. YACs, molecular markers and cDNA clones will be useful for chromosome

  18. Molecular cloning, genomic organization, chromosome mapping, tissues expression pattern and identification of a novel splicing variant of porcine CIDEb gene.

    Science.gov (United States)

    Li, YanHua; Li, AiHua; Yang, Z Q

    2016-09-01

    Cell death-inducing DNA fragmentation factor-α-like effector b (CIDEb) is a member of the CIDE family of apoptosis-inducing factors, CIDEa and CIDEc have been reported to be Lipid droplets (LDs)-associated proteins that promote atypical LD fusion in adipocytes, and responsible for liver steatosis under fasting and obese conditions, whereas CIDEb promotes lipid storage under normal diet conditions [1], and promotes the formation of triacylglyceride-enriched VLDL particles in hepatocytes [2]. Here, we report the gene cloning, chromosome mapping, tissue distribution, genetic expression analysis, and identification of a novel splicing variant of the porcine CIDEb gene. Sequence analysis shows that the open reading frame of the normal porcine CIDEb isoform covers 660bp and encodes a 219-amino acid polypeptide, whereas its alternative splicing variant encodes a 142-amino acid polypeptide truncated at the fourth exon and comprised of the CIDE-N domain and part of the CIDE-C domain. The deduced amino acid sequence of normal porcine CIDEb shows an 85.8% similarity to the human protein and 80.0% to the mouse protein. The CIDEb genomic sequence spans approximately 6KB comprised of five exons and four introns. Radiation hybrid mapping demonstrated that porcine CIDEb is located at chromosome 7q21 and at a distance of 57cR from the most significantly linked marker, S0334, regions that are syntenic with the corresponding region in the human genome. Tissue expression analysis indicated that normal CIDEb mRNA is ubiquitously expressed in many porcine tissues. It was highly expressed in white adipose tissue and was observed at relatively high levels in the liver, lung, small intestine, lymphatic tissue and brain. The normal version of CIDEb was the predominant form in all tested tissues, whereas the splicing variant was expressed at low levels in all examined tissues except the lymphatic tissue. Furthermore, genetic expression analysis indicated that CIDEb mRNA levels were

  19. cDNA cloning, genomic structure, and chromosome mapping of the human epithelial membrane protein CL-20 gene (EMP1), a member of the PMP22 family.

    Science.gov (United States)

    Chen, Y; Medvedev, A; Ruzanov, P; Marvin, K W; Jetten, A M

    1997-04-01

    CL-20 is a novel gene encoding a protein that is structurally related to but distinct from the peripheral myelin protein PMP22. Like PMP22, CL-20 is likely to play important roles in the regulation of cell proliferation, differentiation, and cell death. In this study, we describe the cloning and sequencing of a cDNA encoding the human homologue of CL-20 and characterize the genomic structure of this gene. The hCL-20 gene (HGMW-approved symbol EMP1) encodes a protein of 157 amino acids that exhibits 76% identity to the rabbit CL-20 and to the rat EMP-1, which have been described recently, and 39% identity to human PMP22. CL-20 contains four hydrophobic domains, suggesting that it is an integral membrane protein. In particular the second hydrophobic domain encoded within the fourth exon is highly conserved among CL-20, EMP-1, and PMP22, suggesting a functional role for this region. CL-20 mRNA is abundant in squamous-differentiated bronchial epithelial cells; however, low levels of CL-20 mRNA can be detected in several human tissues by Northern analysis. Retinoic acid, which inhibits squamous differentiation, represses CL-20 expression in normal human bronchial epithelial cells. The genomic structure of the hCL-20 gene was analyzed using a P1 vector containing this gene. The hCL-20 gene contains five exons about 0.2, 0.12, 0.1, 0.14, and 2.2 kb and four introns about 15, 1.9, 0.1, and 0.7 kb. We have mapped the hCL-20 gene to chromosome 12p12 by fluorescence in situ hybridization. PMID:9126480

  20. Plant Artificial Chromosome:The Vector for the Next Generation of Genetic Engineering%植物人工染色体:下一代基因工程的载体

    Institute of Scientific and Technical Information of China (English)

    李晨; 闫晓红; 杨洁; 杨清; 魏文辉

    2011-01-01

    The simultaneous expression of multiple genes and genetically modified (GM) food security research have been considered as the research focus for genetic engineering in plants presently. Unlike conventional gene transformation technologies,plant artificial chromosomes provide one solution to the stable expression of multiple transgenes. As plant artificial chromosome segregation is independent of host chromosomes, they provide a platform for accelerating plant breeding and for studying the specific chromatin domains inserted into them. The generation of artificial plant chromosomes and their applications were reviewed in this article.%植物基因工程技术中的多基因转化及转基因安全已经成为其研究的2个重要方面.植物人工染色体可以在一条不含标记基因的附加染色体上提供稳定的多基因表达,是新一代的转基因载体.由于植物人工染色体独立于宿主染色体,为植物育种提供了便利,同时,也为研究染色质特殊区域的结构与功能提供了平台.本文就植物人工染色体的产生、研究现状及其应用前景等进行了综述和讨论.

  1. Chromosome-specific DNA Repeat Probes

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  2. A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.

    Directory of Open Access Journals (Sweden)

    Teruhiko Suzuki

    Full Text Available Human artificial chromosomes (HACs are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.

  3. IncM Plasmid R1215 Is the Source of Chromosomally Located Regions Containing Multiple Antibiotic Resistance Genes in the Globally Disseminated Acinetobacter baumannii GC1 and GC2 Clones

    OpenAIRE

    Blackwell, Grace A.; Hamidian, Mohammad; Hall, Ruth M.

    2016-01-01

    ABSTRACT Clear similarities between antibiotic resistance islands in the chromosomes of extensively antibiotic-resistant isolates from the two dominant, globally distributed Acinetobacter baumannii clones, GC1 and GC2, suggest a common origin. A close relative of the likely progenitor of both of these regions was found in R1215, a conjugative IncM plasmid from a Serratia marcescens strain isolated prior to 1980. The 37.8-kb resistance region in R1215 lies within the mucB gene and includes aac...

  4. Distribution and uses of legume DNA clone resources

    International Nuclear Information System (INIS)

    Since 1990, my lab has developed and distributed various DNA clone resources for the legumes. In the first several years, the focus was on members of the tropical genus, Vigna, including the widely cultivated species, mungbean (V. radiata) and cowpea (V. unguiculata). Both of these grain legumes play key roles in agriculture in developing countries of Asia (mungbean) and Africa (cowpea). Moreover, because there is substantial genome conservation among legumes, these genetic resources have also been utilized by a wide range of researchers in other crop species. In 1997, my lab began to focus on the development and distribution of a new generation of DNA clone resources; Bacterial Artificial Chromosomes (BAC). A library of these clones was constructed in soybean (Glycine max) the most important legume species worldwide in terms of economic value. Again, the library has become a valuable resource for the legume research community and has been widely used in studies of legume genomics. (author)

  5. A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening

    Directory of Open Access Journals (Sweden)

    Deal Karin R

    2010-12-01

    Full Text Available Abstract Background A five-dimensional (5-D clone pooling strategy for screening of bacterial artificial chromosome (BAC clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones. Results A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~2× genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on Aegilops tauschii chromosome 2D and Ae. tauschii contig maps. Clones in single contigs were successfully assigned to 48 (87% specific SNP markers on the map with 91% precision. Conclusion A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml.

  6. Identification of Chromosomes from Multiple Rice Genomes Using a Universal Molecular Cytogenetic Marker System

    Institute of Scientific and Technical Information of China (English)

    Xiaomin Tang; Weidong Bao; Wenli Zhang; Zhukuan Cheng

    2007-01-01

    To develop reliable techniques for chromosome identification is critical for cytogenetic research, especially for genomes with a large number and smaller-sized chromosomes. An efficient approach using bacterial artificial chromosome (BAG) clones as molecular cytological markers has been developed for many organisms. Herein, we present a set of chromosomal arm-specific molecular cytological markers derived from the gene-enriched regions of the sequenced rice genome. All these markers are able to generate very strong signals on the pachytene chromosomes of Oryza satlva L. (AA genome) when used as fluorescence in situ hybridization (FISH) probes. We further probed those markers to the pachytene chromosomes of O. punctata (BB genome) and O. officinalis (CC genome) and also got very strong signals on the relevant pachytene chromosomes. The signal position of each marker on the related chromosomes from the three different rice genomes was pretty much stable, which enabled us to identify different chromosomes among various rice genomes. We also constructed the karyotype for both O. punctata and O. officinalis with the BB and CC genomes, respectively, by analysis of 10 pachytene cells anchored by these chromosomal arm-specific markers.

  7. Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Arondel, V.; Lemieux, B.; Hwang, I. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1992-11-20

    A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1 tabs.

  8. Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

    DEFF Research Database (Denmark)

    Nilsson, Dan; Hove-Jensen, Bjarne

    1987-01-01

    The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting...

  9. ADVANCES IN REPRODUCTIVE TECHNOLOGIES IN CATTLE: FROM ARTIFICIAL INSEMINATION TO CLONING AVANCES EN BIOTECNOLOÍA REPRODUCTIVA EN BOVINOS: DE LA INSEMINACIÓN ARTIFICIAL A LA CLONACIÓN

    Directory of Open Access Journals (Sweden)

    Bertolini, L.R

    2009-05-01

    Full Text Available The urge for the control of reproductive processes in animals has propelled a great gain in knowledge, also setting off the development of four generations of assisted reproductive technologies (AR T for humans and animals. The use of assisted reproductive techniques has been of great importance in livestock production. In general terms, the main first three generations of ARTs, including 1 artificial insemination (AI and gamete and embryo freezing, 2 multiple ovulation and embryo transfer (MOET and 3 in vitro fertilization (IV F procedures, have matured into successful commercial applications, facilitating the increase in production through genetics, the reduction in generation intervals, the control of diseases, and the cutback in production costs. The fourth generation of AR T encompasses processes that are still more experimental, comprising cloning by nuclear transfer (NT of embryonic or somatic cells, transgenesis, and stem cell biology. Such technologies are intertwined with one another and with currently available molecular tools, being completely dependent upon the previous generations of technologies. However, many reproductive challenges still hinder maximal livestock reproductive performance, affecting productivity and profitability. It is clear that the application of such technologies as lucrative activities will remain questionable if not associated with other components of animal production, such as animal health, nutrition and adequate animal husbandry practices.El afán por controlar los procesos reproductivos en animales ha llevado a una gran ganancia en conocimiento, impulsando el desarrollo de cuatro tecnologías reproductivas asistidas (AR Ts para animales y humanos. El uso de AR Ts ha sido de gran importancia en la producción ganadera. En términos generales, las tres principales generaciones de AR T, incluyendo 1 inseminación artificial (AI y congelación de gametos y embriones, 2 superovulación y transferencia de

  10. Utilization of Super BAC Pools and Fluidigm Access Array Platform for High-Throughput BAC Clone Identification: Proof of Concept

    OpenAIRE

    Peter J Maughan; Smith, Scott M.; Joshua A. Raney

    2012-01-01

    Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooled Amaranthus hypochondriacus BAC library. Ninety...

  11. Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L.and O.granulata Nees et Am.ex Watt.

    Institute of Scientific and Technical Information of China (English)

    XIONG Zhiyong; TAN Guangxuan; YOU Aiqing; HE Guangyuan; SHE Chaowen; LI Lijia; SONG Yunchun

    2004-01-01

    Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice, which are the most valuable for study and utilization in China. In this study, the homology and physical locations of three rice resistance genes, Glh,Bph-3 and xa-5 are comparatively analyzed between O. sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH). The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata. By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O. sativa and O. granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O. sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O. sativa and O. granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.

  12. Identification of a yeast artificial chromosome (YAC) spanning the synovial sarcoma-specific t(X;18)(p11.2;q11.2) breakpoint

    NARCIS (Netherlands)

    de Leeuw, B; Berger, W; Sinke, R J; Suijkerbuijk, R F; Gilgenkrantz, S; Geraghty, M T; Valle, D; Monaco, A P; Lehrach, H; Ropers, H H

    1993-01-01

    A somatic cell hybrid containing the synovial sarcoma-associated t(X;18)(p11.2;q11.2) derivative (der(X)) chromosome was used to characterize the translocation breakpoint region on the X chromosome. By using Southern hybridization of DNA from this der(X) hybrid in conjunction with Xp-region specific

  13. The Chromosome Microdissection and Microcloning Technique.

    Science.gov (United States)

    Zhang, Ying-Xin; Deng, Chuan-Liang; Hu, Zan-Min

    2016-01-01

    Chromosome microdissection followed by microcloning is an efficient tool combining cytogenetics and molecular genetics that can be used for the construction of the high density molecular marker linkage map and fine physical map, the generation of probes for chromosome painting, and the localization and cloning of important genes. Here, we describe a modified technique to microdissect a single chromosome, paint individual chromosomes, and construct single-chromosome DNA libraries. PMID:27511173

  14. 染色体多态性对供精人工授精治疗结局的影响%Influence of chromosomal polymorphism on treatment outcome of artificial insemination by donors

    Institute of Scientific and Technical Information of China (English)

    伍园园; 郑立新; 祝小丽; 舒小妹; 郑炜炜

    2013-01-01

    Objective: To investigate the relationship between the female chromosome polymorphism and the pregnancy outcome of artificial insemination by donors. Methods; The peripheral blood of patients was cultured rountinely for karotype analysis. Clinical pregnancy rate, cumulative pregnancy rate and early obortion rate were observed by treated patients with chromosomal polymorphism as the research group, and patients with normal chromosome as control group. Results: There was no significant difference in the clinical cycle pregnancy rate ( 19. 80% vs 19. 66% ) , cumulative pregnancy rate (44. 82% vs 41. 77% ) , early abortion rate ( 14. 58% vsl2. 07% ) between the two groups ( P > 0. 05 ) . Conclusion: Chromosomal polymorphism carrier status has no impact on treatment outcome of the patients receiving artificial insemination by donors.%目的 探讨女性染色体多态性与供精人工授精妊娠结局的关系.方法 对接受供精人工授精患者常规抽血行染色体检查,将发现有染色体多态的患者作为研究对象,与染色体正常群体对照,观察临床妊娠率、累积妊娠率和早期流产率.结果 染色体多态性组与正常组比较,临床周期妊娠率(19.80% vs 19.66%),累积妊娠率(44.82%vs 41.77%),早期流产率(14.58% vs12.07%),均无统计学差异(P>0.05).结论 染色体多态性携带状态不影响供精人工授精患者的治疗结局.

  15. Protective efficacy of a recombinant bacterial artificial chromosome clone of a very virulent Marek’s disease virus containing a reticuloendotheliosis virus long terminal repeat

    Science.gov (United States)

    Marek’s disease virus (MDV), an alphaherpesvirus, causes Marek’s disease (MD), a lymphoproliferative disease in poultry characterized by T-cell lymphomas, nerve lesions and mortality. Vaccination is used worldwide to control MD, but increasingly virulent field strains can overcome this protection, d...

  16. Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the genes in the FUN38-MAK16-SPO7 region.

    OpenAIRE

    Barton, A B; Kaback, D B

    1994-01-01

    Transcribed regions on a 42-kb segment of chromosome I from Saccharomyces cerevisiae were mapped. Polyadenylated transcripts corresponding to eight previously characterized genes (MAK16, LTE1, CCR4, FUN30, FUN31, TPD3, DEP1, and CYS3) and eight new genes were identified. All transcripts were present at one to four copies per cell except for one which was significantly less abundant. This region has been sequenced, and the sizes, locations, and orientations of the transcripts were in nearly pe...

  17. Diversity of Staphylococcal Cassette Chromosome mec Elements in Predominant Methicillin-Resistant Staphylococcus aureus Clones in a Small Geographic Area ▿

    OpenAIRE

    Basset, Patrick; Senn, Laurence; Vogel, Valérie; Zanetti, Giorgio; Blanc, Dominique S.

    2010-01-01

    Recent population genetic studies suggest that staphylococcal cassette chromosome mec (SCCmec) was acquired much more frequently than previously thought. In the present study, we aimed to investigate the diversity of SCCmec elements in a local methicillin-resistant Staphylococcus aureus (MRSA) population. Each MRSA isolate (one per patient) recovered in the Vaud canton of Switzerland from January 2005 to December 2008 was analyzed by the double-locus sequence typing (DLST) method and SCCmec t...

  18. Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretin receptor superfamily with an unusual extracellular domain

    Energy Technology Data Exchange (ETDEWEB)

    Hamann, J. [Univ. of Amsterdam (Netherlands)]|[Max Planck Society, Berlin-Buch (Germany); Hamann, D.; Lier, R.A.W. [Univ. of Amsterdam (Netherlands)] [and others

    1995-08-15

    CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).

  19. Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis

    Directory of Open Access Journals (Sweden)

    Jinke Lin

    2011-01-01

    Full Text Available We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%, followed by DNA retrotransposons (11.16%–11.69%. Additionally, we found 25 simple sequence repeats (SSRs that could potentially be used as genetic markers.

  20. Cloning of a conserved receptor-like protein kinase gene and its use as a functional marker for homoeologous group-2 chromosomes of the triticeae species.

    Directory of Open Access Journals (Sweden)

    Bi Qin

    Full Text Available Receptor-like kinases (RLKs play broad biological roles in plants. We report on a conserved receptor-like protein kinase (RPK gene from wheat and other Triticeae species. The TaRPK1 was isolated from the Triticum aestivum cv. Prins - Triticum timopheevii introgression line IGVI-465 carrying the powdery mildew resistance gene Pm6. The TaRPK1 was mapped to homoeologous chromosomes 2A (TaRPK1-2A, 2D (TaRPK1-2D and the Pm6-carrier chromosome 2G (TaRPK1-2G of IGVI-465. Under the tested conditions, only the TaRPK1-2G allele was actively transcribed, producing two distinct transcripts via alternative splicing. The predicted 424-amino acid protein of TaRPK1-2G contained a signal peptide, a transmembrane domain and an intracellular serine/threonine kinase domain, but lacked a typical extracellular domain. The expression of TaRPK1-2G gene was up-regulated upon the infection by Blumeria graminis f.sp. tritici (Bgt and treatment with methyl jasmonate (MeJA, but down-regulated in response to treatments of SA and ABA. Over-expression of TaRPK1-2G in the powdery mildew susceptible wheat variety Prins by a transient expression assay showed that it slightly reduced the haustorium index of the infected Bgt. These data indicated that TaRPK1-2G participated in the defense response to Bgt infection and in the JA signaling pathway. Phylogenetic analysis indicated that TaRPK1-2G was highly conserved among plant species, and the amino acid sequence similarity of TaRPK1-2G among grass species was more than 86%. Based on its conservation, the RPK gene-based STS primers were designed, and used to amplify the RPK orthologs from the homoeologous group-2 chromosomes of all the tested Triticeae species, such as chromosome 2G of T. timopheevii, 2R of Secale cereale, 2H of Hordeum vulgare, 2S of Aegilops speltoides, 2S(l of Ae. longissima, 2M(g of Ae. geniculata, 2S(p and 2U(p of Ae. peregrina. The developed STS markers serve as conserved functional markers for the

  1. Mitochondrial HMG to CoA synthase (mHS): cDNA cloning in human, mouse and C. elegans, mapping to human chromosome 1p12-13 and partial human genomic cloning

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal, Quebec (Canada)]|[Kingston General Hospital, Ontario (Canada)] [and others

    1994-09-01

    mHS catalyzes the rate-limiting first step of ketogenesis in the liver. A cytoplasmic HS isozyme, encoded by another gene, catalyzes an early step in cholesterol synthesis. Starting from a rat mHS cDNA obtained by RT-PCR from the published rat cDNA sequence, we obtained and sequenced human and mouse cDNAs spanning the entire coding sequence of natural human and mouse mHS, as well as sequencing C. elegans HS-like cDNA. Consensus sequences for 3 mitochondrial and 4 cytoplasmic HSs were created and compared to invertebrate HS sequences. We found high conversation in the active site and at other regions presumably important for HS function. We mapped the mHS locus, HMGCS2 by in situ hybridization to chromosome 1P12-13, in contrast to the human cHS locus (HMGCS1) known to be on chromosome 5p13. Comparative mapping results suggest that these two chromosomal regions may be contiguous in other species, constant with a recent gene duplication event. Furthermore, we have characterized a human genomic mHS subclone containing 4 mHS exons, and found the position of all splice junctions to be identical to that of the hamster cHS gene except for one site in the 3{prime} nontranslated region. We calculate that the mHS and cHS genes were derived from a common ancestor 400-700 Myrs ago, implying that ketogenesis from fat may have become possible around the time of emergence of vertebrates ({approximately}500 Myr ago). Ketogenesis has evolved into an important pathway of energy metabolism, and we predict the mHS deficiency may prove to be responsible for some as yet explained cases of Reye-like syndromes in humans. This hypothesis can now be tested at the molecular level without the necessity of obtaining hepatic tissue.

  2. Toward a Molecular Cytogenetic Map for Cultivated Sunflower (Helianthus annuus L.) by Landed BAC/BIBAC Clones

    Science.gov (United States)

    Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

    2013-01-01

    Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (∼100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC- fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)−derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources. PMID:23316437

  3. Artificial synthesis and cloning of human obese gene%人肥胖(obese)基因的人工合成及克隆

    Institute of Scientific and Technical Information of China (English)

    杨林; 吴无畏

    2000-01-01

    根据人肥胖基因的cDNA序列,通过合理的引物设计、链延伸反应、PCR反应以及分子克隆等步骤,成功地合成出编码瘦蛋白(Leptin)的肥胖基因(ob基因)全长片段,并将其克隆至pUC18载体质粒上.序列分析和酶切鉴定显示肥胖基因得到了正确合成和克隆.%According to the known sequence of human ob gene cDNA,the gene coding the leptin was synthesized and cloned into pUC18 vector after reasonable primer designing,DNA strain extending,PCR reaction and molecular cloning.Sequence analysis and RE digest results showed the ob gene was synthesized and cloned correctly.

  4. Why Clone?

    Science.gov (United States)

    ... How might cloning be used in medicine? Cloning animal models of disease Much of what researchers learn ... the first place, such as habitat destruction and hunting. But cloning may be one more tool that ...

  5. An integrated linkage, chromosome, and genome map for the yellow fever mosquito Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Vladimir A Timoshevskiy

    Full Text Available BACKGROUND: Aedes aegypti, the yellow fever mosquito, is an efficient vector of arboviruses and a convenient model system for laboratory research. Extensive linkage mapping of morphological and molecular markers localized a number of quantitative trait loci (QTLs related to the mosquito's ability to transmit various pathogens. However, linking the QTLs to Ae. aegypti chromosomes and genomic sequences has been challenging because of the poor quality of polytene chromosomes and the highly fragmented genome assembly for this species. METHODOLOGY/PRINCIPAL FINDINGS: Based on the approach developed in our previous study, we constructed idiograms for mitotic chromosomes of Ae. aegypti based on their banding patterns at early metaphase. These idiograms represent the first cytogenetic map developed for mitotic chromosomes of Ae. aegypti. One hundred bacterial artificial chromosome clones carrying major genetic markers were hybridized to the chromosomes using fluorescent in situ hybridization. As a result, QTLs related to the transmission of the filarioid nematode Brugia malayi, the avian malaria parasite Plasmodium gallinaceum, and the dengue virus, as well as sex determination locus and 183 Mbp of genomic sequences were anchored to the exact positions on Ae. aegypti chromosomes. A linear regression analysis demonstrated a good correlation between positions of the markers on the physical and linkage maps. As a result of the recombination rate variation along the chromosomes, 12 QTLs on the linkage map were combined into five major clusters of QTLs on the chromosome map. CONCLUSION: This study developed an integrated linkage, chromosome, and genome map-iMap-for the yellow fever mosquito. Our discovery of the localization of multiple QTLs in a few major chromosome clusters suggests a possibility that the transmission of various pathogens is controlled by the same genomic loci. Thus, the iMap will facilitate the identification of genomic determinants of

  6. DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Kwan, Johnson; Baumgartner, Adolf; Weier, Jingly F.; Wang, Mei; Escudero, Tomas; Munne' , Santiago; Zitzelsberger, Horst F.; Weier, Heinz-Ulrich

    2009-01-30

    Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome (BAC) clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multi-clone and multi-color mapping experiments do not generate additional information. Our pooling protocol described here with examples from thyroid cancer research and PGD accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as three to four days.

  7. Chromosome region-specific libraries for human genome analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kao, Fa-Ten.

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

  8. Construction of a bacterial artificial chromosome (BAC) library of Lycopersicon esculentum cv. Stevens and its application to physically map the Sw-5 locus

    NARCIS (Netherlands)

    Spassova, MI; Prins, M; Stevens, MR; Hille, J; Goldbach, RW; Spassova, Mariana I.; Stevens, Mikel R.; Goldbach, Rob W.

    1999-01-01

    The Sw-5 gene is a dominantly inherited resistance gene in tomato and functional against a number of tospovirus species. The gene has been mapped on chromosome 9, tightly linked to RFLP markers CT220 and SCAR421. To analyse the Sw-5 locus, a BAC genomic library was constructed of tomato cv. Stevens,

  9. Identification of a yeast artificial chromosome that spans the human papillary renal cell carcinoma-associated t(X;1) breakpoint in Xp11.2

    NARCIS (Netherlands)

    Suijkerbuijk, R F; Meloni, A M; Sinke, R J; de Leeuw, B; Wilbrink, M; Janssen, H A; Geraghty, M T; Monaco, A P; Sandberg, A A; Geurts van Kessel, A

    1993-01-01

    Recently, a specific chromosome abnormality, t(X;1)(p11;q21), was described for a subgroup of human papillary renal cell carcinomas. The translocation breakpoint in Xp11 is located in the same region as that in t(X;18)(p11;q11)-positive synovial sarcoma. We used fluorescence in situ hybridization (F

  10. Genetic and physical mapping of the bovine X chromosome

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Chen Chen; Taylor, J.F.; Sanders, J. O. [Texas A& M Univ., College Station, TX (United States)] [and others

    1996-03-01

    Three hundred eighty reciprocal backcross and F{sub 2} full sib progeny from 33 families produced by embryo transfer from 77 Angus (Bos taurus), Brahman (Bos indicus), and F{sub 1} parents and grandparents were used to construct genetic maps of the bovine X and Y chromosomes. All individuals were scored for 15 microsatellite loci, with an average of 608 informative meioses per locus. The length of the bovine X chromosome genetic map was 118.7 cM (female only) and of the pseudoautosomal region was 13.0 cM (male only). The 15-marker framework map in Kosambi centimorgans is (BM6017-6.1-TGLA89-35.8-TEXAN13-3.4-TGLA128-1.3-BM2713-21.1-BM4604-2.4-BR215-12.9-TGLA68-10.0-BM4321-1.0-HEL14-4.9-TGLA15-2.3-INRA120-12.5-TGLA325-1.6-MAF45-3.2-INRA30), with an average interval of 7.91 cM. Clones containing pseudoautosomal or sex-linked microsatellites were isolated from a bovine bacterial artificial chromosome library and were physically mapped to bovine metaphase chromosomes by fluorescence in situ hybridization to orient the X and Y chromosome maps. BAC57, containing the pseudoautosomal microsatellite INRA30, mapped to the distal end of the long arm of the X chromosome at q42-ter and to the short arm of the Y chromosome at p13-ter. This confirms the published assignment of this region to Yp12-ter, but challenges the published assignment of Xp14-ter and thus reorients the X chromosome physical map. BAC204, containing the X-linked microsatellite BM4604, mapped to the middle of the long arm of the X chromosome at q26-q31. The position of the physically mapped to the middle of the long arm of the X chromosome at q26-q31. The position of the physically mapped markers indicates either a lack of microsatellite markers for a large (30 to 50 cM) region of the short arm of the X chromosome or heterogeneity of recombination along the X chromosome. 46 refs., 2 figs., 3 tabs.

  11. Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

    Science.gov (United States)

    Mollereau, C; Simons, M J; Soularue, P; Liners, F; Vassart, G; Meunier, J C; Parmentier, M

    1996-08-01

    Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

  12. Cloning, expression patterns, and chromosome localization of three human and two mouse homologues of GABA(A) receptor-associated protein.

    Science.gov (United States)

    Xin, Y; Yu, L; Chen, Z; Zheng, L; Fu, Q; Jiang, J; Zhang, P; Gong, R; Zhao, S

    2001-06-15

    Type A receptors of gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, contain alpha, beta, delta, gamma, and rho subunits. The gamma subunit has four subtypes: gamma1, gamma2, gamma3, andgamma4. GABA(A) receptor-associated protein (GABARAP) was previously demonstrated to act as a linker protein between microtubules and the gamma2 subunit of GABA(A) receptors. However, no other linker proteins have been identified as mediating the linkage of microtubules and the remaining subunits of GABA(A) receptors. In this study we identified three human paralogues (GABARAPL1, GABARAPL2, and GABARAPL3) and two mouse orthologues (Gabarapl1 and Gabarapl2) of human GABARAP, all of which encoded 117 amino acids, as does Gabarapl. The expression patterns of GABARAPL1, GABARAPL2, and GABARAP in 16 adult tissues showed that they were expressed ubiquitously. The expression levels of GABARAPL1 as a 2.3-kb transcript were very high in brain, heart, peripheral blood leukocytes, liver, kidney, placenta, and skeletal muscle, very low in thymus and small intestine, and moderate in other tissues tested. The unique 1.35-kb transcript of GABARAPL2 was expressed at high levels in heart, brain, testis, prostate, ovary, spleen, and skeletal muscle, at very low levels in lung, thymus, and small intestine, and moderately in other tissues tested. For GABARAP, a 1.3-kb transcript was abundantly expressed in all tested tissues with small variation. The expression patterns of Gabarapl1 and Gabarapl2 were similar to those of their counterparts in human. In addition, GABARAPL1 was localized to human chromosome 12p12.3 and GABARAPL2 to 16q22.3-q24.1 by RH mapping, while GABARAP and GABARAPL3 were found to be localized at chromosomes 17p13.2 and 15q25.1, respectively, by searching the related databases. Sequence comparison of the cDNAs and their corresponding genomic sequences shows that GABARAP, GABARAPL1, and GABARAPL2 are composed of four exons each, while GABARAPL3 is distributed only at

  13. Estimation of long-terminal repeat element content in the Helicoverpa zea genome from next generation sequencing of reduced representation bacterial artificial chromosome (BAC) pools

    Science.gov (United States)

    The lepidopteran pest insect, Helicoverpa zea, feeds on cultivated corn and cotton crops in North America where control remains challenging due to evolution of resistance to chemical and transgenic insecticidal toxins, yet few genomic resources are available for this species. A bacterial artificial...

  14. Cloning of Rabbit HPRT Gene Using the Recombineering System

    Institute of Scientific and Technical Information of China (English)

    Jianjun SHI; Donghui CAI; Xuejin CHEN; Huizheng SHENG

    2007-01-01

    Hypoxanthine phosphoribosyltransferase (HPRT) plays an important role in the metabolic salvage of purines, and been used as an alternative pathway for mutant selection in many studies. To facilitate its application in rabbits, we have cloned the cDNA and genomic DNA of the rabbit HPRT gene using an approach that combines bioinformatics and recombineering methods. The cDNA is comprised of 1449 bp containing a coding sequence for a protein of 218 amino acids. The deduced amino acid sequence of the rabbit HPRT gene shares 98%, 97%, 98% and 94% identity with human, mouse, pig and cattle HPRT genes, respectively. Reverse transcription-polymerase chain reaction analysis showed that this gene is ubiquitously expressed in tissues of adult rabbit. The rabbit HPRT gene spans approximately 48 kb in length and consists of nine exons. The cloning of the rabbit HPRT gene shows the usefulness of the recombineering system in cloning genes of large size. This system may facilitate the subcloning of DNA from bacterial artificial chromosomes for cloning genes of large size or filling big gaps in genomic sequencing.

  15. Constructing chromosome- and region-specific cosmid maps of the human genome.

    Science.gov (United States)

    Carrano, A V; de Jong, P J; Branscomb, E; Slezak, T; Watkins, B W

    1989-01-01

    A chromosome-specific ordered set of cosmids would be a significant contribution toward understanding human chromosome structure and function. We are developing two parallel approaches for creating an ordered cosmid library of human chromosome 19 and other selected subregions of the human genome. The "bottom up" approach is used to establish sets of overlapping cosmids as islands or "contigs" along the chromosome, while the "top down" approach, using pulsed-field gel electrophoresis and yeast cloning, will establish a large-fragment map and close the inevitable gaps remaining from the "bottom up" approach. Source DNA consists of a single homolog of chromosome 19 from a hamster--human hybrid cell and human fragments cloned in yeast artificial chromosomes. We have constructed cosmid libraries in a vector that facilitates cloning small amounts of DNA, allows transcription of the insert termini, and contains unique sites for partial-digest mapping. Computer simulations of cosmid contig building suggest that near-optimal efficiency can be achieved with high-density restriction fragment digest schemes that can detect 20-30% overlap between cosmids. We developed the chemistry and data analysis tools to compare the ordering efficiencies of several cosmid restriction digest fingerprinting strategies. Restriction fragments from a four-cutter digest are labeled with a fluorochrome, separated by polyacrylamide gel electrophoresis, and detected after laser excitation as they traverse a fixed point in the gel. We have also developed the software to rapidly process the output signal to define and analyze the fragment peaks. Up to three cosmids (or three different digests of the same cosmid) plus a size standard are analyzed simultaneously in a single gel lane.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2698823

  16. Molecular cloning of the interleukin-1 gene cluster: Construction of an integrated YAC/PAC contig and a partial transcriptional map in the region of chromosome 2q13

    Energy Technology Data Exchange (ETDEWEB)

    Nothwang, H.G.; Strahm, B.; Denich, D. [Freiburg Univ. (Germany)] [and others

    1997-05-01

    Genes of the interleukin-1 (IL-1) gene cluster localized on chromosome 2q13 are implicated in many physiological and pathophysiological processes. We present here a high-resolution physical map of this region between markers D2S2008 and D2S43/PAX8. An integrated YAC/PAC contig and a partial transcriptional map were constructed by STS-content mapping using the CEPH YAC library and three PAC libraries. A total of 3 YACs, 34 PACs, and 56 STSs were integrated: 33 newly generated probes to PAC end sequences, 9 polymorphic and 4 nonpolymorphic markers, 5 known genes, 4 expressed sequence tags, and 1 pseudogene. Within the map, a complete PAC contig of > 1 Mb encompasses the IL-1 gene cluster and PAX8, a paired-box-containing gene. This allowed us to define the transcriptional orientation of GLVR1, IL1B, and IL1RN and to show that PAX8 is localized outside the IL-1 gene cluster. FISH analysis localized PAC clones containing the IL-1 gene cluster to 2q12-q13. The data provide the basis for further characterization of the IL-1 gene cluster and for the construction of a sequence-ready PAC contig of this region. 45 refs., 2 figs., 2 tabs.

  17. Genetic and physical mapping of the bovine X chromosome.

    Science.gov (United States)

    Yeh, C C; Taylor, J F; Gallagher, D S; Sanders, J O; Turner, J W; Davis, S K

    1996-03-01

    Three hundred eighty reciprocal backcross and F(2) full sib progeny from 33 families produced by embryo transfer from 77 Angus (Bos taurus), Brahman (Bos indicus), and F1 parents and grandparents were used to construct genetic maps of the bovine X and Y chromosomes. Ml individuals were scored for 15 microsatellite loci, with an average of 608 informative meioses per locus. The length of the bovine X chromosome genetic map was 118.7 cM (female only) and of the pseudoautosomal region was 13.0 cM (male only). The 15-marker framework map in Kosambi centimorgans is [BM6017-6.1 -TGLA89-35.8-TEXAN13-3.4-TGLA128-1.3 -BM2713 -21.1 -BM4604-2.4-BR215 - 12.9-TGLA68-10.0-BM4321 - 1.0-HEL14-4.9-TGLA15-2.3-INRA12O- 12.5-TGLA325- 1.6-MAF45-3.2-INRA3O], with an average interval of 7.91 cM. Clones containing pseudoautosomal or sex-linked microsatellites were isolated from a bovine bacterial artificial chromosome library and were physically mapped to bovine metaphase chromosomes by fluorescence in situ hybridization to orient the X and Y chromosome maps. BAC57, containing the pseudoautosomal microsatellite INRA3O, mapped to the distal end of the long arm of the X chromosome at q42-ter and to the short arm of the Y chromosome at p13-ter. This confirms the published assignment of this region to Ypl2-ter, but challenges the published assignment of Xpl4-ter and thus reorients the X chromosome physical map. BAC2O4, containing the X-linked microsatellite BM4604, mapped to the middle of the long arm of the X chromosome at q26-q31. The position of the physically mapped markers indicates either a lack of microsatellite markers for a large (30 to 50 cM) region of the short arm of the X chromosome or heterogeneity of recombination along the X chromosome. PMID:8833151

  18. Reproductive cloning : can cloning harm the clone?

    OpenAIRE

    Pattinson, S.D.

    2002-01-01

    Since the creation of Dolly the sheep was reported in February 1997, the possibility of a cloned child has elicited powerful declarations of condemnation. A widely held view is that cloning a human being would be immoral and ought to be prohibited by legislation. This paper outlines the regulatory approaches taken in the EU countries (with particular reference to the UK), Canada and the US, before examining the claim that creating a clone would be a wrong to the resultant clone. It is argued ...

  19. Construction of a BAC library and mapping BAC clones to the linkage map of Barramundi, Lates calcarifer

    Directory of Open Access Journals (Sweden)

    Lin Grace

    2008-03-01

    Full Text Available Abstract Background Barramundi (Lates calcarifer is an important farmed marine food fish species. Its first generation linkage map has been applied to map QTL for growth traits. To identify genes located in QTL responsible for specific traits, genomic large insert libraries are of crucial importance. We reported herein a bacterial artificial chromosome (BAC library and the mapping of BAC clones to the linkage map. Results This BAC library consisted of 49,152 clones with an average insert size of 98 kb, representing 6.9-fold haploid genome coverage. Screening the library with 24 microsatellites and 15 ESTs/genes demonstrated that the library had good genome coverage. In addition, 62 novel microsatellites each isolated from 62 BAC clones were mapped onto the first generation linkage map. A total of 86 BAC clones were anchored on the linkage map with at least one BAC clone on each linkage group. Conclusion We have constructed the first BAC library for L. calcarifer and mapped 86 BAC clones to the first generation linkage map. This BAC library and the improved linkage map with 302 DNA markers not only supply an indispensable tool to the integration of physical and linkage maps, the fine mapping of QTL and map based cloning genes located in QTL of commercial importance, but also contribute to comparative genomic studies and eventually whole genome sequencing.

  20. Bacteriophage P22 transduction of integrated plasmids: single-step cloning of Salmonella typhimurium gene fusions.

    OpenAIRE

    Mahan, M. J.; Slauch, J M; Mekalanos, J.J. (John J.)

    1993-01-01

    Transcriptional fusions to Salmonella typhimurium chromosomal genes were constructed by integration of a suicide fusion vector into the chromosome by homologous recombination with random cloned chromosomal fragments. We describe here a transductional method using the generalized transducing phage of S. typhimurium, P22, to clone these fusions directly from the bacterial chromosome, in a single step, without the use of restriction enzymes. In this transduction, the phage packages the chromosom...

  1. [Chromosomal organization of the genomes of small-chromosome plants].

    Science.gov (United States)

    Muravenko, O V; Zelenin, A V

    2009-11-01

    An effective approach to study the chromosome organization in genomes of plants with small chromosomes and/or with low-informative C-banding patterns was developed in the course of investigation of the karyotypes of cotton plant, camomile, flax, and pea. To increase the resolving power of chromosome analysis, methods were worked out for revealing early replication patterns on chromosomes and for artificial impairment of mitotic chromosome condensation with the use of a DNA intercalator, 9-aminoacridine (9-AMA). To estimate polymorphism of the patterns of C-banding of small chromosomes on preparations obtained with the use of 9-AMA, it is necessary to choose a length interval that must not exceed three average sizes of metaphase chromosomes without the intercalator. The use of 9-AMA increases the resolution of differential C- and OR-banding and the precision of physical chromosome mapping by the FISH method. Of particular importance in studying small chromosomes is optimization of the computer-aided methods used to obtain and process chromosome images. The complex approach developed for analysis of the chromosome organization in plant genomes was used to study the karyotypes of 24 species of the genus Linum L. It permitted their chromosomes to be identified for the first time, and, in addition, B chromosomes were discovered and studied in the karyotypes of the species of the section Syllinum. By similarity of the karyotypes, the studied flax species were distributed in eight groups in agreement with the clusterization of these species according to the results of RAPD analysis performed in parallel. Systematic positions and phylogenetic relationships of the studied flax species were verified. Out results can serve as an important argument in favour of the proposal to develop a special program for sequencing the genome of cultivated flax (L. usitatissimum L.), which is a major representative of small-chromosome species. PMID:20058798

  2. EasyClone-MarkerFree

    DEFF Research Database (Denmark)

    Fabre, Mathew Malcolm Jessop; Jakociunas, Tadas; Stovicek, Vratislav;

    2016-01-01

    Clone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained...

  3. A BAC-based physical map of the Hessian fly genome anchored to polytene chromosomes

    Directory of Open Access Journals (Sweden)

    Fellers John P

    2009-07-01

    Full Text Available Abstract Background The Hessian fly (Mayetiola destructor is an important insect pest of wheat. It has tractable genetics, polytene chromosomes, and a small genome (158 Mb. Investigation of the Hessian fly presents excellent opportunities to study plant-insect interactions and the molecular mechanisms underlying genome imprinting and chromosome elimination. A physical map is needed to improve the ability to perform both positional cloning and comparative genomic analyses with the fully sequenced genomes of other dipteran species. Results An FPC-based genome wide physical map of the Hessian fly was constructed and anchored to the insect's polytene chromosomes. Bacterial artificial chromosome (BAC clones corresponding to 12-fold coverage of the Hessian fly genome were fingerprinted, using high information content fingerprinting (HIFC methodology, and end-sequenced. Fluorescence in situ hybridization (FISH co-localized two BAC clones from each of the 196 longest contigs on the polytene chromosomes. An additional 70 contigs were positioned using a single FISH probe. The 266 FISH mapped contigs were evenly distributed and covered 60% of the genome (95,668 kb. The ends of the fingerprinted BACs were then sequenced to develop the capacity to create sequenced tagged site (STS markers on the BACs in the map. Only 3.64% of the BAC-end sequence was composed of transposable elements, helicases, ribosomal repeats, simple sequence repeats, and sequences of low complexity. A relatively large fraction (14.27% of the BES was comprised of multi-copy gene sequences. Nearly 1% of the end sequence was composed of simple sequence repeats (SSRs. Conclusion This physical map provides the foundation for high-resolution genetic mapping, map-based cloning, and assembly of complete genome sequencing data. The results indicate that restriction fragment length heterogeneity in BAC libraries used to construct physical maps lower the length and the depth of the contigs, but is

  4. Radiation-induced chromosomal instability

    Energy Technology Data Exchange (ETDEWEB)

    Ritter, S. [GSI, Biophysics, Darmstadt (Germany)

    1999-03-01

    Recent studies on radiation-induced chromosomal instability in the progeny of exposed mammalian cells were briefly described as well as other related studies. For the analysis of chromosomal damage in clones, cells were seeded directly after exposure in cell well-dish to form single cell clones and post-irradiation chromosome aberrations were scored. Both exposure to isoeffective doses of X-ray or 270 MeV/u C-ions (13 keV/{mu}m) increased the number of clones with abnormal karyotype and the increase was similar for X-ray and for C-ions. Meanwhile, in the progeny of cells for mass cultures, there was no indication of a delayed expression of chromosomal damage up to 40 population doublings after the exposure. A high number of aberrant cells were only observed directly after exposure to 10.7 MeV/u O-ions, i.e. in the first cycle cells and decreased with subsequent cell divisions. The reason for these differences in the radiation-induced chromosomal instability between clonal isolates and mass culture has not been clarified. Recent studies indicated that genomic instability occurs at a high frequency in the progeny of cells irradiated with both sparsely and densely ionizing radiation. Such genomic instability is thought likely to increase the risk of carcinogenesis, but more data are required for a well understanding of the health risks resulting from radiation-induced delayed instability. (M.N.)

  5. A BAC clone of MDV strain GX0101 with REV-LTR integration retained its pathogenicity

    Institute of Scientific and Technical Information of China (English)

    SUN AiJun; LAWRENCE Petherbridge; ZHAO YuGuang; LI YanPeng; NAIR Venugopal K; CUI ZhiZhong

    2009-01-01

    The complete genome of Marek's disease virus (MDV) strain GX0101,which was integrated with the LTR sequences of REV,was cloned in Escherichia coli as a bacterial artificial chromosome (BAC).BAC vector sequences were introduced into the US2 locus of the MDV genome by homologous recombination.The viral DNA containing the BAC vector was used to transform Escherichia coli strain of DH10B.Then the recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast (CEF).This BAC viral clone was named bac-GX0101.When the reconstituted virus was inoculated into 1-day-old birds,visceral tumors could be detected as early as 62 d post infection.There was no difference in growth ability and pathogenicity to birds between the BAC derived virus and its parental virus.The BAC derived virus maintained its oncogenicity and immunosuppressive effects.In conclusion,the complete genome of GX0101 strain was successfully cloned into BAC and the infectious clone was rescued.With the powerful BAC manipulation system,the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV.

  6. A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo and chicken (Gallus gallus genomes

    Directory of Open Access Journals (Sweden)

    Delany Mary E

    2011-09-01

    Full Text Available Abstract Background A robust bacterial artificial chromosome (BAC-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. Results The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. Conclusion The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and

  7. Construction of the Primary Physical Map of Rice Chromosome 12

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A primary physical map of rice chromosome 12 was constructed using marker-based chromosome landing and chromosome walking. A BAC library from IR64 was screened using 84 RFLP markers, 4 STS markers and 6 microsatellite markers on chromosome 12 by colony hybridization and polymerase chain reaction (PCR) amplification. A total of 59 contigs consisting of 419 BAC clones including 5 single-clones were physically aligned on rice chromosome 12 with the largest BAC contig covering 855 kb. The whole physical map had a size of ~16 Mb and covered about 52% of rice chromosome 12. This physical map will be certainly helpful for map-based gene cloning of agronomically and biological important genes and understanding the genome structure of the chromosome.

  8. Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

    Science.gov (United States)

    Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

    2015-12-01

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.

  9. Walking, cloning, and mapping with YACs in 3q27: Localization of five ESTs including three members of the cystatin gene family and identification of CpG islands

    Energy Technology Data Exchange (ETDEWEB)

    James, L.A.; Ogilvie, D.J.; Anand, R. [Zeneca Pharmaceuticals, Cheshire (United Kingdom)] [and others

    1996-03-05

    Using yeast artificial chromosomes, we have generated a high-resolution physical map for 2.7 Mb of human chromosomal region 3q27. The YAC clones group into three contigs, one of which has also been linked to the CEPH YAC contig map of human chromosome 3. Fluorescence in situ hybridization has been used to order the contigs on the chromosome and to estimate the distance between them. Expressed sequence tags for five genes, including three members of the cystatin gene family and a gene thought to be involved in B-cell non-Hodgkin lymphoma, have been placed within the YAC contigs, and 12 putative CpG islands have been identified. These YACs provide a useful resource to complete the physical mapping of 3q27 and to begin identification and characterization of further genes that are located there. 27 refs., 1 fig., 1 tab.

  10. Construction and characterization of two Citrus BAC libraries and identification of clones containing the phytoene synthase gene.

    Science.gov (United States)

    Baig, M N R; Yu, An; Guo, Wenwu; Deng, Xiuxin

    2009-05-01

    Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

  11. Optical mapping of a rice B AC clone using restriction endonuclease and imaging with fluorescent microscopy at single molecule level

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A method of constructing restriction map by optical mapping and single molecule fluorescent microscopy is described. DNA molecules were aligned and adsorbed on a glass coverslip surface by a mbdified "molecular combing"technique, and then the surface-immobilized DNAs were cleaved in situ with a restriction endonuclease. Individual DNA molecules digested by the endonuclease EcoR I were observable with fluorescent microscopy. Using optical mapping, a physical map of a rice bacterial artificial chromosome clone was constructed. This method will facilitate genomic mapping and tracing the dynamic process in real time at a single molecule level with fluorescence microscopy.

  12. The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

    Science.gov (United States)

    Miner, J H; Patton, B L; Lentz, S I; Gilbert, D J; Snider, W D; Jenkins, N A; Copeland, N G; Sanes, J R

    1997-05-01

    Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously

  13. High-resolution mapping, cloning and molecular characterization of the Pi-k ( h ) gene of rice, which confers resistance to Magnaporthe grisea.

    Science.gov (United States)

    Sharma, T R; Madhav, M S; Singh, B K; Shanker, P; Jana, T K; Dalal, V; Pandit, A; Singh, A; Gaikwad, K; Upreti, H C; Singh, N K

    2005-12-01

    In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ( h )) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F(2) individuals, we mapped the Pi-k ( h ) gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ( h ) gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ( h ) gene revealed that its expression is pathogen inducible. PMID:16228246

  14. A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

    OpenAIRE

    Noskov, Vladimir; Kouprina, Natalay; Leem, Sun-Hee; Koriabine, Maxim; Barrett, J. Carl; Larionov, Vladimir

    2002-01-01

    Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ∼0.5%; the positive clones are identifi...

  15. Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

    Science.gov (United States)

    Kamboj, Aman; Saini, Mohini; Rajan, Lekshmi S; Patel, Chhabi Lal; Chaturvedi, V K; Gupta, Praveen K

    2015-12-15

    To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector. PMID:26478540

  16. Consensus maps of cloned plant cuticle genes

    Institute of Scientific and Technical Information of China (English)

    Eviatar; Nevo

    2010-01-01

    Plant cuticle,which covers the plant surface,consists of waxes and cutins,and is associated with plant drought,cold,and salt resistance.Hitherto,at least 47 genes participating in the formation of plant cuticle have been cloned from Arabidopsis thaliana,Oryza sativa,Zea mays,Ricinus communis,Brassica napus,and Medicago truncatula;and about 85% of them encode proteins sharing above 50% identities with their rice homologous sequences.These cloned cuticle genes were mapped in silico on different chromosomes of rice and Arabidopsis,respectively.The mapping results revealed that plant cuticle genes were not evenly distributed in both genomes.About 40% of the mapped cuticle genes were located on chromosome 1 in Arabidopsis,while 20% of the mapped cuticle genes were located on chromosome 2 but none on chromosome 12 in rice.Some cloned plant cuticle genes have several rice homologous sequences,which might be produced by chromosomal segment duplication.The consensus map of cloned plant cuticle genes will provide important clues for the selection of candidate genes in a positional cloning of an unknown cuticle gene in plants.

  17. Clones above the unary clone

    OpenAIRE

    Goldstern, Martin; Sági, Gábor; Shelah, Saharon

    2011-01-01

    Let c be the cardinality of the continuum. We give a family of pairwise incomparable clones (on a countable base set) 2^c members, all with the same unary fragment, namely the set of all unary operations. We also give, for each n, a family of 2^c clones all with the same n-ary fragment, and all containing the set of all unary operations.

  18. Physical Mapping and Refinement of the Painted Turtle Genome (Chrysemys picta) Inform Amniote Genome Evolution and Challenge Turtle-Bird Chromosomal Conservation.

    Science.gov (United States)

    Badenhorst, Daleen; Hillier, LaDeana W; Literman, Robert; Montiel, Eugenia Elisabet; Radhakrishnan, Srihari; Shen, Yingjia; Minx, Patrick; Janes, Daniel E; Warren, Wesley C; Edwards, Scott V; Valenzuela, Nicole

    2015-06-24

    Comparative genomics continues illuminating amniote genome evolution, but for many lineages our understanding remains incomplete. Here, we refine the assembly (CPI 3.0.3 NCBI AHGY00000000.2) and develop a cytogenetic map of the painted turtle (Chrysemys picta-CPI) genome, the first in turtles and in vertebrates with temperature-dependent sex determination. A comparison of turtle genomes with those of chicken, selected nonavian reptiles, and human revealed shared and novel genomic features, such as numerous chromosomal rearrangements. The largest conserved syntenic blocks between birds and turtles exist in four macrochromosomes, whereas rearrangements were evident in these and other chromosomes, disproving that turtles and birds retain fully conserved macrochromosomes for greater than 300 Myr. C-banding revealed large heterochromatic blocks in the centromeric region of only few chromosomes. The nucleolar-organizing region (NOR) mapped to a single CPI microchromosome, whereas in some turtles and lizards the NOR maps to nonhomologous sex-chromosomes, thus revealing independent translocations of the NOR in various reptilian lineages. There was no evidence for recent chromosomal fusions as interstitial telomeric-DNA was absent. Some repeat elements (CR1-like, Gypsy) were enriched in the centromeres of five chromosomes, whereas others were widespread in the CPI genome. Bacterial artificial chromosome (BAC) clones were hybridized to 18 of the 25 CPI chromosomes and anchored to a G-banded ideogram. Several CPI sex-determining genes mapped to five chromosomes, and homology was detected between yet other CPI autosomes and the globally nonhomologous sex chromosomes of chicken, other turtles, and squamates, underscoring the independent evolution of vertebrate sex-determining mechanisms.

  19. Utilization of Super BAC Pools and Fluidigm Access Array Platform for High-Throughput BAC Clone Identification: Proof of Concept

    Directory of Open Access Journals (Sweden)

    Peter J. Maughan

    2012-01-01

    Full Text Available Bacterial artificial chromosome (BAC libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs from an Amplicon Express seven-plate super pooled Amaranthus hypochondriacus BAC library. Ninety-six SNP loci, spanning the length of A. hypochondriacus linkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC. Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of ~$0.05 per reaction.

  20. Fertile Garlic Clones in the Gatersleben Collection

    OpenAIRE

    ETOU, Takeomi; Keller, E. R. Joachim; SENULA, Angelika

    2001-01-01

    In the Gatersleben collection, fertile garlic accessions were surveyed. It was clarified that in total, 15 accessions were pollen fertile, and that 14 of them came from Central Asia, Caucasus or Russia. These accessions are useful for garlic breeding. Meiosis was examined in five accessions. All of them showed regular chromosome pairing at meiosis, eight bivalent chromosomes. Two of the five accessions came from Europe, Italy and France. It was confirmed that there exist some garlic clones wi...

  1. Radiation induced chromosome instability in human fibroblasts

    International Nuclear Information System (INIS)

    Evidence has been arising that some biological effects can manifest many cell divisions after irradiation. We have demonstrated that de novo chromosome instability can be detected 10- 15 mean population doubling after heavy ion irradiations. This chromosome instability is characterized by end to end fusions between specific chromosomes. The specificity of the instability may differ from one donor to another but for the same donor, the same instability should be observed after irradiation, during the senescence process and after SV40 transfection (before crisis). In irradiated primary culture fibroblasts, the expression of the delayed chromosomal instability lasts for several cell divisions without inducing cell death. Several rounds of fusions- breakage-fusions can be performed and unbalanced clones emerge (gain or loss of chromosomes with the shorter telomeres would become unstable first.. The difference in the chromosomal instability among donors could be due to a polymorphism in telomere lengths. This could induce large variation in long term response to irradiation among individuals. (author)

  2. Artificial intelligence

    CERN Document Server

    Hunt, Earl B

    1975-01-01

    Artificial Intelligence provides information pertinent to the fundamental aspects of artificial intelligence. This book presents the basic mathematical and computational approaches to problems in the artificial intelligence field.Organized into four parts encompassing 16 chapters, this book begins with an overview of the various fields of artificial intelligence. This text then attempts to connect artificial intelligence problems to some of the notions of computability and abstract computing devices. Other chapters consider the general notion of computability, with focus on the interaction bet

  3. Evidence for different origin of sex chromosomes in snakes, birds, and mammals and step-wise differentiation of snake sex chromosomes.

    Science.gov (United States)

    Matsubara, Kazumi; Tarui, Hiroshi; Toriba, Michihisa; Yamada, Kazuhiko; Nishida-Umehara, Chizuko; Agata, Kiyokazu; Matsuda, Yoichi

    2006-11-28

    All snake species exhibit genetic sex determination with the ZZ/ZW type of sex chromosomes. To investigate the origin and evolution of snake sex chromosomes, we constructed, by FISH, a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 109 cDNA clones. Eleven of the 109 clones were localized to the Z chromosome. All human and chicken homologues of the snake Z-linked genes were located on autosomes, suggesting that the sex chromosomes of snakes, mammals, and birds were all derived from different autosomal pairs of the common ancestor. We mapped the 11 Z-linked genes of E. quadrivirgata to chromosomes of two other species, the Burmese python (Python molurus bivittatus) and the habu (Trimeresurus flavoviridis), to investigate the process of W chromosome differentiation. All and 3 of the 11 clones were localized to both the Z and W chromosomes in P. molurus and E. quadrivirgata, respectively, whereas no cDNA clones were mapped to the W chromosome in T. flavoviridis. Comparative mapping revealed that the sex chromosomes are only slightly differentiated in P. molurus, whereas they are fully differentiated in T. flavoviridis, and E. quadrivirgata is at a transitional stage of sex-chromosome differentiation. The differentiation of sex chromosomes was probably initiated from the distal region on the short arm of the protosex chromosome of the common ancestor, and then deletion and heterochromatization progressed on the sex-specific chromosome from the phylogenetically primitive boids to the more advanced viperids.

  4. A new approach for molecular cloning in cyanobacteria: cloning of an anacystis nidulans met gene using a Tn 907-induced mutant

    NARCIS (Netherlands)

    Tandeau de Marsac, N.; Borrias, W.E.; Kuhlemeijer, C.J.; Castets, A.M.; Arkel, G.A. van; Hondel, C.A.M.J.J. van den

    1982-01-01

    A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn 901 mutant was isolated. Chromosomal DNA fragments were cloned in the

  5. Nephropathic cystinosis (CTNS-LSB): construction of a YAC contig comprising the refined critical region on chromosome 17p13.

    Science.gov (United States)

    Peters, U; Senger, G; Rählmann, M; Du Chesne, I; Stec, I; Köhler, M R; Weissenbach, J; Leal, S M; Koch, H G; Deufel, T; Harms, E

    1997-01-01

    A yeast artificial chromosome (YAC) contig was constructed encompassing the entire region on chromosome 17p13 where the autosomal recessive disorder infantile nephropathic cystinosis (MIM 21980, CTNS-LSB) has been genetically mapped. It comprises seven clones ordered by their content of a series of six sequence-tagged sites (STSs). Fluorescence in situ hybridisation (FISH) revealed two chimaeric clones. The order of four polymorphic STSs mapped with the contig was consistent with that of the known genetic map with the exception of markers D17S1583 (AFMb307zg5) and D17S1798 (AFMa202xf5) where a telomeric location of D17S1583 was inferred from the contig; two non-polymorphic STSs were localised within the marker frame-work. From the analysis of recombination events in an unaffected individual as defined by leucocyte cystine levels we support the high-resolution mapping of this region to a small genetic interval and show that it is entirely represented on a single, non-chimaeric YAC clone in the contig.

  6. Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements.

    Energy Technology Data Exchange (ETDEWEB)

    Leem, Sun-Hee; Kouprina, Natalay; Grimwood, Jane; Kim, Jung-Hyun; Mullokandov, Michael; Yoon, Young-Ho; Chae, Ji-Youn; Morgan, Jenna; Lucas, Susan; Richardson, Paul; Detter, Chris; Glavina, Tijana; Rubin, Eddy; Barrett, J. Carl; Larionov, Vladimir

    2003-09-01

    The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to {approx} 1 percent of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.

  7. Chromosome region-specific libraries for human genome analysis. Progress report, September 1, 1991--August 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Kao, Fa-Ten

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

  8. Artificial Limbs

    Science.gov (United States)

    ... you are missing an arm or leg, an artificial limb can sometimes replace it. The device, which ... activities such as walking, eating, or dressing. Some artificial limbs let you function nearly as well as ...

  9. Reproductive Cloning

    OpenAIRE

    Neves, D.

    2010-01-01

    ABSTRACT Man desires to perpetuate himself and thereby will cross ethical barriers in the attempt to preserve his genetic heritage and thus to immortalize himself. To create a being in one’s own image has been a dream of man since the dawn of civilization and since the advent of the cloned sheep Dolly (05/07/1996) it now seems possible. The great challenge now is too reconcile human knowledge and scientific knowledge in the search for happiness, as the fron...

  10. The molecular characterization of maize B chromosome specific AFLPs

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The origin and evolution of B chromosomes could be explained by the specific DNA sequence on them.But the specific sequences known were quite limited. To investigate maize B chromosome sqicific DNA sequeces, maize genomes with and without B chromosomes were analyzed by AFLP. Only 5 markers were found specific to genomes with B chromosomes among about 2000 AFLP markers. Southern hybridization and sequence analysis revealed that only the sequence of M8-2D was a B chromosome specific sequence.This sequence contained the telomeric repeat unit AGGGTTT conserved in plant chromosome telomeres.In addition, the sequence of M8-2D shared low homology to clones from maize chromosome 4 centromere as well. M8-2D were localized to B chromosome centromeric and telomeric regions.

  11. Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

    Directory of Open Access Journals (Sweden)

    Robyn N. Hall

    2012-02-01

    Full Text Available Bacterial artificial chromosome (BAC vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses.

  12. Chromosome Microarray.

    Science.gov (United States)

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed. PMID:27276104

  13. Occurrence and type of chromosomal abnormalities in consecutive malignant monoclonal gammopathies: correlation with survival

    DEFF Research Database (Denmark)

    Lisse, I M; Drivsholm, A; Christoffersen, P

    1988-01-01

    Chromosome studies were done on 73 patients with multiple myeloma and three patients with plasma cell leukemia. Eighteen of 76 patients (24%) had chromosomally abnormal clones, including all three patients with PCL. The most common anomalous chromosomes were #1, #14, and #12. In addition, i(17q...

  14. Asexual Reproduction Does Not Apparently Increase the Rate of Chromosomal Evolution: Karyotype Stability in Diploid and Triploid Clonal Hybrid Fish (Cobitis, Cypriniformes, Teleostei.

    Directory of Open Access Journals (Sweden)

    Zuzana Majtánová

    Full Text Available Interspecific hybridization, polyploidization and transitions from sexuality to asexuality considerably affect organismal genomes. Especially the last mentioned process has been assumed to play a significant role in the initiation of chromosomal rearrangements, causing increased rates of karyotype evolution. We used cytogenetic analysis and molecular dating of cladogenetic events to compare the rate of changes of chromosome morphology and karyotype in asexually and sexually reproducing counterparts in European spined loach fish (Cobitis. We studied metaphases of three sexually reproducing species and their diploid and polyploid hybrid clones of different age of origin. The material includes artificial F1 hybrid strains, representatives of lineage originated in Holocene epoch, and also individuals of an oldest known age to date (roughly 0.37 MYA. Thereafter we applied GISH technique as a marker to differentiate parental chromosomal sets in hybrids. Although the sexual species accumulated remarkable chromosomal rearrangements after their speciation, we observed no differences in chromosome numbers and/or morphology among karyotypes of asexual hybrids. These hybrids possess chromosome sets originating from respective parental species with no cytogenetically detectable recombinations, suggesting their integrity even in a long term. The switch to asexual reproduction thus did not provoke any significant acceleration of the rate of chromosomal evolution in Cobitis. Asexual animals described in other case studies reproduce ameiotically, while Cobitis hybrids described here produce eggs likely through modified meiosis. Therefore, our findings indicate that the effect of asexuality on the rate of chromosomal change may be context-dependent rather than universal and related to particular type of asexual reproduction.

  15. Cloning of a Chryseobacterium (Flavobacterium) meningosepticum Chromosomal Gene (blaACME) Encoding an Extended-Spectrum Class A β-Lactamase Related to the Bacteroides Cephalosporinases and the VEB-1 and PER β-Lactamases

    OpenAIRE

    Rossolini, Gian Maria; Franceschini, Nicola; Lauretti, Laura; Caravelli, Berardo; Riccio, Maria Letizia; Galleni, Moreno; Frère, Jean-Marie; Amicosante, Gianfranco

    1999-01-01

    In addition to the BlaB metallo-β-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine β-lactamase, named CME-1, with an alkaline isoelectric pH. The blaACME gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A β-lactamase ...

  16. Genetic modification of mammalian genome at chromosome level

    Directory of Open Access Journals (Sweden)

    OLEG L. SEROV

    2000-09-01

    Full Text Available The review is concerned with a progress in genetic modification of a mammalian genome in vitro and in vivo at chromosomal level. Recently three new approaches for the chromosome biotechnology have been developed: Using Cre/loxP-system a researcher is able to produce targeted rearrangements of whole chromosomes or their segments or particular genes within the genome, and therefore to modify the set, position and copy number of the endogenous elements of the genome. Mammalian artificial chromosomes (MACs provide a possibility to introduce into genome relatively large segments of alien chromosome material, either artificially constructed or derived from the genome of different species. Using ES-somatic cell hybrids allows to transfer whole chromosomes or their fragments between different genomes within and between species. Advantages and limitations of these approaches are discussed.

  17. 细菌人工染色体及其在分子克隆化病毒中的应用%The application of bacterial artificial chromosome in molecular cloned virus

    Institute of Scientific and Technical Information of China (English)

    丁家波; 崔治中; 王建新

    2003-01-01

    细菌人工染色(BAC)是一种新的用于大分子DNA克隆的载体系统,它具有容量大,遗传稳定,操作简单等优点.本文综述了近几年来BAC的发展,及其常用的分析和修饰方法.同时,介绍了BAC在分子克隆化病毒中的应用.

  18. Complementation of a DNA repair defect in xeroderma pigmentosum cells by transfer of human chromosome 9

    International Nuclear Information System (INIS)

    Complementation of the repair defect in xeroderma pigmentosum cells of complementation group A was achieved by the transfer of human chromosome 9. A set of mouse-human hybrid cell lines, each containing a single Ecogpt-marked human chromosome, was used as a source of donor chromosomes. Chromosome transfer to XPTG-1 cells, a hypoxanthine/guanine phosphoribosyltransferase-deficient mutant of simian virus 40-transformed complementation group A cells, was achieved by microcell fusion and selection for Ecogpt. Chromosome-transfer clones of XPTG-1 cells, each containing a different human donor chromosome, were analyzed for complementation of sensitivity to UV irradiation. Among all the clones, increased levels of resistance to UV was observed only in clones containing chromosome 9. Since our recipient cell line XPTG-1 is hypoxanthine/guanine phosphoribosyltransferase deficient, cultivation of Ecogpt+ clones in medium containing 6-thioguanine permits selection of cells for loss of the marker and, by inference, transferred chromosome 9. Clones isolated for growth in 6-thioguanine, which have lost the Ecogpt-marked chromosome, exhibited a UV-sensitive phenotype, confirming the presence of the repair gene(s) for complementation group A on chromosome 9

  19. The mapping of novel genes to human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Buenaventura, J.M. [Sarah Lawrence College, Bronxville, NY (United States)

    1994-12-01

    The principle goal of our laboratory is the discovery of new genes on human chromosome 19. One of the strategies to achieve this goal is through the use of cDNA clones known as {open_quotes}expressed sequence tags{close_quotes} (ESTs). ESTs, short segments of sequence from a cDNA clone that correspond to the mRNA, occur as unique regions in the genome and, therefore, can be used as markers for specific positions. In collaboration with researchers from Genethon in France, fifteen cDNA clones from a normalized human infant brain cDNA library were tested and determined to map to chromosome 19. A verification procedure is then followed to confirm assignment to chromosome 19. First, primers for each cDNA clone are developed and then amplified by polymerase chain reaction from genomic DNA. Next, a {sup 32}P-radiolabeled probe is made by polymerase chain reaction for each clone and then hybridized against filters containing an LLNL chromosome 19-specific cosmid library to find putative locations on the chromosome. The location is then verified by running a polymerase chain reactions from the positive cosmids. With the Browser database at LLNL, additional information about the positive cosmids can be found. Through use of the BLAST database at the National Library of Medicine, homologous sequences to the clones can be found. Among the fifteen cDNA clones received from Genethon, all have been amplified by polymerase chain reaction. Three have turned out as repetitive elements in the genome. Ten have been mapped to specific locations on chromosome 19. Putative locations have been found for the remaining two clones and thus verification testing will proceed.

  20. Molecular mapping of chromosomes 17 and X

    Energy Technology Data Exchange (ETDEWEB)

    Barker, D.F.

    1991-01-15

    Progress toward the construction of high density genetic maps of chromosomes 17 and X has been made by isolating and characterizing a relatively large set of polymorphic probes for each chromosome and using these probes to construct genetic maps. We have mapped the same polymorphic probes against a series of chromosome breakpoints on X and 17. The probes could be assigned to over 30 physical intervals on the X chromosome and 7 intervals on 17. In many cases, this process resulted in improved characterization of the relative locations of the breakpoints with respect to each other and the definition of new physical intervals. The strategy for isolation of the polymorphic clones utilized chromosome specific libraries of 1--15 kb segments from each of the two chromosomes. From these libraries, clones were screened for those detecting restriction fragment length polymorphisms. The markers were further characterized, the chromosomal assignments confirmed and in most cases segments of the original probes were subcloned into plasmids to produce probes with improved signal to noise ratios for use in the genetic marker studies. The linkage studies utilize the CEPH reference families and other well-characterized families in our collection which have been used for genetic disease linkage work. Preliminary maps and maps of portions of specific regions of 17 and X are provided. We have nearly completed a map of the 1 megabase Mycoplasma arthritidis genome by applying these techniques to a lambda phage library of its genome. We have found bit mapping to be an efficient means to organize a contiguous set of overlapping clones from a larger genome.

  1. Artificial blood

    Directory of Open Access Journals (Sweden)

    Sarkar Suman

    2008-01-01

    Full Text Available Artificial blood is a product made to act as a substitute for red blood cells. While true blood serves many different functions, artificial blood is designed for the sole purpose of transporting oxygen and carbon dioxide throughout the body. Depending on the type of artificial blood, it can be produced in different ways using synthetic production, chemical isolation, or recombinant biochemical technology. Development of the first blood substitutes dates back to the early 1600s, and the search for the ideal blood substitute continues. Various manufacturers have products in clinical trials; however, no truly safe and effective artificial blood product is currently marketed. It is anticipated that when an artificial blood product is available, it will have annual sales of over $7.6 billion in the United States alone.

  2. A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B

    OpenAIRE

    Kobayashi, F; Wu, J. Z.; Kanamori, H; Tanaka, T.; Katagiri, S.; Karasawa, W.; Kaneko, S.; Watanabe, S; Sakaguchi, T; Šafář, J. (Jan); Šimková, H. (Hana); Mukai, Y.; M. Hamada; Saito, M; Hayakawa, K

    2015-01-01

    Background: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with t...

  3. A YAC contig of approximately 3 Mb from human chromosome 5q31 [yields] q33

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiang; Wang Jabs, E.; Hawkins, A.L.; Griffin, C.A. (John Hopkins School of Medicine, Baltimore, MD (United States)); Wise, C.A.; Lovett, M. (Univ. of Texas Southwestern Medical Center, Dallas, TX (United States)); Le Paslier, D. (CEPH, Paris (France)); Pittler, S.J. (Univ. of South Alabama College of Medicine, Mobile, AL (United States))

    1994-02-01

    The human chromosome 5q31-q33 region contains an interesting cluster of growth factor and receptor genes. In addition, several genetic disease loci have been localized within this region, but have not as yet been isolated as molecular clones. These include those loci involved in autosomal dominant limb-girdle muscular dystrophy, diastrophic dysplasia, Treacher Collins syndrome, and myeloid disorders associated with the 5q-syndrome. A yeast artificial chromosome (YAC) contig of this region would assist in the further localization and isolation of these genes. The authors have used YACs isolated from the Washington University and Centre d'Etude du Polymorphisme Humain YAC libraries, including YACs from the large insert (mega) YAC library to build a contig greater than 3 Mb in size. An STS content strategy coupled with limited walking from YAC ends was used to isolate 22 overlapping YACs with as much as sixfold coverage. A total of 20 STSs, derived from genes, anonymous sequences, and vector Alu-PCR or inverse PCR products, were used to compile this contig. The order of loci, centromere-GRL-D5S207-D5S70-D5S545-D5S546-D5S547-D5S68-D5S548-D5S210-D5S549-D5S686- ADRB2-D5S559-CSF1R-D5S551-RPS14-D5S519-SPARC-telomere, was derived from the overlapping clones. This contig and clones derived from it will be useful substrates in selecting candidate cDNAs for the disease loci in this interval. 45 refs., 1 fig., 2 tabs.

  4. Isolation, characterization, and chromosomal mapping of the human Nkx6.1 gene (NKX6A), a new pancreatic islet homeobox gene

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Hiroshi; Permutt, M.A.; Veile, R. [Washington Univ. School of Medicine, St. Louis, MO (United States)] [and others

    1997-03-01

    Nkx6.1 (gene symbol NKX6A), a new member of the NK homeobox gene family, was recently identified in rodent pancreatic islet 13-cell lines. The pattern of expression suggested that this gene product might be important for control of islet development and/or regulation of insulin biosynthesis. We now report cloning of human NKX6A, characterization of its genomic structure, and its chromosomal localization. The predicted protein of human NKX6A contained 367 amino acids and had 97% identity to the hamster protein. The highly conserved NK decapeptide and homeodomain regions were identical between human and hamster, suggesting functional importance of these domains. The coding region spanned approximately 4.8 kb and was composed of three exons. The gene was localized to four CEPH {open_quotes}B{close_quotes} yeast artificial chromosome clones (914B4, 951G9, 981D6, and 847133), and a nearby polymorphic marker (D4S1538) on chromosome 4 was identified <1270 kb from the gene. Using fluorescence in situ hybridization, we also determined that NKX6A maps to 4q21.2-q22. 11 refs., 2 figs.

  5. Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene

    DEFF Research Database (Denmark)

    Andresen, B S; Bross, P; Vianey-Saban, C;

    1996-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE......) based strategy to obtain the sequence of cDNAs encoding human VLCAD from placenta and fibroblasts. Alignment of the predicted amino acid sequence of human VLCAD with those of the other human ACD enzymes revealed extensive sequence homology. Moreover, human VLCAD and human acyl-CoA oxidase showed...... extensive sequence homology corroborating the notion that these genes are evolutionarily related. Southern blot analysis of genomic DNA from hybrid cell lines was used to localize the VLCAD gene to human chromosome 17p11.2-p11.13105. Using Northern and Western blot analysis to investigate the tissue...

  6. Different subfamilies of alphoid repetitive DNA are present on the human and chimpanzee homologous chromosomes 21 and 22.

    OpenAIRE

    Jørgensen, A L; Jones, C; Bostock, C J; Bak, A L

    1987-01-01

    The alphoid repeat DNA on chimpanzee chromosome 22 was compared with alphoid repeat DNA on its human homologue, chromosome 21. Hybridization of different alphoid probes under various conditions of stringency show that the alphoid repeats of chimpanzee chromosome 22 are not closely related to those of human chromosome 21. Sequence analysis of cloned dimer and tetramer EcoRI fragments from chimpanzee chromosome 22 confirm the low overall level of homology, but reveal the presence of several nuc...

  7. Efficient transfer of chromosome-based DNA constructs into mammalian cells

    NARCIS (Netherlands)

    Oberle, [No Value; de Jong, G; Drayer, JI; Hoekstra, D

    2004-01-01

    Artificial chromosomes, engineered minichromosomes and other chromosome-based DNA constructs are promising new vectors for use in gene therapy, protein production and transgenics. However, a major drawback in the application of chromosome-based DNA is the lack of a suitable and convenient procedure

  8. DNA methylation patterns of Brachypodium distachyon chromosomes and their alteration by 5-azacytidine treatment

    OpenAIRE

    Borowska, Natalia; Idziak, Dominika; Hasterok, Robert

    2011-01-01

    Sequential immunolocalisation of 5-methylcytosine (5-MeC) and fluorescence in situ hybridisation with chromosome-specific BAC clones were performed on Brachypodium distachyon mitotic metaphase chromosomes to determine specific DNA methylation patterns of each chromosome in the complement. In the majority of cells examined, chromosomes Bd4 and Bd5, which bear the loci of 5S and 35S ribosomal DNA, respectively, had characteristic 5-MeC patterns. In contrast, the distribution of 5-MeC along the ...

  9. Localization of Sry gene on Y chromosome of Muntjac munticus vaginalis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The chromosomes 1, Y1, Y2 of Muntjac munticus vaginalis were isolated by fluorescence activated chromosome sorting and amplified by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). A primer pair within human Sry HMG box was designed and the Sry gene of the male M. m vaginalis was amplified. The product was cloned and sequenced. The result proved that Sry is located on chromosome Y2, which is the sex-determining chromosome in the male M. m vaginalis.

  10. Artificial urushi.

    Science.gov (United States)

    Kobayashi, S; Uyama, H; Ikeda, R

    2001-11-19

    A new concept for the design and laccase-catalyzed preparation of "artificial urushi" from new urushiol analogues is described. The curing proceeded under mild reaction conditions to produce the very hard cross-linked film (artificial urushi) with a high gloss surface. A new cross-linkable polyphenol was synthesized by oxidative polymerization of cardanol, a phenol derivative from cashew-nut-shell liquid, by enzyme-related catalysts. The polyphenol was readily cured to produce the film (also artificial urushi) showing excellent dynamic viscoelasticity. PMID:11763444

  11. Artificial intelligence

    CERN Document Server

    Ennals, J R

    1987-01-01

    Artificial Intelligence: State of the Art Report is a two-part report consisting of the invited papers and the analysis. The editor first gives an introduction to the invited papers before presenting each paper and the analysis, and then concludes with the list of references related to the study. The invited papers explore the various aspects of artificial intelligence. The analysis part assesses the major advances in artificial intelligence and provides a balanced analysis of the state of the art in this field. The Bibliography compiles the most important published material on the subject of

  12. Quantum cloning

    International Nuclear Information System (INIS)

    It is impossible to make perfect copies or 'clones' of unknown quantum states, but approximate copies could still have many uses in quantum computing. A computer is a physical device that consists of components that are all subject to the laws of physics. Since computers deal exclusively in information, there is a close connection between information and physical systems. But what happens if the components inside the computer become so small that they must be described by quantum mechanics rather than classical physics? The seemingly unstoppable decrease in the size of transistors and other components will force the computer industry to confront this question in the near future. However, a small band of far-sighted physicists has been thinking about these problems for almost two decades. Starting with the work of Paul Benioff, Richard Feynman, David Deutsch and Charles Bennett in the mid-1980s, the field of 'quantum information' has grown to become one of the most exciting areas of modern physics. These early pioneers realized that the representation of information by quantum systems, such as single electrons or photons, was an opportunity rather than a problem. (U.K.)

  13. Correlation of chromosome patterns in human leukemic cells with exposure to chemicals and/or radiation

    International Nuclear Information System (INIS)

    This project seeks to defining the chromosome segments associated with radiation induced leukemogenesis (treatment-related acute myeloid leukemia, or t-AML). Towards these goals genetic analysis of human chromosomes 5 and 7 continues to investigate correlation of treatment with balanced and unbalanced chromosomal translocations. Progress is being made in cloning the breakpoints in balanced translocations in t-AML, that is to clone the t(9;11) and t(11;19) breakpoints, to clone the t(3;21)(q26;q22) breakpoints and to determine the relationship of these translocations to prior exposure to topoisomerase II inhibitors. 11 figs. 3 figs

  14. Artificial Reefs

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — An artificial reef is a human-made underwater structure, typically built to promote marine life in areas with a generally featureless bottom, control erosion, block...

  15. The Clone Factory

    Science.gov (United States)

    Stoddard, Beryl

    2005-01-01

    Have humans been cloned? Is it possible? Immediate interest is sparked when students are asked these questions. In response to their curiosity, the clone factory activity was developed to help them understand the process of cloning. In this activity, students reenact the cloning process, in a very simplified simulation. After completing the…

  16. Physical mapping of a large plant genome using global high-information-content-fingerprinting: the distal region of the wheat ancestor Aegilops tauschii chromosome 3DS

    Directory of Open Access Journals (Sweden)

    You Frank M

    2010-06-01

    Full Text Available Abstract Background Physical maps employing libraries of bacterial artificial chromosome (BAC clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum, Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat. Results We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs. Conclusions The physical map reported here is the first physical map using fingerprinting of a complete

  17. Reproductive Incompatibility Involving Senegalese Aedes aegypti (L) Is Associated with Chromosome Rearrangements.

    Science.gov (United States)

    Dickson, Laura B; Sharakhova, Maria V; Timoshevskiy, Vladimir A; Fleming, Karen L; Caspary, Alex; Sylla, Massamba; Black, William C

    2016-04-01

    Aedes aegypti, the primary vector of dengue, yellow fever and Zika flaviviruses, consists of at least two subspecies. Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf), has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane's rule modified for species, like Aedes, that have an autosomal sex determining locus (SDL). Egg-pupal survival was predicted to be low in females mated to hybrid F1 males but average when a male mates with a hybrid F1 female. Survival was in fact significantly reduced when females mated to hybrid males but egg-pupal survival was significantly increased when males were mated to hybrid F1 females. These observations are therefore inconclusive with regards to Haldane's rule. Basic cytogenetic analyses and Fluorescent In Situ Hybridization (FISH) experiments were performed to compare SenAae strains with the IB12 strain of Aaa that was used for genome sequencing and physical mapping. Some SenAae strains had longer chromosomes than IB12 and significantly different centromeric indices on chromosomes 1 and 3. DAPI staining was

  18. Reproductive Incompatibility Involving Senegalese Aedes aegypti (L) Is Associated with Chromosome Rearrangements

    Science.gov (United States)

    Dickson, Laura B.; Sharakhova, Maria V.; Timoshevskiy, Vladimir A.; Fleming, Karen L.; Caspary, Alex; Sylla, Massamba; Black, William C.

    2016-01-01

    Aedes aegypti, the primary vector of dengue, yellow fever and Zika flaviviruses, consists of at least two subspecies. Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf), has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane’s rule modified for species, like Aedes, that have an autosomal sex determining locus (SDL). Egg-pupal survival was predicted to be low in females mated to hybrid F1 males but average when a male mates with a hybrid F1 female. Survival was in fact significantly reduced when females mated to hybrid males but egg-pupal survival was significantly increased when males were mated to hybrid F1 females. These observations are therefore inconclusive with regards to Haldane’s rule. Basic cytogenetic analyses and Fluorescent In Situ Hybridization (FISH) experiments were performed to compare SenAae strains with the IB12 strain of Aaa that was used for genome sequencing and physical mapping. Some SenAae strains had longer chromosomes than IB12 and significantly different centromeric indices on chromosomes 1 and 3. DAPI staining

  19. Reproductive Incompatibility Involving Senegalese Aedes aegypti (L) Is Associated with Chromosome Rearrangements.

    Science.gov (United States)

    Dickson, Laura B; Sharakhova, Maria V; Timoshevskiy, Vladimir A; Fleming, Karen L; Caspary, Alex; Sylla, Massamba; Black, William C

    2016-04-01

    Aedes aegypti, the primary vector of dengue, yellow fever and Zika flaviviruses, consists of at least two subspecies. Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf), has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane's rule modified for species, like Aedes, that have an autosomal sex determining locus (SDL). Egg-pupal survival was predicted to be low in females mated to hybrid F1 males but average when a male mates with a hybrid F1 female. Survival was in fact significantly reduced when females mated to hybrid males but egg-pupal survival was significantly increased when males were mated to hybrid F1 females. These observations are therefore inconclusive with regards to Haldane's rule. Basic cytogenetic analyses and Fluorescent In Situ Hybridization (FISH) experiments were performed to compare SenAae strains with the IB12 strain of Aaa that was used for genome sequencing and physical mapping. Some SenAae strains had longer chromosomes than IB12 and significantly different centromeric indices on chromosomes 1 and 3. DAPI staining was

  20. Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer

    Institute of Scientific and Technical Information of China (English)

    TomohikoICHIKAWA; ShigeruHOSOKI; HiroyoshiSUZUKI; KoichiroAKAKURA; TatsuoIGARASHI; YuzoFURUYA; MitsuoOSHIMURA; CarrieW.RINKER-SCHAEFFER; NaokiNIHEI; JohnT.ISAACS; HaruoITO

    2000-01-01

    Aim: To identify the metastasis suppressor genes for prostate cancer. Methods: A copy of human chromosomes was introduced into the highly metastatic Dunning R-3327 rat prostate cancer cells by the use of microcell-mediated chromosome transfer. Relationships between the size of human chromosomes introduced into microcell hybrid clones and the number of lung metastases produced by the clones were analyzed to determine which part of human chromosomes contained the metastasis suppressor gene (s) for prostate cancer. To determine portions of human chromosomes introduced, G-banding chromosomal analysis, fluorescence in situ hybridization analysis, and polymerase chain reaction analysis were performed. Results: Each of microcell hybrid clones containing human chromosomes 7, 8, 10, 11, 12, or 17 showed decreased ability to metastasize to the lung without any loss of ttmaorigenicity. This demonstrates that these human chromosomes contain metastasis suppressor genes for prostate cancer. Spontaneous deletion of portions of human chromosomes was observed in the human chromosome 7, 10, 11, 12, and 17 studies. In the human chromosome 8 study, irradiated microcell-mediated chromosome transfer was performed to enrich chromosomal ann deletions of human chromosome 8. Molecular and cytogenetic analyses of microcell hybrid clones demonstrated that metastasis suppressor genes on human chromosomes were located on 7q21-22, 7q31.2-32, 8p21-12, 10q11-22, 11p13-11.2, 12p11-q13, 12q24-ter, and 17pter-q23. KAI1 and MKK4/SEKI were identified as metastasis suppressor genes from 11p11.2 and 17p12, respectively. Conclusion: This assay system is useful to identify metastasis suppressor gene (s) for prostate cancer.

  1. Cloning and disruption of Ustilago maydis genes.

    OpenAIRE

    Fotheringham, S.; Holloman, W K

    1989-01-01

    We have demonstrated that genes from Ustilago maydis can be cloned by direct complementation of mutants through the use of genomic libraries made in a high-frequency transformation vector. We isolated a gene involved in amino acid biosynthesis as an illustrative example and showed that integrative and one-step disruption methods can be used to create null mutations in the chromosomal copy of the gene by homologous recombination. The results of this investigation make it clear that one-step ge...

  2. Conversion of BAC clones into binary BAC (BIBAC) vectors and their delivery into basidiomycete fungal cells using Agrobacterium tumefaciens.

    Science.gov (United States)

    Ali, Shawkat; Bakkeren, Guus

    2015-01-01

    The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi. PMID:25239747

  3. Construction, Characterization, and Chromosomal Mapping of a Fosmid Library of the White-Cheeked Gibbon (Nomascus leucogenys)

    Institute of Scientific and Technical Information of China (English)

    Liping; Chen; Jianping; Ye; Yan; Liu; Jinghuan; Wang; Weiting; Su; Fengtang; Yang; Wenhui; Nie

    2007-01-01

    Gibbons have experienced extensive karyotype rearrangements during evolution and represent an ideal model for studying the underlying molecular mechanism of evolutionary chromosomal rearrangements. It is anticipated that the cloning and sequence characterization of evolutionary chromosomal breakpoints will provide vital insights into the molecular force that has driven such a radical karyotype reshuffle in gibbons. We constructed and characterized a high-quality fosmid li- brary of the white-cheeked gibbon (Nomascus leucogenys) containing 192,000 non- redundant clones with an average insert size of 38 kb and 2.5-fold genome coverage. By end sequencing of 100 randomly selected fosmid clones, we generated 196 se- quence tags for the library. These end-sequenced fosmid clones were then mapped onto the chromosomes of the white-cheeked gibbon by fluorescence in situ hy- bridization, and no spurious chimeric clone was detected. BLAST search against the human genome showed a good correlation between the number of hit clones and the number of chromosomes, an indication of unbiased chromosomal distribu- tion of the fosmid library. The chromosomal distribution of the mapped clones is also consistent with the BLAST search result against human and white-cheeked gibbon genomes. The fosmid library and the mapped clones will serve as a valu- able resource for further studying gibbons' chromosomal rearrangements and the underlying molecular mechanism as well as for comparative genomic study in the lesser apes.

  4. Mapping of the human dentin matrix acidic phosphoprotein gene (DMP1) to the dentinogenesis imperfecta type II critical region at chromosome 4q21

    Energy Technology Data Exchange (ETDEWEB)

    Aplin, H.M.; Hirst, K.L.; Crosby, A.H.; Dixon, M.J. [Univ. of Manchester (United Kingdom)

    1995-11-20

    Dentinogenesis imperfecta type II (DGI1) is an autosomal dominant disorder of dentin formation, which has been mapped to human chromosome 4q12-q21. The region most likely to contain the DGI1 locus is a 3.2-cM region surrounding the osteopontin (SPP1) locus. Recently, a novel dentin-specific acidic phosphoprotein (dmp1) has been cloned in the rat and mapped to mouse chromosome 5q21. In the current investigation, we have isolated a cosmid containing the human DMP1 gene. The isolation of a short tandem repeat polymorphism at this locus has allowed us to map the DMP1 locus to human chromosome 4q21 and demonstrate that it is tightly linked to DGI1 in two families (Z{sub max} = 11.01, {theta} = 0.001). The creation of a yeast artificial chromosome contig around SPP1 has further allowed us to demonstrate that DMP1 is located within 150 kb of the bone sialoprotein and 490 kb of the SPP1 loci, respectively. DMP1 is therefore a strong candidate for the DGI1 locus. 12 refs., 2 figs., 1 tab.

  5. Duplication and loss of chromosome 21 in two children with Down syndrome and acute leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Rogan, P.K.; Close, P.; Gannutz, L. [Pennsylvania State Univ., Hershey, PA (United States)] [and others

    1995-11-06

    Acute leukemia in Down syndrome (DS) is often associated with additional changes in the number of structure of chromosome 21. We present two DS patients whose leukemic karyotypes were associated with changes in chromosome 21 ploidy. Patient 1 developed acute lymphocytic leukemia (type L1); disomy for chromosome 21 was evident in all blast cells examined. Loss of the paternal chromosome in the leukemic clone produced maternal uniparental disomy with isodisomy over a 25-cM interval. The second patient had acute monoblastic leukemia (type M5) with tetrasomy 21 in all leukemic cells. DNA polymorphism analysis showed duplicate paternal chromosomes in the constitutional genotype. The maternal chromosome was subsequently duplicated in the leukemic clone. The distinct inheritance patterns of chromosome 21 in the blast cells of these patients would appear to indicate that leukemogenesis occurred by different genetic mechanisms in each individual. 57 refs., 2 figs., 3 tabs.

  6. MOLECULAR CLONING OF HUMAN NEUROTROPHIN-4 GENE

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Cloning and sequencing of the human neurotrophin-4(hNT-4) gene.Methods With the chromosomal DNA of human blood lymphocytes as template,hNT-4 coding genes were amplified by polymerase chain reaction(PCR) and recombinated into phage vector pGEM-T Easy,which were sequenced by using Sanger's single stranded DNA terminal termination method.Results The sequence of the cloned gene is completely the same as that reported in the literature(GenBank data base,M86528).Conclusion This study successfully cloning and sequenced the gene of mhNT-4,and it would be convenient for us to study the expression of mhNT-4 in eukaryote,and to continue the research on the gene therapy of Alzheimer's disease intensively.This study indicate that the hNT-4 is conservative in different races and individuals.

  7. Caracterização citogenética, viabilidade de pólen e hibridação artificial em gérbera Chromosome number, pollen viability and gerbera hybridization

    Directory of Open Access Journals (Sweden)

    Raquel DL Cardoso

    2009-03-01

    Full Text Available Este trabalho foi conduzido com o objetivo de confirmar o número de cromossomos em cultivares de Gerbera hybrida Hort., determinar o número de cromossomos em acessos não comerciais de Gerbera sp., avaliar a viabilidade de pólen e a possibilidade de cruzamentos entre cultivares e acessos não comerciais. Foram coletados ápices de raízes e pólen de seis cultivares e de sete acessos não comerciais. O material coletado foi corado com carmim acético a 45%. A contagem dos cromossomos foi realizada em células metafásicas intactas e a estimativa de viabilidade de pólen realizada por meio da contagem do número de grãos de pólen viáveis e não viáveis. A possibilidade de cruzamento entre as cultivares e entre as cultivares e acessos não comerciais foi avaliada por meio da hibridação entre os genitores femininos, cv. Terra Fame e acesso A8, e masculinos, cvs. Cariba e Azteca. Todos os acessos contiveram cinqüenta cromossomos, indicando que a variação morfológica nos capítulos (simples, semidobrado e dobrado não é devida a mutações cromossômicas numéricas ou a poliploidia. A viabilidade do pólen variou de 87,67% a 99,27%. A formação de sementes foi de 4,46% nos cruzamentos entre cultivares, e de 50% entre o A8 e as cultivares. A compatibilidade genômica entre os acessos, a alta viabilidade do pólen e o sucesso na obtenção de sementes entre acessos comercias e não comerciais, revela a possibilidade de produção de híbridos com novas combinações alélicas e transferência de caracteres desejáveis dos acessos não comerciais para os comerciaisThis work was conducted to confirm the chromosomes number of Gerbera hybrida Hort. cultivars, to determine the chromosomes number in the non commercial accessions of Gerbera sp., and to estimate the pollen viability and the possibility of crossings among different accessions. Root-tip and pollen were collected from six cultivars and seven non commercial accessions. The collected

  8. Artificial intelligence

    International Nuclear Information System (INIS)

    A vivid example of the growing need for frontier physics experiments to make use of frontier technology is in the field of artificial intelligence and related themes. This was reflected in the second international workshop on 'Software Engineering, Artificial Intelligence and Expert Systems in High Energy and Nuclear Physics' which took place from 13-18 January at France Telecom's Agelonde site at La Londe des Maures, Provence. It was the second in a series, the first having been held at Lyon in 1990

  9. Artificial Intelligence

    CERN Document Server

    Warwick, Kevin

    2011-01-01

    if AI is outside your field, or you know something of the subject and would like to know more then Artificial Intelligence: The Basics is a brilliant primer.' - Nick Smith, Engineering and Technology Magazine November 2011 Artificial Intelligence: The Basics is a concise and cutting-edge introduction to the fast moving world of AI. The author Kevin Warwick, a pioneer in the field, examines issues of what it means to be man or machine and looks at advances in robotics which have blurred the boundaries. Topics covered include: how intelligence can be defined whether machines can 'think' sensory

  10. The microcell mediated transfer of human chromosome 8 into highly metastatic rat liver cancer cell line C5F

    Institute of Scientific and Technical Information of China (English)

    Hu Liu; Sheng-Long Ye; Jiong Yang; Zhao-You Tang; Yin-Kun Liu; Lun-Xiu Qin; Shuang-Jian Qiu; Rui-Xia Sun

    2003-01-01

    AIM: Our previous research on the surgical samples of primary liver cancer with CGH showed that the loss of human chromosome 8p had correlation with the metastatic phenotype of liver cancer. In order to seek the functional evidence that there could be a metastatsis suppressor gene (s) for liver cancer on human chromosome 8, we tried to transfer normal human chromosome 8 into rat liver cancer cell line C5F, which had high metastatic potential to lung.METHODS: Human chromosome 8 randomly marked with neo gene was introduced into C5F cell line by MMCT and positive microcell hybrids were screened by double selections of G418 and HAT. Single cell isolation cloning was applied to clone microcell hybrids. Finally, STS-PCR and WCP-FISH were used to confirm the introduction.RESULTS: Microcell hybrids resistant to HAT and G418 were obtained and 15 clones were obtained by single-cell isolation cloning. STS-PCR and WCP-FISH proved that human chromosome 8 had been successfully introduced into rat liver cancer cell line C5F. STS-PCR detected a random loss in the chromosome introduced and WCP-FISH found a consistent recombination of the introduced human chromosome with the rat chromosome.CONCLUSION: The successful introduction of human chromosome 8 into highly metastatic rat liver cancer cell line builds the basis for seeking functional evidence of a metastasis suppressor gene for liver cancer harboring on human chromosome 8 and its subsequent cloning.

  11. Artificial sweeteners

    DEFF Research Database (Denmark)

    Raben, Anne Birgitte; Richelsen, Bjørn

    2012-01-01

    Artificial sweeteners can be a helpful tool to reduce energy intake and body weight and thereby risk for diabetes and cardiovascular diseases (CVD). Considering the prevailing diabesity (obesity and diabetes) epidemic, this can, therefore, be an important alternative to natural, calorie-containin...

  12. Construction of a DNA library from chromosome 4 of rice (Oryza sativa) by microdissection

    Institute of Scientific and Technical Information of China (English)

    MAOYINGWEI; SIYUANLIANG; 等

    1998-01-01

    A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.

  13. Ethical issues in cloning.

    Science.gov (United States)

    Satris, S

    2000-01-01

    There is great public concern with the ethics of human cloning. This paper briefly examines some of what I identify as pseudo-problems or myths associated with cloning, and some of the more substantial ethical concerns.

  14. Undetected sex chromosome aneuploidy by chromosomal microarray.

    Science.gov (United States)

    Markus-Bustani, Keren; Yaron, Yuval; Goldstein, Myriam; Orr-Urtreger, Avi; Ben-Shachar, Shay

    2012-11-01

    We report on a case of a female fetus found to be mosaic for Turner syndrome (45,X) and trisomy X (47,XXX). Chromosomal microarray analysis (CMA) failed to detect the aneuploidy because of a normal average dosage of the X chromosome. This case represents an unusual instance in which CMA may not detect chromosomal aberrations. Such a possibility should be taken into consideration in similar cases where CMA is used in a clinical setting.

  15. Cloning and regulation of Erwinia herbicola pigment genes.

    OpenAIRE

    Perry, K L; Simonitch, T A; Harrison-Lavoie, K J; Liu, S T

    1986-01-01

    The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink-yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. c...

  16. Chromatin structure and ionizing-radiation-induced chromosome aberrations

    International Nuclear Information System (INIS)

    The possible influence of chromatic structure or activity on chromosomal radiosensitivity was studied. A cell line was isolated which contained some 105 copies of an amplified plasmid in a single large mosquito artificial chromosome (MAC). This chromosome was hypersensitive to DNase I. Its radiosensitivity was some three fold greater than normal mosquito chromosomes in the same cell. In cultured human cells irradiated during G0, the initial breakage frequency in chromosome 4, 19 and the euchromatic and heterochromatic portions of the Y chromosome were measured over a wide range of doses by inducing Premature Chromosome Condensation (PCC) immediately after irradiation with Cs-137 gamma rays. No evidence was seen that Y heterochromatin or large fragments of it remained unbroken. The only significant deviation from the expected initial breakage frequency per Gy per unit length of chromosome was that observed for the euchromatic portion of the Y chromosome, with breakage nearly twice that expected. The development of aberrations involving X and Y chromosomes at the first mitosis after irradation was also studied. Normal female cells sustained about twice the frequency of aberrations involving X chromosomes for a dose of 7.3 Gy than the corresponding male cells. Fibroblasts from individuals with supernumerary X chromosomes did not show any further increase in X aberrations for this dos. The frequency of aberrations involving the heterochromatic portion of the long arm of the Y chromosome was about what would be expected for a similar length of autosome, but the euchromatic portion of the Y was about 3 times more radiosensitive per unit length. 5-Azacytidine treatment of cultured human female fibroblasts or fibroblasts from a 49,XXXXY individual, reduced the methylation of cytosine residues in DNA, and resulted in an increased chromosomal radiosensitivity in general, but it did not increase the frequency of aberrations involving the X chromosomes

  17. Pathogenesis of vestibular schwannoma in ring chromosome 22

    Directory of Open Access Journals (Sweden)

    Debiec-Rychter Maria

    2009-09-01

    Full Text Available Abstract Background Ring chromosome 22 is a rare human constitutional cytogenetic abnormality. Clinical features of neurofibromatosis type 1 and 2 as well as different tumour types have been reported in patients with ring chromosome 22. The pathogenesis of these tumours is not always clear yet. Methods We report on a female patient with a ring chromosome 22 presenting with severe mental retardation, autistic behaviour, café-au-lait macules and facial dysmorphism. Peripheral blood lymphocytes were karyotyped and array CGH was performed on extracted DNA. At the age of 20 years she was diagnosed with a unilateral vestibular schwannoma. Tumour cells were analyzed by karyotyping, array CGH and NF2 mutation analysis. Results Karyotype on peripheral blood lymphocytes revealed a ring chromosome 22 in all analyzed cells. A 1 Mb array CGH experiment on peripheral blood DNA showed a deletion of 5 terminal clones on the long arm of chromosome 22. Genetic analysis of vestibular schwannoma tissue revealed loss of the ring chromosome 22 and a somatic second hit in the NF2 gene on the remaining chromosome 22. Conclusion We conclude that tumours can arise by the combination of loss of the ring chromosome and a pathogenic NF2 mutation on the remaining chromosome 22 in patients with ring chromosome 22. Our findings indicate that patients with a ring 22 should be monitored for NF2-related tumours starting in adolescence.

  18. Role of SV40 Integration Site at Chromosomal Interval 1q21.1 in Immortalized CRL2504 Cells

    Science.gov (United States)

    Liu, Jinglan; Kaur, Gurpreet; Zhawar, Vikramjit K.; Zimonjic, Drazen B.; Popescu, Nicholas C.; Kandpal, Raj P.; Athwal, Raghbir S.

    2009-01-01

    We have applied a functional gene transfer strategy to demonstrate the importance of viral integration site in cellular immortalization. The large tumor antigen of SV40 is capable of extending the cellular life span by sequestering tumor suppressor proteins pRB and p53 in virus-transformed human cells. Although SV40-LT is essential, it is not sufficient for cellular immortalization, suggesting that additional alterations in cellular genes are required to attain infinite proliferation. We demonstrate here that the disruption of human chromosomal interval at 1q21.1, by SV40 integration, can be an essential step for cellular immortalization. The transfer of a 150Kb bacterial artificial chromosome (BAC) clone, RP364B14, corresponding to viral integration site in CRL2504 cells, reverted their immortal phenotype. Interestingly, the BAC transfer clones of CRL-2504 cells displayed characteristics of either senescence as shown by β-galactosidase activity or apoptosis as revealed by positive staining with M30 cytoDeath antibody. The SV40 integration at 1q21.1, in the vicinity of epidermal differentiation complex genes, resulted in the down-regulation of the filaggrin (FLG) gene that is part of the epidermal differentiation complex. FLG gene expression was restored to its normal levels in BAC transfer senescent and apoptotic clones. Our results suggest that the disruption of native genomic sequence by SV40 may alter expression of genes involved in senescence and apoptosis by modulating chromatin structure. These studies imply that identification of genes located in the vicinity of viral integration sites in human cancers may be helpful in developing new diagnostic and therapeutic strategies. PMID:19789346

  19. A physical map of Brassica oleracea shows complexity of chromosomal changes following recursive paleopolyploidizations

    Directory of Open Access Journals (Sweden)

    Giattina Emily

    2011-09-01

    Full Text Available Abstract Background Evolution of the Brassica species has been recursively affected by polyploidy events, and comparison to their relative, Arabidopsis thaliana, provides means to explore their genomic complexity. Results A genome-wide physical map of a rapid-cycling strain of B. oleracea was constructed by integrating high-information-content fingerprinting (HICF of Bacterial Artificial Chromosome (BAC clones with hybridization to sequence-tagged probes. Using 2907 contigs of two or more BACs, we performed several lines of comparative genomic analysis. Interspecific DNA synteny is much better preserved in euchromatin than heterochromatin, showing the qualitative difference in evolution of these respective genomic domains. About 67% of contigs can be aligned to the Arabidopsis genome, with 96.5% corresponding to euchromatic regions, and 3.5% (shown to contain repetitive sequences to pericentromeric regions. Overgo probe hybridization data showed that contigs aligned to Arabidopsis euchromatin contain ~80% of low-copy-number genes, while genes with high copy number are much more frequently associated with pericentromeric regions. We identified 39 interchromosomal breakpoints during the diversification of B. oleracea and Arabidopsis thaliana, a relatively high level of genomic change since their divergence. Comparison of the B. oleracea physical map with Arabidopsis and other available eudicot genomes showed appreciable 'shadowing' produced by more ancient polyploidies, resulting in a web of relatedness among contigs which increased genomic complexity. Conclusions A high-resolution genetically-anchored physical map sheds light on Brassica genome organization and advances positional cloning of specific genes, and may help to validate genome sequence assembly and alignment to chromosomes. All the physical mapping data is freely shared at a WebFPC site (http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/; Temporarily password-protected: account

  20. 小麦中与白粉病抗性相关的两个新基因序列的克隆、特征分析及染色体定位%Cloning, Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat

    Institute of Scientific and Technical Information of China (English)

    于玲; 牛吉山; 马正强; 陈佩度; 齐莉莉; 刘大钧

    2002-01-01

    Reverse-transcription Polymerase Chain Reaction (RT-PCR) was performed using cDNAs as templates from wheat-Haynaldia villosa 6VS/6AL translocation line and "Yangmai 5" induced with fungus Erysiphe graminis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. The cDNA sequences encoding cyclophilin-like and H+-ATPase-like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H+-ATPases isolated from other plants. Thus they were designated as Ta-Cyp and Ta-MAH. The obvious expression differences could be observed between wheat-H. villosa 6VS/6AL translocation line and susceptible wheat cultivar "Yangmai 5", implying that the two genes may be related with the resistance of wheat-H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta-Cyp gene and one copy of the Ta-MAH gene. Chinese Spring nulli-tetrasomic line analysis located the Ta-Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta-Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa, amphiploid of Triticum durum-H. villosa, wheat-H. villosa 6VS/6AL translocation line and "Yangmai 5", suggesting that Ta-Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa.%根据抗病基因保守结构域设计简并性引物,以被白粉菌(Erysiphe graminis)诱导后的小麦-簇毛麦6VS/6AL易位系和"扬麦5号"cDNA为模板进行反转录PCR(Reverse-transcription Polymerase Chain Reaction, RT-PCR)筛选,从小麦(Triticum aestivum Linn.)中分离到2个cDNA片段.蛋白质一级结构分析表明,它们分别与植物中已分离的cyclophilin蛋白和H+-ATP酶高度同源,将小麦中这2个基因分别定名为:Ta-Cyp 和Ta-MAH.经Northern杂交分析表明,这2个

  1. Artificial Intelligence.

    Science.gov (United States)

    Lawrence, David R; Palacios-González, César; Harris, John

    2016-04-01

    It seems natural to think that the same prudential and ethical reasons for mutual respect and tolerance that one has vis-à-vis other human persons would hold toward newly encountered paradigmatic but nonhuman biological persons. One also tends to think that they would have similar reasons for treating we humans as creatures that count morally in our own right. This line of thought transcends biological boundaries-namely, with regard to artificially (super)intelligent persons-but is this a safe assumption? The issue concerns ultimate moral significance: the significance possessed by human persons, persons from other planets, and hypothetical nonorganic persons in the form of artificial intelligence (AI). This article investigates why our possible relations to AI persons could be more complicated than they first might appear, given that they might possess a radically different nature to us, to the point that civilized or peaceful coexistence in a determinate geographical space could be impossible to achieve.

  2. Artificial intelligence

    OpenAIRE

    Duda, Antonín

    2009-01-01

    Abstract : Issue of this work is to acquaint the reader with the history of artificial inteligence, esspecialy branch of chess computing. Main attention is given to progress from fifties to the present. The work also deals with fighting chess programs against each other, and against human opponents. The greatest attention is focused on 1997 and duel Garry Kasparov against chess program Deep Blue. The work is divided into chapters according to chronological order.

  3. Clones on regular cardinals

    OpenAIRE

    Goldstern, Martin; Shelah, Saharon

    2000-01-01

    We investigate the structure of the lattice of clones on an infinite set X. We first observe that ultrafilters naturally induce clones; this yields a simple proof of Rosenberg's theorem: "there are 2^2^kappa many maximal (=precomplete) clones on a set of size kappa." The clones we construct here do not contain all unary functions. We then investigate clones that do contain all unary functions. Using a strong negative partition theorem we show that for many cardinals kappa there are 2^2^kappa ...

  4. Artificial vision.

    Science.gov (United States)

    Zarbin, M; Montemagno, C; Leary, J; Ritch, R

    2011-09-01

    A number treatment options are emerging for patients with retinal degenerative disease, including gene therapy, trophic factor therapy, visual cycle inhibitors (e.g., for patients with Stargardt disease and allied conditions), and cell transplantation. A radically different approach, which will augment but not replace these options, is termed neural prosthetics ("artificial vision"). Although rewiring of inner retinal circuits and inner retinal neuronal degeneration occur in association with photoreceptor degeneration in retinitis pigmentosa (RP), it is possible to create visually useful percepts by stimulating retinal ganglion cells electrically. This fact has lead to the development of techniques to induce photosensitivity in cells that are not light sensitive normally as well as to the development of the bionic retina. Advances in artificial vision continue at a robust pace. These advances are based on the use of molecular engineering and nanotechnology to render cells light-sensitive, to target ion channels to the appropriate cell type (e.g., bipolar cell) and/or cell region (e.g., dendritic tree vs. soma), and on sophisticated image processing algorithms that take advantage of our knowledge of signal processing in the retina. Combined with advances in gene therapy, pathway-based therapy, and cell-based therapy, "artificial vision" technologies create a powerful armamentarium with which ophthalmologists will be able to treat blindness in patients who have a variety of degenerative retinal diseases.

  5. A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast

    OpenAIRE

    Barrett J Carl; Ouspenski Ilia; Leem Sun-Hee; Kouprina Natalay; Noskov Vladimir N; Larionov Vladimir

    2003-01-01

    Abstract Background Transformation-associated recombination (TAR) cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes. The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (~ 60 bp) 5' and 3' gene targeting sequences (hooks). Result TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating seque...

  6. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.

    1982-09-01

    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

  7. Molecular cloning and functional characterization of avian interleukin-19

    Science.gov (United States)

    The present study describes the cloning and functional characterization of avian interleukin (IL)-19, a cytokine that, in mammals, alters the balance of Th1 and Th2 cells in favor of the Th2 phenotype. The full-length avian IL-19 gene, located on chromosome 26, was amplified from LPS-stimulated chi...

  8. 一种用高拷贝质粒载体制备BAC载体基本功能基因的新方法%Research on the Construction of Bacterial Artificial Chromosome Vector DNA and the Potentials in Application

    Institute of Scientific and Technical Information of China (English)

    崔红玉; 王亚萍; 徐明举; 薛永志; 石星明; 兰德松; 王云峰; 童光志

    2008-01-01

    [Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119-Bluelox BAG. [Method] With 8electing a proper single restriction site, sequences of a single copy BAC vector plasmid were inserted into proper site of High-copy plasmid pUC119 vector. [Result] The gene sequence of BAG vector lost control function of single copy number in new plasmid pUC119-BAC and was copied through High-copy form. The gene sequence of BAC vector basic function was completely cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection. [ Conclusion ] The High-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAG vector, besides thai it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.

  9. Numerical Chromosome Errors in Day 7 Somatic Nuclear Transfer Bovine Blastocysts

    DEFF Research Database (Denmark)

    Booth, Paul J.; VIUFF, Dorte; Tan, Shijian;

    2002-01-01

    amino acids, myoinositol, sodium citrate, and 5% cattle serum in microwells for 7 days, at which time nuclei from all blastocysts were extracted and chromosome aberrations were evaluated using dual-color fluorescent in situ hybridization with bovine chromosome 6- and 7-specific probes. Five embryo clone...

  10. Fetal chromosome analysis: screening for chromosome disease?

    DEFF Research Database (Denmark)

    Philip, J; Tabor, Ann; Bang, J;

    1983-01-01

    The aim of the study was to investigate the rationale of the current indications for fetal chromosome analysis. 5372 women had 5423 amniocentesis performed, this group constituting a consecutive sample at the chromosome laboratory, Rigshospitalet, Copenhagen from March 1973 to September 1980 (Group...... A + B). Pregnant women 35 years of age, women who previously had a chromosomally abnormal child, families with translocation carriers or other heritable chromosomal disease, families where the father was 50 years or more and women in families with a history of Down's syndrome (group A), were compared...... to women having amniocentesis, although considered not to have any increased risk of fetal chromosome abnormality (1390 pregnancies, group B). They were also compared with 750 consecutive pregnancies in women 25-34 years of age, in whom all heritable diseases were excluded (group C). The risk of unbalanced...

  11. Chromosome Disorder Outreach

    Science.gov (United States)

    ... BLOG Join Us Donate You are not alone. Chromosome Disorder Outreach, Inc. is a non-profit organization, ... Support For all those diagnosed with any rare chromosome disorder. Since 1992, CDO has supported the parents ...

  12. Physical organization of mixed protease inhibitor gene clusters, coordinated expression and association with resistance to late blight at the StKI locus on potato chromosome III.

    Science.gov (United States)

    Odeny, Damaris Achieng; Stich, Benjamin; Gebhardt, Christiane

    2010-12-01

    Protease inhibitors (PIs) play a role in plant defence against pests and pathogens as well as in plant development. Potato (Solanum tuberosum) contains abundant levels of diverse PIs. Most potato Kunitz-type inhibitor (KTI) genes map to the StKI locus on potato chromosome III, which is linked to a quantitative trait locus (QTL) for resistance to Phytophthora infestans. To elucidate the physical organization of PIs at the StKI locus, we screened bacterial artificial chromosome (BAC) libraries with KTI probes. Ten different clones were selected, sequenced and annotated. Of 100 putative genes, 22 corresponded to five PI classes. Expression analysis by quantitative real-time PCR (qRT-PCR) using PI class-specific primers in different tissues of the tetraploid potato cultivars 'Nikita' and 'Baltica' revealed different transcript levels, depending on PI type and genotype. During the compatible interaction with a complex race of P. infestans, four PI classes showed coordinated expression over 3 d after infection, a strong decrease in infected leaves and a transient induction in systemic leaves. Basal transcript levels in non-infected leaves differed strongly between the two genotypes examined. Two microsatellite markers located within the PI gene cluster were associated with resistance to P. infestans in a population of potato varieties and breeding clones. PMID:20716067

  13. ZEBRAFISH CHROMOSOME-BANDING

    NARCIS (Netherlands)

    PIJNACKER, LP; FERWERDA, MA

    1995-01-01

    Banding techniques were carried out on metaphase chromosomes of zebrafish (Danio rerio) embryos. The karyotypes with the longest chromosomes consist of 12 metacentrics, 26 submetacentrics, and 12 subtelocentrics (2n = 50). All centromeres are C-band positive. Eight chromosomes have a pericentric C-b

  14. Chromosome painting in plants.

    NARCIS (Netherlands)

    Schubert, I.; Fransz, P.F.; Fuchs, J.; Jong, de J.H.

    2001-01-01

    The current 'state-of-art' as to chromosome painting in plants is reviewed. We define different situations described as painting so far: i) Genomic in situ hybridisation (GISH) with total genomic DNA to distinguish alien chromosomes on the basis of divergent dispersed repeats, ii) 'Chromosomal in si

  15. Red-mediated transposition and final release of the mini-F vector of a cloned infectious herpesvirus genome.

    Directory of Open Access Journals (Sweden)

    Felix Wussow

    Full Text Available Bacterial artificial chromosomes (BACs are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors.

  16. Dicentric chromosome in the bone marrow of a child with megakaryoblastic leukaemia and Down's syndrome.

    OpenAIRE

    Wilkie, A O; Kitchen, C.; Oakhill, A; Howell, R T; Berry, P J

    1988-01-01

    A two year old girl with Down's syndrome (constitutional karyotype: 47 + 21), presenting with pancytopenia, developed acute megakaryoblastic leukaemia (AMKL). Her bone marrow contained an abnormal clone with a novel dicentric chromosome derived from chromosomes 5 and 7 (karyotype 46, XX, -5, -7, +dic (5;7) (p 13; p 11.2), +21. This case provides further evidence for a connection between chromosome 21 and this unusual form of childhood leukaemia, and raises questions about the loss of short ar...

  17. Genetic stability of pestivirus genomes cloned into BACs

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Uttenthal, Åse;

    Infectious cDNA clones are a prerequisite for directed genetic manipulations of pestivirus genomes to obtain attenuated pestiviruses designed as new modified live DIVA vaccine candidates against classical swine fever. However, the construction of new infectious pestivirus cDNA clones has been...... hampered due to the large size of the pestivirus genome and due to genetic instability of the cloned cDNA, which in combination with plasmid vectors tend to be unstable and deleterious in the bacterial host. Therefore, new strategies are needed to facilitate construction of stable infectious cDNA clones...... of pestivirus strains. In a collaborative research project, between DTU Vet and FLI, on the establishment of genetically modified pestiviruses engineered specifically for the DIVA principle, we cloned a series of complete pestivirus genomes, obtained by full-length RT-PCR, directly into the bacterial artificial...

  18. Genome-wide epigenetic alterations in cloned bovine fetuses.

    Science.gov (United States)

    Cezar, Gabriela Gebrin; Bartolomei, Marisa S; Forsberg, Erik J; First, Neal L; Bishop, Michael D; Eilertsen, Kenneth J

    2003-03-01

    To gain a better understanding of global methylation differences associated with development of nuclear transfer (NT)-generated cattle, we analyzed the genome-wide methylation status of spontaneously aborted cloned fetuses, cloned fetuses, and adult clones that were derived from transgenic and nontransgenic cumulus, genital ridge, and body cell lines. Cloned fetuses were recovered from ongoing normal pregnancies and were morphologically normal. Fetuses generated by artificial insemination (AI) were used as controls. In vitro fertilization (IVF) fetuses were compared with AI controls to assess effects of in vitro culture on the 5-methylcytosine content of fetal genomes. All of the fetuses were female. Skin biopsies were obtained from cloned and AI-generated adult cows. All of the adult clones were phenotypically normal and lactating and had no history of health or reproductive disorders. Genome-wide cytosine methylation levels were monitored by reverse-phase HPLC, and results indicated reduced levels of methylated cytosine in NT-generated fetuses. In contrast, no differences were observed between adult, lactating clones and similarly aged lactating cows produced by AI. These data imply that survivability of cloned cattle may be closely related to the global DNA methylation status. This is the first report to indicate that global methylation losses may contribute to the developmental failure of cloned bovine fetuses. PMID:12604655

  19. Establishment of Sequence-Tagged Sites on 15q11-q13 by Alu-Vector PCR Cloning of Yac-Generated Fragments

    Directory of Open Access Journals (Sweden)

    W. S. Kim

    1996-01-01

    Full Text Available Angelman syndrome (AS is caused by the loss of function of undefined gene(s on human chromosome 15. The majority of subjects have deletions involving maternally-derived chromosome 15q II-q 13, and the shortest region of deletion overlap (SRO has been localized to the region between D15S10 and D15S113. In this study, yeast artificial chromosomes (YACs, 6G-D4, 9H-D2 and 37D-F9, mapping within the AS SRO, were isolated from the ICI Y AC library. Alu-vector PCR products were amplified from the YACs and from YACs A229A2 and A33FI 0 which had been obtained from the St. Louis Y AC library. The PCR products were cloned and sequenced, and three new sequence-tagged sites were generated within the AS SRO, facilitating the characterization of gene(s involved in the Angelman syndrome.

  20. Cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene from the Treacher Collins syndrome candidate region at 5q32-q33.1

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, J.; Loftus, S.K.; Gladwin, A.J. [Univ. of Manchester (United Kingdom)] [and others

    1995-03-20

    Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. Previous studies have shown that the Treacher Collins syndrome locus is flanked by D5S519 and SPARC, and a yeast artificial chromosome contig encompassing this {open_quotes}critical region{close_quotes} has been completed. In the current investigation a cosmid containing D5S519 has been used to screen a human placental cDNA library. This has resulted in the cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene. Two different mRNA species that have identical protein coding sequences but that differ in the size and sequence of the 3{prime} untranslated regions (3{prime}UTR) have been identified. The smaller species has a 3{prime}UTR of 1035 bp, whereas that of the larger is 4878 bp. 24 refs., 3 figs.

  1. Statement on Human Cloning

    Science.gov (United States)

    ... as our understanding of this technology advances. Support Stem Cell Research (including Research Cloning) AAAS supports stem cell research, including the use of nuclear transplantation techniques (also ...

  2. Mapping genes on human chromosome 20

    Energy Technology Data Exchange (ETDEWEB)

    Keith, T.; Phipps, P.; Serino, K. [Collaborative Research, Inc., Waltham, MA (United States)] [and others

    1994-09-01

    While a substantial number of genes have been physically localized to human chromosome 20, few have been genetically mapped. In the process of developing a genetic linkage map of chromosome 20, we have mapped microsatellite polymorphisms associated with six genes. Three of these had highly informative polymorphisms (greater than 0.70) that were originally identified by other investigators. These include avian sarcoma oncogene homolog (SRC), ribophorin II (RPN2), and phosphoenolpyruvate carboxykinase (PCK1). Polymorphisms associated with two genes were determined following a screen of their DNA sequences in GenBank. These include dinucleotide polymorphisms in introl II of cystatin c (CST3) and in the promoter region of neuroendocrine convertase 2 (NEC2) with heterozygosities of 0.52 and 0.54, respectively. A sixth gene, prodynorphin (PDYN) was mapped following the identification of a dinucleotide repeat polymorphism (heterozygosity of 0.35) in a cosmid subclone from a YAC homologous to the original phage clone. CA-positive cosmid subclones from a YAC for an additional gene, guanine nucleotide binding protein, alpha (GNAS10), have been identified and sequencing is in progress. Similar efforts were utilized to identify a microsatellite polymorphism from a half-YAC cloned by W. Brown and localized by FISH to 20pter. This polymorphism is highly informative, with a heterozygosity of 0.83, and serves to delimit the genetic map of the short arm of this chromosome.

  3. Chimpanzee chromosome 12 is homologous to human chromosome 2q

    Energy Technology Data Exchange (ETDEWEB)

    Sun, N. C.; Sun, C. R.Y.; Ho, T.

    1977-01-01

    Most of the 46 human chromosomes find their counterparts in the 48 chimpanzee chromosomes except for chromosome 2 which has been hypothesized to have been derived from a centric fusion of two chimpanzee acrocentric chromosomes. These two chromosomes correspond to the human chromosomes 2p and 2g. This conclusion is based primarily on chromosome banding techniques, and the somatic cell hybridization technique has also been used. (HLW)

  4. Cloning, expression, and chromosome mapping of human galectin-7

    DEFF Research Database (Denmark)

    Madsen, Peder; Rasmussen, H H; Flint, T;

    1995-01-01

    comigrated with IEF 17 as determined by two-dimensional (two-dimensional gel electrophoresis) analysis of proteins expressed by transiently transfected COS-1 cells, and bound lactose. Alignment of the amino acid sequences with other members of the family showed that the amino acids central to the beta...

  5. Stochasticity or the fatal `imperfection' of cloning

    Indian Academy of Sciences (India)

    Reiner A Veitia

    2005-02-01

    The concept of clone is analysed with the aim of exploring the limits to which a phenotype can be said to be determined geneticaly. First of all, mutations that result from the replication, topological manipulation or lesion of DNA introduce a source of heritable variation in an otherwise identical genetic background. But more important, stochastic effects in many biological processes may superimpose a phenotypic variation which is not encoded in the genome. The source of stochasticity ranges from the random selection of alleles or whole chromosomes to be expressed in small cell populations, to fluctuations in processes such as gene expression, due to limiting amounts of the players involved. The picture emerging is that the term clone is a statistical over-simplification representing a series of individuals having essentially the same genome but capable of exhibiting wide phenotypic variation. Finally, to what extent fluctuations in biological processes, usually thought of as noise, are in fact signal is also discussed.

  6. Acentric chromosome ends are prone to fusion with functional chromosome ends through a homology-directed rearrangement.

    Science.gov (United States)

    Ohno, Yuko; Ogiyama, Yuki; Kubota, Yoshino; Kubo, Takuya; Ishii, Kojiro

    2016-01-01

    The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.

  7. The Trypanosoma cruzi Genome Project: Nuclear Karyotype and Gene Mapping of Clone CL Brener

    Directory of Open Access Journals (Sweden)

    Marcia RM Santos

    1997-11-01

    Full Text Available By using improved pulsed field gel electrophoresis conditions, the molecular karyotype of the reference clone CL Brener selected for Trypanosoma cruzi genome project was established. A total of 20 uniform chromosomal bands ranging in size from 0.45 to 3.5 Megabase pairs (Mbp were resolved in a single run. The weighted sum of the chromosomal bands was approximately 87 Mbp. Chromoblots were hybridized with 39 different homologous probes, 13 of which identified single chromosomes. Several markers showed linkage and four different linkage groups were identified, each comprising two markers. Densitometric analysis suggests that most of the chromosomal bands contain two or more chromosomes representing either homologous chromosomes and/or heterologous chromosomes with similar sizes

  8. Molecular mapping of chromosomes 17 and X. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Barker, D.F.

    1991-01-15

    Progress toward the construction of high density genetic maps of chromosomes 17 and X has been made by isolating and characterizing a relatively large set of polymorphic probes for each chromosome and using these probes to construct genetic maps. We have mapped the same polymorphic probes against a series of chromosome breakpoints on X and 17. The probes could be assigned to over 30 physical intervals on the X chromosome and 7 intervals on 17. In many cases, this process resulted in improved characterization of the relative locations of the breakpoints with respect to each other and the definition of new physical intervals. The strategy for isolation of the polymorphic clones utilized chromosome specific libraries of 1--15 kb segments from each of the two chromosomes. From these libraries, clones were screened for those detecting restriction fragment length polymorphisms. The markers were further characterized, the chromosomal assignments confirmed and in most cases segments of the original probes were subcloned into plasmids to produce probes with improved signal to noise ratios for use in the genetic marker studies. The linkage studies utilize the CEPH reference families and other well-characterized families in our collection which have been used for genetic disease linkage work. Preliminary maps and maps of portions of specific regions of 17 and X are provided. We have nearly completed a map of the 1 megabase Mycoplasma arthritidis genome by applying these techniques to a lambda phage library of its genome. We have found bit mapping to be an efficient means to organize a contiguous set of overlapping@ clones from a larger genome.

  9. Cloning-free CRISPR

    NARCIS (Netherlands)

    Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

    2015-01-01

    We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each targ

  10. Measurement of background translocation frequencies in individuals with clones

    Energy Technology Data Exchange (ETDEWEB)

    Wade, M.J.

    1996-08-01

    In the leukemia case the unseparated B and T lymphocytes had a high translocation frequency even after 0.0014, respectively. After purging all clones from the data, the translocation frequencies for Bio 8 and Bio 23 were 0.00750.0014 and 0.0073 metaphases were scored for chromosomal aberrations,, specifically reciprocal translocations, using fluorescence in situ hybridization (FISH). Metaphase spreads were used from two healthy, unexposed individuals (not exposed to radiation, chemotherapy or radiotherapy) and one early B- precursor acute lymphocytic leukemia (ALL) patient (metaphase spreads from both separated T lymphocytes and unseparated B and T lymphocytes were scored). All three individuals had an abnormally high translocation frequency. The high translocation frequencies resulted from clonal expansion of specific translocated chromosomes. I show in this thesis that by purging (discounting or removing) clones from the data of unexposed individuals, one can obtain true background translocation frequencies. In two cases, Bio 8 and Bio 23, the measured translocation frequency for chromosomes 1, 2 and 4 was 0.0124 purging all of the clones from the data. This high translocation frequency may be due to a low frequency of some clones and may not be recognized. The separated T lymphocytes had a higher translocation frequency than expected.

  11. The Precarious Prokaryotic Chromosome

    OpenAIRE

    Kuzminov, Andrei

    2014-01-01

    Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the t...

  12. A sex chromosomal restriction-fragment-length marker linked to melanoma-determining Tu loci in Xiphophorus

    OpenAIRE

    Schartl, Manfred

    2012-01-01

    In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restriction-fragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from vari...

  13. Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes

    Directory of Open Access Journals (Sweden)

    SEROV OLEG

    2001-01-01

    Full Text Available The properties of embryonic hybrid cells obtained by fusion of embryonic stem (ES or teratocarcinoma (TC cells with differentiated cells are reviewed. Usually, ES-somatic or TC-somatic hybrids retain pluripotent capacity at high levels quite comparable or nearly identical with those of the pluripotent partner. When cultured in vitro, ES-somatic- and TC-somatic hybrid cell clones, as a rule, lose the chromosomes derived from the somatic partner; however, in some clones the autosomes from the ES cell partner were also eliminated, i.e. the parental chromosomes segregated bilaterally in the ES-somatic cell hybrids. This opens up ways for searching correlation between the pluripotent status of the hybrid cells and chromosome segregation patterns and therefore for identifying the particular chromosomes involved in the maintenance of pluripotency. Use of selective medium allows to isolate in vitro the clones of ES-somatic hybrid cells in which "the pluripotent" chromosome can be replaced by "the somatic" counterpart carrying the selectable gene. Unlike the TC-somatic cell hybrids, the ES-somatic hybrids with a near-diploid complement of chromosomes are able to contribute to various tissues of chimeric animals after injection into the blastocoel cavity. Analysis of the chimeric animals showed that the "somatic" chromosome undergoes reprogramming during development. The prospects for the identification of the chromosomes that are involved in the maintenance of pluripotency and its cis- and trans-regulation in the hybrid cell genome are discussed.

  14. Ring chromosome 13

    DEFF Research Database (Denmark)

    Brandt, C A; Hertz, Jens Michael; Petersen, M B;

    1992-01-01

    A stillborn male child with anencephaly and multiple malformations was found to have the karyotype 46,XY,r(13) (p11q21.1). The breakpoint at 13q21.1, determined by high resolution banding, is the most proximal breakpoint ever reported in patients with ring chromosome 13. In situ hybridisation...... with the probe L1.26 confirmed the derivation from chromosome 13 and DNA polymorphism analysis showed maternal origin of the ring chromosome. Our results, together with a review of previous reports of cases with ring chromosome 13 with identified breakpoints, could neither support the theory of distinct clinical...

  15. Characterization of a chromosome-specific chimpanzee alpha satellite subset: Evolutionary relationship to subsets on human chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Warburton, P.E.; Gosden, J.; Lawson, D. [Western General Hospital, Edinburgh (United Kingdom)] [and others

    1996-04-15

    Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.

  16. Molecular mapping of chromosomes 17 and X

    Energy Technology Data Exchange (ETDEWEB)

    Barker, D.F.

    1989-01-01

    The basic aims of this project are the construction of high density genetic maps of chromosomes 17 and X and the utilization of these maps for the subsequent isolation of a set of physically overlapping DNA segment clones. The strategy depends on the utilization of chromosome specific libraries of small (1--15 kb) segments from each of the two chromosomes. Since the time of submission of our previous progress report, we have refined the genetic map of markers which we had previously isolated for chromosome 17. We have completed our genetic mapping in CEPH reference and NF1 families of 15 markers in the pericentric region of chromosome 17. Physical mapping results with three probes, were shown be in very close genetic proximity to the NF1 gene, with respect to two translocation breakpoints which disrupt the activity of the gene. All three of the probes were found to lie between the centromere and the most proximal translocation breakpoint, providing important genetic markers proximal to the NF1 gene. Our primary focus has shifted to the X chromosome. We have isolated an additional 30 polymorphic markers, bringing the total number we have isolated to over 80. We have invested substantial effort in characterizing the polymorphisms at each of these loci and constructed plasmid subclones which reveal the polymorphisms for nearly all of the loci. These subclones are of practical value in that they produce simpler and stronger patterns on human genomic Southern blots, thus improving the efficiency of the genetic mapping experiments. These subclones may also be of value for deriving DNA sequence information at each locus, necessary for establishing polymerase chain reaction primers specific for each locus. Such information would allow the use of each locus as a sequence tagged site.

  17. Chromosomal localization of 5S rDNA in Chinese shrimp (Fenneropenaeus chinensis):a chromosome-specific marker for chromosome identification

    Institute of Scientific and Technical Information of China (English)

    郇聘; 张晓军; 李富花; 赵翠; 张成松; 相建海

    2010-01-01

    Chinese shrimp(Fenneropenaeus chinensis)is an economically important aquaculture species in China.However,cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to isolate and analyze.In this study,fluorescence in-situ hybridization(FISH) was used to identify the chromosomes of F.chinensis.The 5S ribosomal RNA gene(rDNA)of F. chinensis was isolated,cloned and then used as a hybridization probe.The results show that the 5S rDNA was located on one pair of homologo...

  18. Artificial Economy

    Directory of Open Access Journals (Sweden)

    Alexandru JIVAN

    2011-08-01

    Full Text Available This paper proposes to eliminate, a routine in the economic thinking, claimed to be responsible for the negative essence of economic developments, from the point of view, of the ecological implications (employment in the planetary ecosystem. The methodological foundations start from the natural origins of the functionality of the human economic society according to the originary physiocrat liberalism, and from specific natural characteristics of the humankind. This paper begins with a comment-analysis of the difference between natural and artificial within the economy, and then explains some of the most serious diversions from the natural essence of economic liberalism. It shall be explained the original (heterodox interpretation of the Classical political economy (economics, by making calls to the Romanian economic thinking from aggravating past century. Highlighting the destructive impact of the economy - which, under the invoked doctrines, we call unnatural - allows an intuitive presentation of a logical extension of Marshall's market price, based on previous research. Besides the doctrinal arguments presented, the economic realities inventoried along the way (major deficiencies and effects, determined demonstrate the validity of the hypothesis of the unnatural character and therefore necessarily to be corrected, of the concept and of the mechanisms of the current economy.The results of this paper consist of original heterodox methodspresented, intuitive or developed that can be found conclusively within the key proposals for education and regulation.

  19. Banding studies of chromosomes in a patient with mycosis fungoides

    Energy Technology Data Exchange (ETDEWEB)

    Fukuhara, S.; Rowley, J.D.; Variakojis, D.

    1978-11-01

    Chromosomes from a patient with mycosis fungoides were examined in detail with banding techniques. Hyperdiploid cells from a lymph node had common anomalies of certain chromosomes which formed three similar clones. The abnormalities involved chromosomes Nos. 1, 2, 5, 8, 9, 10, 14, and 18, in addition to an unknown small metacentric marker (M3). Although there were a number of mitotic cells in peripheral blood cultured both with and without PHA, none of the few cells with abnormal karyotypes was similar to the clonal cells of the lymph node. One of the abnormalities in the lymph node was a 14q rearrangement, which could be the result of a translocation of Nos. 8 and 14 involving a third chromosome, No. 2. An abnormality in the blood resulted from a translocation between the long arms of Nos. 1 and 14. These findings could be useful for studies in which mycosis fungoides is compared with the Sezary syndrome and other lymphoid malignancies.

  20. Electochemical detection of chromosome translocation

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Dimaki, Maria; Silahtaroglu, Asli;

    2014-01-01

    Cytogenetics is a study of the cell structure with a main focus on chromosomes content and their structure. Chromosome abnormalities, such as translocations may cause various genetic disorders and heametological malignancies. Chromosome translocations are structural rearrangements of two chromoso...

  1. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  2. RELATIVE EXPRESSION AND STABILITY OF A CHROMOSOMALLY INTEGRATED AND PLASMID-BORNE MARKER GENE FUSION IN ENVIRONMENTALLY COMPETENT BACTERIA

    Science.gov (United States)

    A xyIE-iceC transcriptional fusion was created by ligating a DNA fragment harboring the cloned xyIE structural gene from the TOL plasmid of Pseudomonas putida mt-2 into the cloned iceC gene of Pseudomonas syringae Cit7. This fusion construct was integrated into chromosome of Pseu...

  3. Frequent occurrence of highly expanded but unrelated B-cell clones in patients with multiple myeloma.

    Science.gov (United States)

    Kriangkum, Jitra; Motz, Sarah N; Debes Marun, Carina S; Lafarge, Sandrine T; Gibson, Spencer B; Venner, Christopher P; Johnston, James B; Belch, Andrew R; Pilarski, Linda M

    2013-01-01

    Clonal diversity in multiple myeloma (MM) includes both MM-related and MM-unrelated clonal expansions which are subject to dominance exerted by the MM clone. Here we show evidence for the existence of minor but highly expanded unrelated B-cell clones in patients with MM defined by their complementary determining region 3 (CDR3) peak. We further characterize these clones over the disease and subsequent treatment. Second clones were identified by their specific IgH-VDJ sequences that are distinct from those of dominant MM clones. Clonal frequencies were determined through semi-quantitative PCR, quantitative PCR and single-cell polymerase chain reaction of the clone-specific sequence. In 13/74 MM patients, more than one dominant CDR3 peak was identified with 12 patients (16%) being truly biclonal. Second clones had different frequencies, were found in different locations and were found in different cell types from the dominant MM clone. Where analysis was possible, they were shown to have chromosomal characteristic distinct from those of the MM clone. The frequency of the second clone also changed over the course of the disease and often persisted despite treatment. Molecularly-defined second clones are infrequent in monoclonal gammopathy of undetermined significance (MGUS, 1/43 individuals or 2%), suggesting that they may arise at relatively late stages of myelomagenesis. In further support of our findings, biclonal gammopathy and concomitant MM and CLL (chronic lymphocytic leukemia) were confirmed to originate from two unrelated clones. Our data supports the idea that the clone giving rise to symptomatic myeloma exerts clonal dominance to prevent expansion of other clones. MM and second clones may arise from an underlying niche permissive of clonal expansion. The clinical significance of these highly expanded but unrelated clones remains to be confirmed. Overall, our findings add new dimensions to evaluating related and unrelated clonal expansions in MM and the

  4. Frequent occurrence of highly expanded but unrelated B-cell clones in patients with multiple myeloma.

    Directory of Open Access Journals (Sweden)

    Jitra Kriangkum

    Full Text Available Clonal diversity in multiple myeloma (MM includes both MM-related and MM-unrelated clonal expansions which are subject to dominance exerted by the MM clone. Here we show evidence for the existence of minor but highly expanded unrelated B-cell clones in patients with MM defined by their complementary determining region 3 (CDR3 peak. We further characterize these clones over the disease and subsequent treatment. Second clones were identified by their specific IgH-VDJ sequences that are distinct from those of dominant MM clones. Clonal frequencies were determined through semi-quantitative PCR, quantitative PCR and single-cell polymerase chain reaction of the clone-specific sequence. In 13/74 MM patients, more than one dominant CDR3 peak was identified with 12 patients (16% being truly biclonal. Second clones had different frequencies, were found in different locations and were found in different cell types from the dominant MM clone. Where analysis was possible, they were shown to have chromosomal characteristic distinct from those of the MM clone. The frequency of the second clone also changed over the course of the disease and often persisted despite treatment. Molecularly-defined second clones are infrequent in monoclonal gammopathy of undetermined significance (MGUS, 1/43 individuals or 2%, suggesting that they may arise at relatively late stages of myelomagenesis. In further support of our findings, biclonal gammopathy and concomitant MM and CLL (chronic lymphocytic leukemia were confirmed to originate from two unrelated clones. Our data supports the idea that the clone giving rise to symptomatic myeloma exerts clonal dominance to prevent expansion of other clones. MM and second clones may arise from an underlying niche permissive of clonal expansion. The clinical significance of these highly expanded but unrelated clones remains to be confirmed. Overall, our findings add new dimensions to evaluating related and unrelated clonal expansions in

  5. Molecular localization of the t(11;22)(q24;q12) translocation of Ewing sarcoma by chromosomal in situ suppression hybridization

    International Nuclear Information System (INIS)

    Chromosome translocations are associated with a variety of human leukemias, lymphomas, and solid tumors. To localize molecular markers flanking the t(11;22)(q24;q12) breakpoint that occurs in virtually all cases of Ewing sarcoma and peripheral neuroepithelioma, high-resolution chromosomal in situ suppression hybridization was carried out using a panel of cosmid clones localized and ordered on chromosome 11q. The location of the Ewing sarcoma translocation breakpoint was determined relative to the nearest two cosmid markers on 11q, clones 23.2 and 5.8, through the analysis of metaphase chromosome hybridization. By in situ hybridization to interphase nuclei, the approximate physical separation of these two markers was determined. In both Ewing sarcoma and peripheral neuroepithelioma, cosmid clone 5.8 is translocated from chromosome 11q24 to the derivative chromosome 22 and a portion of chromosome 22q12 carrying the leukemia inhibitory factor gene is translocated to the derivative chromosome 11. The physical distance between the flanking cosmid markers on chromosome 11 was determined to be in the range of 1,000 kilobases, and genomic analysis using pulsed-field gel electrophoresis showed no abnormalities over a region of 650 kilobases in the vicinity of the leukemia inhibitory factor gene on chromosome 22. This approach localizes the Ewing sarcoma breakpoint to a small region on chromosome 11q24 and provides a rapid and precise technique for the molecular characterization of chromosomal aberrations

  6. Main: Clone Detail [KOME

    Lifescience Database Archive (English)

    Full Text Available Clone Detail Mapping Pseudomolecule data detail Detail information Mapping to the TIGR japonica Pseudomolecu...les kome_mapping_pseudomolecule_data_detail.zip kome_mapping_pseudomolecule_data_detail ...

  7. Placentation in cloned cattle

    DEFF Research Database (Denmark)

    Miglino, M A; Pereira, F T V; Visintin, J A;

    2007-01-01

    To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies...

  8. Do Managers Clone Themselves?

    Science.gov (United States)

    Baron, Alma S.

    1981-01-01

    A recent questionnaire survey provides statistics on male managers' views of female managers. The author recommends that male managers break out of their cloning behavior and that the goal ought to be a plurality in management. (Author/WD)

  9. Radiation-induced chromosomal instability in human mammary epithelial cells

    Science.gov (United States)

    Durante, M.; Grossi, G. F.; Yang, T. C.

    1996-01-01

    Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

  10. Asymmetric quantum cloning machines

    International Nuclear Information System (INIS)

    A family of asymmetric cloning machines for quantum bits and N-dimensional quantum states is introduced. These machines produce two approximate copies of a single quantum state that emerge from two distinct channels. In particular, an asymmetric Pauli cloning machine is defined that makes two imperfect copies of a quantum bit, while the overall input-to-output operation for each copy is a Pauli channel. A no-cloning inequality is derived, characterizing the impossibility of copying imposed by quantum mechanics. If p and p' are the probabilities of the depolarizing channels associated with the two outputs, the domain in (√p,√p')-space located inside a particular ellipse representing close-to-perfect cloning is forbidden. This ellipse tends to a circle when copying an N-dimensional state with N→∞, which has a simple semi-classical interpretation. The symmetric Pauli cloning machines are then used to provide an upper bound on the quantum capacity of the Pauli channel of probabilities px, py and pz. The capacity is proven to be vanishing if (√px, √py, √pz) lies outside an ellipsoid whose pole coincides with the depolarizing channel that underlies the universal cloning machine. Finally, the tradeoff between the quality of the two copies is shown to result from a complementarity akin to Heisenberg uncertainty principle. (author)

  11. Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina.

    Science.gov (United States)

    McLachlan, Elizabeth; White, Thomas W; Ugonabo, Chioma; Olson, Carl; Nagy, James I; Valdimarsson, Gunnar

    2003-09-15

    The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.

  12. CHROMOSOMES OF AMERICAN MARSUPIALS.

    Science.gov (United States)

    BIGGERS, J D; FRITZ, H I; HARE, W C; MCFEELY, R A

    1965-06-18

    Studies of the chromosomes of four American marsupials demonstrated that Caluromys derbianus and Marmosa mexicana have a diploid number of 14 chromosomes, and that Philander opossum and Didelphis marsupialis have a diploid number of 22. The karyotypes of C. derbianus and M. mexicana are similar, whereas those of P. opossum and D. marsupialis are dissimilar. If the 14-chromosome karyotype represents a reduction from a primitive number of 22, these observations suggest that the change has occurred independently in the American and Australasian forms.

  13. Molecular cloning and expression of a stress-inducible gene highly homologous to human structural maintenance of chromosome 6%与人染色体结构维持基因6高度同源应力可诱导基因克隆及其表达

    Institute of Scientific and Technical Information of China (English)

    王光平; 蔡维君

    2008-01-01

    laboratory of Department of Experimental Cardiology of Max-Planck Institute for Physiological and Clinical Research at Bad Nauheim in Germany and the Department of Hematology, Xiangya Hospital of Central South University from July in 1998 to May in 2007. All the animals were anesthetized and thoracotomized. Following 30 minutes of stabilization, the left anterior descending coronary artery (LAD) was occluded for 10 minutes followed by 30-minute reperfusion and then another 10-minute reocclusion. The porcines were killed immediately at corresponding time points. The sham-operated animals were killed without occlusion. Experimental tissue was removed from the LAD area and control tissue from the region of the left circumflex coronary artery area. Firstly, a porcine heart cDNA library was screened, DNA and deduced amino acid sequences were then analyzed. Meanwhile, short periods of myocardium ischemia/reperfusion was performed by occluding porcine LAD followed by reperfusion as mentioned above, and total RNAs isolated from myocardium or a variety of other tissues were used for Northern blotting. Quantitation of mRNA levels was accomplished by using a PhosphorImager and Image Quant software, the ration of mRNA/18S rRNA was suggested as the level of gene expression.MAIN OUTCOME MEASURES: ① DNA and amino acid sequences analysis of the cloned gene; ② Analysis of the cloned gene expression.RESULTS: All the 16 porcines were involved in the final analysis of results. After screening, a cDNA fragment with 3 461 base pairs was obtained. DNA sequencing and searching revealed that this cDNA shared 86% identity to human structural maintenance of chromosome 6 gene (hSMC6), and 84% identity to Mus musculus SMC6 (mSMC6). Furthermore, a largest polypeptide deduced from this cDNA contained 1007 amino acid residues, and protein homology searching indicated that the predicted polypeptide carried 92% and 89% identity to hSMC6 or mSMC6 protein respectively. Thus, the cloned cDNA was referred to

  14. Comparative sequence analysis of a highly oncogenic but horizontal spread-defective clone of Marek's disease virus.

    Science.gov (United States)

    Spatz, Stephen J; Zhao, Yuguang; Petherbridge, Lawrence; Smith, Lorraine P; Baigent, Susan J; Nair, Venugopal

    2007-12-01

    Marek's disease virus (MDV) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. MDV isolates vary greatly in pathogenicity. While some of the strains such as CVI988 are non-pathogenic and are used as vaccines, others such as RB-1B are highly oncogenic. Molecular determinants associated with differences in pathogenicity are not completely understood. Comparison of the genome sequences of phenotypically different strains could help to identify molecular determinants of pathogenicity. We have previously reported the construction of bacterial artificial chromosome (BAC) clones of RB-1B from which fully infectious viruses could be reconstituted upon DNA transfection into chicken cells. MDV reconstituted from one of these clones (pRB-1B-5) showed similar in vitro and in vivo replication kinetics and oncogenicity as the parental virus. However, unlike the parental RB-1B virus, the BAC-derived virus showed inability to spread between birds. In order to identify the unique determinants for oncogenicity and the ''non-spreading phenotype'' of MDV derived from this clone, we determined the full-length sequence of pRB-1B-5. Comparative sequence analysis with the published sequences of strains such as Md5, Md11, and CVI988 identified frameshift mutations in RLORF1, protein kinase (UL13), and glycoproteins C (UL44) and D (US6). Comparison of the sequences of these genes with the parental virus indicated that the RLORF1, UL44, and US6 mutations were also present in the parental RB-1B stock of the virus. However with regard to UL13 mutation, the parental RB-1B stock appeared to be a mixture of wild type and mutant viruses, indicating that the BAC cloning has selected a mutant clone. Although further studies are needed to evaluate the role of these genes in the horizontal-spreading defective phenotype, our data clearly indicate that mutations in these genes do not affect the oncogenicity of MDV.

  15. Chromosomal abnormalities and autism

    Directory of Open Access Journals (Sweden)

    Farida El-Baz

    2016-01-01

    Conclusion: Chromosomal abnormalities were not detected in the studied autistic children, and so the relation between the genetics and autism still needs further work up with different study methods and techniques.

  16. Chromosome condensation and segmentation

    International Nuclear Information System (INIS)

    Some aspects of chromosome condensation in mammalians -humans especially- were studied by means of cytogenetic techniques of chromosome banding. Two further approaches were adopted: a study of normal condensation as early as prophase, and an analysis of chromosome segmentation induced by physical (temperature and γ-rays) or chemical agents (base analogues, antibiotics, ...) in order to show out the factors liable to affect condensation. Here 'segmentation' means an abnormal chromosome condensation appearing systematically and being reproducible. The study of normal condensation was made possible by the development of a technique based on cell synchronization by thymidine and giving prophasic and prometaphasic cells. Besides, the possibility of inducing R-banding segmentations on these cells by BrdU (5-bromodeoxyuridine) allowed a much finer analysis of karyotypes. Another technique was developed using 5-ACR (5-azacytidine), it allowed to induce a segmentation similar to the one obtained using BrdU and identify heterochromatic areas rich in G-C bases pairs

  17. Chromosomal Abnormalties with Epilepsy

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2005-02-01

    Full Text Available The correlation between specific chromosome abnormalties and various epilepsies was investigated by a study of 76 patients’ records obtained by questionnaires distributed to members of Kyoto Multi-institutional Study Group of Pediatric Neurology.

  18. Self-Compatibility Crosses of Several Cocoa Clones

    OpenAIRE

    Agung Wahyu Susilo

    2006-01-01

    Self compatibility cross in cocoa is a useful criterion in utilizing germplasm collection. Evaluation of self-compatibility crosses on cocoa clones has been carried out in Kaliwining Experimental Station of ICCRI by treating artificial self-pollination. The observed clones were TSH 858, ICS 60, ICS 13, UIT 1, KW 162, KW 165, KW 163, DR 1, DR 2, DRC 16, DRC 15, KKM 22, Na 32, Na 33 and DR 38. Self-compatibility crosses were identified by percentage of fruit set evaluated during 6 consecutive w...

  19. Chimpanzee chromosome 13 is homologous to human chromosome 2p

    Energy Technology Data Exchange (ETDEWEB)

    Sun, N. C.; Sun, C. R.Y.; Ho, T.

    1977-01-01

    Similarities between human and chimpanzee chromosomes are shown by chromosome banding techniques and somatic cell hybridization techniques. Cell hybrids were obtained from the chimpanzee lymphocyte LE-7, and the Chinese hamster mutant cell, Gal-2. Experiments showed that the ACPL, MDHs, and Gal-Act genes could be assigned to chimpanzee chromosome 13, and since these genes have been assigned to human chromosme 2p, it is suggested that chimpanzee chromosome 13 is homologous to human chromosome 2p. (HLW)

  20. [Advances in Molecular Cloning].

    Science.gov (United States)

    Ashwini, M; Murugan, S B; Balamurugan, S; Sathishkumar, R

    2016-01-01

    "Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems. PMID:27028806

  1. Chromosome doubling method

    Science.gov (United States)

    Kato, Akio

    2006-11-14

    The invention provides methods for chromosome doubling in plants. The technique overcomes the low yields of doubled progeny associated with the use of prior techniques for doubling chromosomes in plants such as grasses. The technique can be used in large scale applications and has been demonstrated to be highly effective in maize. Following treatment in accordance with the invention, plants remain amenable to self fertilization, thereby allowing the efficient isolation of doubled progeny plants.

  2. Strategy for Cloning Large Gene Assemblages as Illustrated Using the Phenylacetate and Polyhydroxyalkanoate Gene Clusters

    OpenAIRE

    García, Belén; Olivera, Elías R.; Sandoval, Ángel; Arias-Barrau, Elsa; Arias, Sagrario; Naharro, Germán; Luengo, José M.

    2004-01-01

    We report an easy procedure for isolating chromosome-clustered genes. By following this methodology, the entire set of genes belonging to the phenylacetic acid (PhAc; 18-kb) pathway as well as those required for the synthesis and mobilization of different polyhydroxyalkanoates (PHAs; 6.4 kb) in Pseudomonas putida U were recovered directly from the bacterial chromosome and cloned into a plasmid for the first time. The transformation of different bacteria with these genetic constructions confer...

  3. Micromechanics of human mitotic chromosomes

    International Nuclear Information System (INIS)

    Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed

  4. A physical map of the hyperthermophilic bacterium Aquifex pyrophilus chromosome.

    OpenAIRE

    Shao, Z; Mages, W; Schmitt, R.

    1994-01-01

    A genomic map of the hyperthermophilic hydrogen-oxidizing bacterium Aquifex pyrophilus was established with NotI (GC/GGCCGC), SpeI (A/CTAGT), and XbaI (T/CTAGA). Linking clones and cross-hybridization of restriction fragments revealed a single circular chromosome of 1.6 Mbp. A single flagellin gene and six rRNA gene units were located on this map by Southern hybridization.

  5. An improved method for producing radiation hybrids applied to human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, C.L.

    1992-01-01

    At the initiation of the grant we had just produced radiation hybrids from a monochromosomal microcell hybrid containing human chromosome 19 as its only human component. Radiation hybrids were produced using doses of radiation ranging from 1000--8000 rads. Lethally irradiated cells were then fused to hamster recipients (CHTG49) and selected for growth in histidinol. Approximately 240 clones were isolated and 75 clones were expanded for the isolation of DNA. This report describes in situ hybridization studies and the introduction of markers into human chromosome 19.

  6. Cloning-free CRISPR

    OpenAIRE

    Mandana Arbab; Sharanya Srinivasan; Tatsunori Hashimoto; Niels Geijsen; Richard I. Sherwood

    2015-01-01

    Summary We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produ...

  7. Trends in Artificial Intelligence.

    Science.gov (United States)

    Hayes, Patrick

    1978-01-01

    Discusses the foundations of artificial intelligence as a science and the types of answers that may be given to the question, "What is intelligence?" The paradigms of artificial intelligence and general systems theory are compared. (Author/VT)

  8. Structural organization of the inactive X chromosome in the mouse.

    Science.gov (United States)

    Giorgetti, Luca; Lajoie, Bryan R; Carter, Ava C; Attia, Mikael; Zhan, Ye; Xu, Jin; Chen, Chong Jian; Kaplan, Noam; Chang, Howard Y; Heard, Edith; Dekker, Job

    2016-07-28

    X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions,remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes indifferent neural progenitor clones are associated with the presence of different TAD

  9. Structural organization of the inactive X chromosome in the mouse.

    Science.gov (United States)

    Giorgetti, Luca; Lajoie, Bryan R; Carter, Ava C; Attia, Mikael; Zhan, Ye; Xu, Jin; Chen, Chong Jian; Kaplan, Noam; Chang, Howard Y; Heard, Edith; Dekker, Job

    2016-07-28

    X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions,remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes indifferent neural progenitor clones are associated with the presence of different TAD

  10. Affected chromosome homeostasis and genomic instability of clonal yeast cultures.

    Science.gov (United States)

    Adamczyk, Jagoda; Deregowska, Anna; Panek, Anita; Golec, Ewelina; Lewinska, Anna; Wnuk, Maciej

    2016-05-01

    Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events-disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair.

  11. Artificial Inteligence and Law

    OpenAIRE

    Fuková, Kateřina

    2012-01-01

    Submitted diploma work Artificial Intelligence and Law deals with the rule of law and its position in the process of new advanced technologies in computer cybernetics and further scientific disciplines related with artificial intelligence and its creation. The first part of the work introduces the history of the first imagines about artificial intelligence and concerns with its birth. This chapter presents main theoretical knowledge and hypotheses defined artificial intelligence and progre...

  12. Development of a novel HAC-based "gain of signal" quantitative assay for measuring chromosome instability (CIN) in cancer cells

    OpenAIRE

    Kim, Jung Hyun; Lee, Hee Sheung; Lee, Nicholas C.O.; Goncharov, Nikolay V.; Kumeiko, Vadim; Masumoto, Hiroshi; Earnshaw, William C.; Kouprina, Natalay; Larionov, Vladimir

    2016-01-01

    Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene ("loss of signal" a...

  13. A Seminar on Human Cloning: Cloning in Reproductive Medicine

    OpenAIRE

    Illmensee, Karl

    2001-01-01

    This review article summarizes the historical development of mammalian cloning, presents current advances and presumed risk factors in the field of reproductive cloning, discusses possible clinical applications of therapeutic and diagnostic cloning and outlines prospective commercial trends in pharmacytical cloning. Predictable progress in biotechnology and stem cell engineering should prove to be advantageous for patients' health and for novel benefits in reproductive and regenerative medicine.

  14. Laser microdissection-based analysis of the Y sex chromosome of the Antarctic fish Chionodraco hamatus (Notothenioidei, Channichthyidae

    Directory of Open Access Journals (Sweden)

    Ennio Cocca

    2015-02-01

    Full Text Available Microdissection, DOP-PCR amplification and microcloning were used to study the large Y chromosome of Chionodraco hamatus, an Antarctic fish belonging to the Notothenioidei, the dominant component of the Southern Ocean fauna. The species has evolved a multiple sex chromosome system with digametic males showing an X1YX2 karyotype and females an X1X1X2X2 karyotype. Fluorescence in situ hybridization, performed with a painting probe made from microdissected Y chromosomes, allowed a deeper insight on the chromosomal rearrangement, which underpinned the fusion event that generated the Y. Then, we used a DNA library established by microdissection and microcloning of the whole Y chromosome of Ch. hamatus for searching sex-linked sequences. One clone provided preliminary information on the presence on the Y chromosome of the CHD1 gene homologue, which is sex-linked in birds but in no other vertebrates. Several clones from the Y-chromosome mini-library contained microsatellites and transposable elements, one of which mapped to the q arm putative fusion region of the Y chromosome. The findings confirm that interspersed repetitive sequences might have fostered chromosome rearrangements and the emergence of the Y chromosome in Ch. hamatus. Detection of the CHD1 gene in the Y sex-determining region could be a classical example of convergent evolution in action.

  15. Secure the Clones

    Science.gov (United States)

    Jensen, Thomas; Kirchner, Florent; Pichardie, David

    Exchanging mutable data objects with untrusted code is a delicate matter because of the risk of creating a data space that is accessible by an attacker. Consequently, secure programming guidelines for Java stress the importance of using defensive copying before accepting or handing out references to an internal mutable object. However, implementation of a copy method (like clone()) is entirely left to the programmer. It may not provide a sufficiently deep copy of an object and is subject to overriding by a malicious sub-class. Currently no language-based mechanism supports secure object cloning. This paper proposes a type-based annotation system for defining modular copy policies for class-based object-oriented programs. A copy policy specifies the maximally allowed sharing between an object and its clone. We present a static enforcement mechanism that will guarantee that all classes fulfill their copy policy, even in the presence of overriding of copy methods, and establish the semantic correctness of the overall approach in Coq. The mechanism has been implemented and experimentally evaluated on clone methods from several Java libraries.

  16. Clip, Connect, Clone

    DEFF Research Database (Denmark)

    Fujima, Jun; Lunzer, Aran; Hornbæk, Kasper Anders Søren;

    2010-01-01

    using three mechanisms: clipping of input and result elements from existing applications to form cells on a spreadsheet; connecting these cells using formulas, thus enabling result transfer between applications; and cloning cells so that multiple requests can be handled side by side. We demonstrate...

  17. Asian Yellow Goat Cloned

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ It was released on August 24,2005 by Prof. CHEN Dayuan (Da-Yuan Chen) from the CAS Institute of Zoology that the first success in cloning the Asian Yellow Goat by nuclear transfer had recently been achieved in east China's Shandong Province.

  18. Animal Cloning and Food Safety

    Science.gov (United States)

    ... Products For Consumers Home For Consumers Consumer Updates Animal Cloning and Food Safety Share Tweet Linkedin Pin it ... This conclusion stems from an extensive study of animal cloning and related food safety, culminating in the release ...

  19. The First Human Cloned Embryo.

    Science.gov (United States)

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  20. Cloning and Orientation of the Gene Encoding Polynucleotide Phosphorylase in Escherichia coli

    OpenAIRE

    Crofton, S; Dennis, P P

    1983-01-01

    Mutations which affect the activity of polynucleotide phosphorylase (PNPase) map near 69 min on the bacterial chromosome. This region of the chromosome has been cloned by inserting the kanamycin-resistant transposon Tn5 near the argG and mtr loci at 68.5 min. Large SalI fragments of chromosomal DNA containing the Tn5 element were inserted into pBR322, and selection was made for kanamycin-resistant recombinant plasmids. Two of these plasmids were found to produce high levels of PNPase activity...

  1. Chromosome duplication in Lolium multiflorum Lam.

    Directory of Open Access Journals (Sweden)

    Roselaine Cristina Pereira

    2014-11-01

    Full Text Available Artificial chromosome duplication of diploid genotypes of Lolium multiflorum (2n=2x=14 is worthy to breeding, and aims to increase the expression of traits with agronomic interest. The purpose of this study was to obtain polyploid plants of L. multiflorum from local diploid populations in order to exploit adaptation and future verification of the effects of polyploidy in agronomic traits. Seedlings were immersed in different colchicine solutions for an exposure time of 3h and 24h. Ploidy determination was made by the DNA content and certified by chromosomes counts. The plants confirmed as tetraploids were placed in a greenhouse, and, at flowering, pollen viability was evaluated, and seeds were harvested to assess the stability of the progenies. The percentage of polyploids obtained was 20%. Pollen viability of the tetraploids generated ranged from 58% to 69%. The tetraploid plants obtained in the experiment generated 164 progenies, of which 109 presented DNA content compatible with the tetraploid level, showing stability of chromosome duplication in the filial generation.

  2. Sex determination by chromosome manipulation in fish

    International Nuclear Information System (INIS)

    Since it is impossible to artificially remove only sex chromosomes in sperm, gamma- or UV-irradiation has been used in destroying all chromosomes without loss of abilities of sperm movement and egg activation. It has been shown that a dose of gamma rays required for this purpose is 105 rad in any species of fish. For UV-irradiation, a 15 W lamp is used and irradiation for 60 to 120 seconds is required. With such an irradiation technique, gynogenetic haploid embryogenesis is induced. In developing normal diploid embryos of eggs inseminated with irradiated sperm (gynogenetic diploid embryogenesis with XX type), it is furthermore necessary to use physical procedures, such as low or high temperature and hydrostatic pressure. Irradiated sperm of different species of fish has also been used in inducing gynogenesis. As the most desirable technique, it is proposed to physiologically convert the sex of gynogenetic diploid embryos into males and to use sperm from those physiological males with XX chromosomes. Theoretical possibility of developing androgenetic haploid embryogenesis has been suggested. (Namekawa, K.)

  3. HUMAN CLONING AND FDA REGULATION

    OpenAIRE

    Vosk, Ted

    1998-01-01

    In the February 27, 1997 issue of the journal Nature scientists from Scotland's Roslin Institute reported their successful efforts to clone an adult sheep using differentiated somatic cells from the animal. The clone, named Dolly, was the first instance of the successful cloning of an adult mammal. The shock waves created by the possible ramifications of this development were immediately felt around the world. For the first time, the cloning of an adult human being was no longer being conside...

  4. Probabilistic Cloning and Quantum Computation

    Institute of Scientific and Technical Information of China (English)

    GAO Ting; YAN Feng-Li; WANG Zhi-Xi

    2004-01-01

    @@ We discuss the usefulness of quantum cloning and present examples of quantum computation tasks for which the cloning offers an advantage which cannot be matched by any approach that does not resort to quantum cloning.In these quantum computations, we need to distribute quantum information contained in the states about which we have some partial information. To perform quantum computations, we use a state-dependent probabilistic quantum cloning procedure to distribute quantum information in the middle of a quantum computation.

  5. Construction of a chromosome specific library of human MARs and mapping of matrix attachment regions on human chromosome 19.

    Science.gov (United States)

    Nikolaev, L G; Tsevegiyn, T; Akopov, S B; Ashworth, L K; Sverdlov, E D

    1996-01-01

    Using a novel procedure a representative human chromosome 19-specific library was constructed of short sequences, which bind preferentially to the nuclear matrix (matrix attachment regions, or MARs). Judging by 20 clones sequenced so far, the library contains > 50% of human inserts, about 90% of which are matrix-binding by the in vitro test. Computer analysis of sequences of eight human MARs did not reveal any significant homologies with the EMBL Nucleotide Data Base entries as well as between MARs themselves. Eight MARs were assigned to individual positions on the chromosome 19 physical map. The library constructed can serve as a good source of MAR sequences for comparative analysis and classification and for further chromosome mapping of MARs as well. PMID:8614638

  6. Physical Characterization of human centromeric regions using transformation-associated recombination cloning technology

    Energy Technology Data Exchange (ETDEWEB)

    Vladimir Larionov, Ph D

    2007-06-05

    A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that can be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii

  7. Chromosome numbers in Bromeliaceae

    Directory of Open Access Journals (Sweden)

    Cotias-de-Oliveira Ana Lúcia Pires

    2000-01-01

    Full Text Available The present study reports chromosome numbers of 17 species of Bromeliaceae, belonging to the genera Encholirium, Bromelia, Orthophytum, Hohenbergia, Billbergia, Neoglaziovia, Aechmea, Cryptanthus and Ananas. Most species present 2n = 50, however, Bromelia laciniosa, Orthophytum burle-marxii and O. maracasense are polyploids with 2n = 150, 2n = 100 and 2n = 150, respectively, while for Cryptanthus bahianus, 2n = 34 + 1-4B. B chromosomes were observed in Bromelia plumieri and Hohenbergia aff. utriculosa. The chromosome number of all species was determined for the first time, except for Billbergia chlorosticta and Cryptanthus bahianus. Our data supports the hypothesis of a basic number of x = 25 for the Bromeliaceae family and decreasing aneuploidy in the genus Cryptanthus.

  8. Those amazing dinoflagellate chromosomes

    Institute of Scientific and Technical Information of China (English)

    PETER J RIZZO

    2003-01-01

    Dinoflagellates are a very large and diverse group of eukaryotic algae that play a major role in aquatic food webs of both fresh water and marine habitats. Moreover, the toxic members of this group pose a health threat in the form of red tides. Finally, dinoflagellates are of great evolutionary importance,because of their taxonomic position, and their unusual chromosome structure and composition. While the cytoplasm of dinoflagellates is typically eukaryotic, the nucleus is unique when compared to the nucleus of other eukaryotes. More specifically, while the chromosomes of all other eukaryotes contain histones,dinoflagellate chromosomes lack histones completely. There are no known exceptions to this observation: all dinoflagellates lack histones, and all other eukaryotes contain histones. Nevertheless, dinoflagellates remain a relatively unstudied group of eukaryotes.

  9. Unique inclusions of maximal C-clones in maximal clones

    OpenAIRE

    Behrisch, Mike; Vargas-García, Edith

    2014-01-01

    $\\mathit{C}$-clones are polymorphism sets of so-called clausal relations, a special type of relations on a finite domain, which first appeared in connection with constraint satisfaction problems in [Creignou et al. 2008]. We completely describe the relationship w.r.t. set inclusion between maximal $\\mathit{C}$-clones and maximal clones. As a main result we obtain that for every maximal $\\mathit{C}$-clone there exists exactly one maximal clone in which it is contained. A precise description of...

  10. Comparative mapping of DNA probes derived from the V{sub k} immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, N.; Wienberg, J.; Ermert, K. [Universitaet Muenchen (Germany)] [and others

    1995-03-01

    Fluorescence in situ hybridization (FISH) of cosmid clones of human V{sub K} gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the K locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo Pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore, be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V{sub K}I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V{sub K} region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution. 18 refs., 2 figs.

  11. Chromosomal assignments of the genes coding for human types II, III, and IV collagen: a dispersed gene family.

    OpenAIRE

    Solomon, E; Hiorns, L R; Spurr, N; Kurkinen, M.; Barlow, D; Hogan, B L; Dalgleish, R.

    1985-01-01

    The human type II collagen gene, COL2A1, has been assigned to chromosome 12, the type III gene, COL3A1, to chromosome 2, and one of the type IV genes, COL4A1, to chromosome 13. These assignments were made by using cloned genes as probes on Southern blots of DNA from a panel of mouse/human somatic cell hybrids. The two genes of type I collagen, COL1A1 and COL2A1, have been mapped previously to chromosomes 17 and 7, respectively. This family of conserved genes seems therefore to be dispersed th...

  12. Quality and safety of bovine clones and their products.

    Science.gov (United States)

    Heyman, Y; Chavatte-Palmer, P; Fromentin, G; Berthelot, V; Jurie, C; Bas, P; Dubarry, M; Mialot, J P; Remy, D; Richard, C; Martignat, L; Vignon, X; Renard, J P

    2007-08-01

    A multidisciplinary research programme was developed to get a scientific expertise for the quality assessment of products obtained from cloned livestock. Thirty-seven bovine Holstein female clones of five different genotypes and their products were analysed in comparison with 38 control animals obtained by conventional artificial insemination and raised under the same conditions at the same experimental farm. Animal evaluation included over 150 criteria and more than 10 000 measurements to check the physiological status and health over a 3-year period. All the parameters studied were in the normal range for age and breed, but some significant differences were detected between clone and control groups in terms of delayed onset of puberty in clones, higher neutrophil counts in haematology or lower biochemical plasma concentrations of gamma glutamyl transferase. Milk and meat analyses were conformable to expected values. We, however, found some differences in fatty acid (FA) composition of milk and muscle suggesting a possible deviation in lipid metabolism as assessed by higher delta-9 desaturase activity indexes in both milk and muscles from clones compared with controls. Repeated muscle biopsies in the semitendinosus muscle of the same animals demonstrated a higher oxidative activity in muscle of young clones (8 months of age) compared with controls, suggesting a delayed muscle maturation in clones. Nutritional evaluation of milk and meat using the rat feeding trials did not show any difference between clone and control products for food intake, growth rate, body composition of the rats, nor for possible allergenicity. Possible reactivation of bovine endogenous retroviruses (BERVs) was analysed and compared between normal and cloned cattle. As expected, these BERV sequences are not transcribed and no RNA was detected in the blood of clones, donor animals or controls; therefore, it may be assumed that the sanitary risk associated with BERV sequences is not higher in

  13. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    Energy Technology Data Exchange (ETDEWEB)

    Rubin, Edward M.

    1980-01-01

    Bacillus subtilis carries two genes, thyA and thyB, each encoding different protein products, with thymidylate synthetase (TSase) activity. Either of these genes alone is sufficient for thymidine independence in B. subtilis. In addition there exist two B. subtilis temperate bacteriophages which upon infection of thymine requiring auxotrophs results in conversion of the organism to thymine independence. Chimeric plasmids selected for Thy/sup +/ transforming activity in E. coli were constructed and then used as a source of defined highly enriched DNA with which to transform competent B. subtilis. These plasmids were studied for their: (1) abiility to transform B. subtilis to thymine independence; (2) site of integration within the B. subtilis chromosome upon transformation; (3) phenotype of Thy/sup +/ plasmid generated transformants; and (4) nucleotide sequence homology among the cloned DNA fragments conferring thymine independence. Plasmids containing the two bacteriophage thy genes displayed the phenotype associated with thyA, whereas the plasmids containing the cloned B. subtilis chromosomal genes displayed the phenotype associated with thyB. Utilizing similar technology, the ability of an entirely foreign hybred bacterial plasmiid to transform B. subtilis was examined. In this case the gene from E. coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid was effective in transforming both E. coli and B. subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine requiring strains of B. subtilis to thymine independence. Although the Thy/sup +/ transformants of E. coli contained plasmid DNA, the Thy/sup +/ transformants derived from the transformation of B. subtilis did not contain detectable extrachromosomal DNA. Instead the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis. (ERB)

  14. The Staurotypus turtles and aves share the same origin of sex chromosomes but evolved different types of heterogametic sex determination.

    Directory of Open Access Journals (Sweden)

    Taiki Kawagoshi

    Full Text Available Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY and female heterogametic (ZZ/ZW sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (Staurotypus triporcatus, Kinosternidae, Testudines and the giant musk turtle (Staurotypus salvinii have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (Pelodiscus sinensis revealed that the X and Y chromosomes of S. triporcatus have homology with P. sinensis chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of S. triporcatus homologs of 16 chicken Z-linked genes and mapped them to S. triporcatus and S. salvinii chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (Struthio camelus Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of Staurotypus turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.

  15. Localization of the casein gene family to a single mouse chromosome

    OpenAIRE

    1982-01-01

    A series of mouse-hamster somatic cell hybrids containing a variable number of mouse chromosomes and a constant set of hamster chromosomes have been used to determine the chromosomal location of a family of hormone-inducible genes, the murine caseins. Recombinant mouse cDNA clones encoding the alpha-, beta-, and gamma-caseins were constructed and used in DNA restriction mapping experiments. All three casein cDNAs hybridized to the same set of somatic cell hybrid DNAs isolated from cells conta...

  16. 用高分辨G带和人工细菌染色体荧光原位杂交技术分析中国儿童孤独症患者的染色体改变%Detection of chromosome aberrations in Chinese children with autism using G-banding and BAC FISH

    Institute of Scientific and Technical Information of China (English)

    刘青杰; 陈晓宁; 沈岩; 马芬; 李丹; 王晓维; 田文雁; 陈艳; 封江彬; 陆雪; 陈德清

    2005-01-01

    Objective To detect the characteristic chromosomal changes in Chinese children with infantile autism. Methods Chromosome aberrations in 68 cases of infantile autism were analyzed by high-resolution G-banding and fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) clones. Results Chromosomal changes were detected in 4 cases by high-resolution G-banding: one case with t(4;6)(q23-24;p21), one case with longer p arm of chromosome 21 (21p+), and two cases with pericentric inversion of chromosome 9 (inv(9)) which was confirmed by C-banding. BAC FISH analysis was performed to confirm these observations and changes in chromosomes 2, 7 and 15, which are often found in autistic children. There could exist the translocation of t(4;6) (q25-26;p21.1). Chromosome changes often reported previously in chromosomes 2, 7 and 15 were not detected in this study. Inv(9) and 21p+ were not confirmed with present BAC clones. Conclusion Chromosomal changes were detected in four cases of infantile autism, with a detectability of 5.9%, far lower than that (10% to 48%) reported in literature. The breakpoint of translocation could be detected more accurately using BAC FISH method.%目的检测中国儿童孤独症患者的特征性染色体改变.方法应用高分辨G带和人工细菌染色体(bacterial artificial chromosome, BAC)荧光原位杂交(flourescence in situ hybridization, FISH)分析68例中国儿童孤独症患者的染色体改变.结果用G带分析观察到有染色体改变的4例患者,分别为1例t(4;6)(q23-24;p21)、1例21p+和2例9号染色体臂间倒位.BAC FISH进一步证实易位病例,而且更精确[t(4;6)(q25-26;p21.1)];涉及7号、15号、2号染色体的BAC FISH均未观察到文献中报道的染色体改变;而9号染色体的臂间倒位和21p+因无BAC克隆而无法证实.结论用G带和BAC FISH发现少数中国孤独症患者有染色体改变,但远没有文献中报道的10%~48%那么高.BAC FISH有助于精确地确定染色体易位断裂点.

  17. Altered metaphase chromosome structure in xrs-5 cells is not related to its radiation sensitivity or defective DNA break rejoining

    International Nuclear Information System (INIS)

    The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive derivative of CHO-K1 cells. The xrs-5 cells have a defect in DNA double-strand break rejoining and show alterations in chromosome structure and nuclear morphology. The relationship between radiation sensitivity and metaphase chromosome morphology was examined in 12 'revertant' xrs-5 clones isolated following treatment with 5-azacytidine. Nine of the clones were radioresistant while the other three retained xrs-5-like radiation sensitivity. Chromosome morphology reverted to CHO-K1-like characteristics in three of the radioresistant clones and one of the radiosensitive clones suggesting that the over-condensed metaphase chromosome morphology of xrs-5 cells does not underlie its radiation sensitivity. Radiation sensitivity did correlate with DNA double-strand break rejoining ability. The radioresistant clones showing the over-condensed xrs-5-like chromosome morphology were also slightly more sensitive to the topoisomerase II inhibitor etoposide (VP-16) than CHO-K1, suggesting that the over-condensed morphology might be due to alterations in the phosphorylation of chromatin proteins

  18. A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

    Directory of Open Access Journals (Sweden)

    Zhou Li

    2011-03-01

    Full Text Available Abstract Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo

  19. Post-death cloning of endangered Jeju black cattle (Korean native cattle): fertility and serum chemistry in a cloned bull and cow and their offspring.

    Science.gov (United States)

    Kim, Eun Young; Song, Dong Hwan; Park, Min Jee; Park, Hyo Young; Lee, Seung Eun; Choi, Hyun Yong; Moon, Jeremiah Jiman; Kim, Young Hoon; Mun, Seong Ho; Oh, Chang Eon; Ko, Moon Suck; Lee, Dong Sun; Riu, Key Zung; Park, Se Pill

    2013-12-17

    To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC. PMID:23955237

  20. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  1. Overlap extension PCR cloning.

    Science.gov (United States)

    Bryksin, Anton; Matsumura, Ichiro

    2013-01-01

    Rising demand for recombinant proteins has motivated the development of efficient and reliable cloning methods. Here we show how a beginner can clone virtually any DNA insert into a plasmid of choice without the use of restriction endonucleases or T4 DNA ligase. Chimeric primers encoding plasmid sequence at the 5' ends and insert sequence at the 3' ends are designed and synthesized. Phusion(®) DNA polymerase is utilized to amplify the desired insert by PCR. The double-stranded product is subsequently employed as a pair of mega-primers in a PCR-like reaction with circular plasmids. The original plasmids are then destroyed in restriction digests with Dpn I. The product of the overlap extension PCR is used to transform competent Escherichia coli cells. Phusion(®) DNA polymerase is used for both the amplification and fusion reactions, so both steps can be monitored and optimized in the same way. PMID:23996437

  2. The Y Chromosome

    Science.gov (United States)

    Offner, Susan

    2010-01-01

    The Y chromosome is of great interest to students and can be used to teach about many important biological concepts in addition to sex determination. This paper discusses mutation, recombination, mammalian sex determination, sex determination in general, and the evolution of sex determination in mammals. It includes a student activity that…

  3. Chromosomes, cancer and radiosensitivity

    International Nuclear Information System (INIS)

    Some specific chromosomal abnormalities are associated with certain cancers. The earliest description of such a specific association is the one of the Philadelphia chromosome and myelogenous leukemia (1960). Other congenital karyotype abnormalities are associated with specific cancers. Examples of these are Down's syndrome with leukemia and Klinefelter's syndrome with male breast cancer. Genetic diseases of increased chromosome breakage, or of defective chromosome repair, are associated with greatly increased cancer incidence. Three such diseases have been recognized: 1) Fanconi's anemia, associated with leukemias and lymphomas, 2) Bloom's syndrome, associated with acute leukemias and lymphosarcoma, and 3) ataxia telangiectasia, associated with Hodgkin's disease, leukemia, and lymphosarcomas. Ten percent of individuals with ataxia telangiectasia will develop one of these neoplasms. Individuals with certain of these syndromes display an unusually high radiosensitivity. Radiation therapy for cancers has been fatal in patients who received as low as 3000 rad. This remarkable radiosensitivity has been quantitated in cell cultures from such cases. Evidence suggests that the apparent sensitivity may reflect subnormal ability to repair radiation damage. The rapid proliferation of information in this field stems from the interdigitation of many disciplines and specialties, including cytogenetics, cell biology, molecular biology, epidemiology, radiobiology, and several others. This paper is intended for clinicians; it presents a structured analytic scheme for correlating and classifying this multidisciplinary information as it becomes available

  4. Chromosomes, cancer and radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Samouhos, E.

    1983-08-01

    Some specific chromosomal abnormalities are associated with certain cancers. The earliest description of such a specific association is the one of the Philadelphia chromosome and myelogenous leukemia (1960). Other congenital karyotype abnormalities are associated with specific cancers. Examples of these are Down's syndrome with leukemia and Klinefelter's syndrome with male breast cancer. Genetic diseases of increased chromosome breakage, or of defective chromosome repair, are associated with greatly increased cancer incidence. Three such diseases have been recognized: 1) Fanconi's anemia, associated with leukemias and lymphomas, 2) Bloom's syndrome, associated with acute leukemias and lymphosarcoma, and 3) ataxia telangiectasia, associated with Hodgkin's disease, leukemia, and lymphosarcomas. Ten percent of individuals with ataxia telangiectasia will develop one of these neoplasms. Individuals with certain of these syndromes display an unusually high radiosensitivity. Radiation therapy for cancers has been fatal in patients who received as low as 3000 rad. This remarkable radiosensitivity has been quantitated in cell cultures from such cases. Evidence suggests that the apparent sensitivity may reflect subnormal ability to repair radiation damage. The rapid proliferation of information in this field stems from the interdigitation of many disciplines and specialties, including cytogenetics, cell biology, molecular biology, epidemiology, radiobiology, and several others. This paper is intended for clinicians; it presents a structured analytic scheme for correlating and classifying this multidisciplinary information as it becomes available.

  5. Ethical issues in livestock cloning.

    Science.gov (United States)

    Thompson, P B

    1999-01-01

    Although cloning may eventually become an important technology for livestock production, four ethical issues must be addressed before the practice becomes widespread. First, researchers must establish that the procedure is not detrimental to the health or well-being of affected animals. Second, animal research institutions should evaluate the net social benefits to livestock producers by weighing the benefits to producers against the opportunity cost of research capacity lost to biomedical projects. Third, scientists should consider the indirect effects of cloning research on the larger ethical issues surrounding human cloning. Finally, the market structure for products of cloned animals should protect individual choice, and should recognize that many individuals find the prospect of cloning (or consuming cloned animals) repugnant. Analysis of these four issues is complicated by spurious arguments alleging that cloning will have a negative impact on environment and genetic diversity.

  6. Cloning-free CRISPR

    Science.gov (United States)

    Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

    2015-01-01

    Summary We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. PMID:26527385

  7. Biodiversity versus cloning

    International Nuclear Information System (INIS)

    The announcement has been made on the cloning of mice in these days and he doesn't stop to miss, because the world lives a stage where conscience of the protection is creating that should be given to the biodiversity. It is known that alone we won't subsist and the protection of the means and all that contains that environment is of vital importance for the man. But it is also known that the vegetables and animal transgenic that they come to multiply the species have appeared that we prepare. The transgenic has been altered genetically, for substitution of one or more genes of other species, inclusive human genes. This represents an improvement compared with the investigations that gave origin to the cloning animal. But it is necessary to notice that to it you arrived through the cloning. This year 28 million hectares have been sowed in cultivations of transgenic seeds and there is around 700 bovine transgenic whose milk contains a necessary protein in the treatment of the man's illnesses

  8. Cloning-free CRISPR

    Directory of Open Access Journals (Sweden)

    Mandana Arbab

    2015-11-01

    Full Text Available We present self-cloning CRISPR/Cas9 (scCRISPR, a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.

  9. Telomere dysfunction and chromosome instability

    Energy Technology Data Exchange (ETDEWEB)

    Murnane, John P., E-mail: jmurnane@radonc.ucsf.edu [Department of Radiation Oncology, University of California San Francisco, 2340 Sutter Street, San Francisco, CA 94143-1331 (United States)

    2012-02-01

    The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is

  10. A sex chromosomal restriction-fragment-length marker linked to melanoma-determining Tu loci in Xiphophorus.

    Science.gov (United States)

    Schartl, M

    1988-07-01

    In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restriction-fragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from various Xiphophorus strains and hybrids including those bearing different Tu wild-type, deletion and translocation chromosomes, were screened for the presence of random RFLMs using homologous or heterologous sequences as hybridization probes. We find an EcoRI restriction fragment which shows limited crosshybridization to the v-erb B gene--but not representing the authentic c-erb B gene of Xiphophorus--to be polymorphic with respect to different sex chromosomes. Linkage analysis revealed that a 5-kb fragment is linked to the Tu-Sd locus on the X chromosome, a 7-kb fragment is linked to the Tu-Sr locus on the Y chromosome, both of Xiphophorus maculatus, and that a 12-kb fragment is linked to the Tu-Li locus on the X chromosome of Xiphophorus variatus. Using different chromosomal mutants this RFLM has been mapped to a frequent deletion/translocation breakpoint of the X chromosome, less than 0.3 cM apart from the Tu locus. PMID:2841190

  11. 3p22.1p21.31 microdeletion identifies CCK as Asperger syndrome candidate gene and shows the way for therapeutic strategies in chromosome imbalances

    OpenAIRE

    Iourov, Ivan Y; Vorsanova, Svetlana G; Voinova, Victoria Y.; Yurov, Yuri B.

    2015-01-01

    Background In contrast to other autism spectrum disorders, chromosome abnormalities are rare in Asperger syndrome (AS) or high-functioning autism. Consequently, AS was occasionally subjected to classical positional cloning. Here, we report on a case of AS associated with a deletion of the short arm of chromosome 3. Further in silico analysis has identified a candidate gene for AS and has suggested a therapeutic strategy for manifestations of the chromosome rearrangement. Results Using array c...

  12. Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin.

    Directory of Open Access Journals (Sweden)

    Penghua Yang

    Full Text Available Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79 x 10(-2 percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale.

  13. Chromosomal assignment of canine THADA gene to CFA 10q25

    Directory of Open Access Journals (Sweden)

    Dolf Gaudenz

    2008-06-01

    Full Text Available Abstract Background Chromosomal translocations affecting the chromosome 2p21 cluster in a 450 kb breakpoint region are frequently observed in human benign thyroid adenomas. THADA (thyroid adenoma associated was identified as the affected gene within this breakpoint region. In contrast to man tumours of the thyroid gland of dogs (Canis lupus familiaris constitute mainly as follicular cell carcinomas, with malignant thyroid tumours being more frequent than benign thyroid adenomas. In order to elucidate if the THADA gene is also a target of chromosomal rearrangements in thyroid adenomas of the dog we have physically mapped the canine THADA gene to canine chromosome 10. A PCR was established to screen a canine genome library for a BAC clone containing the gene sequence of canine THADA. Further PCR reactions were done using the identified BAC clone as a template in order to verify the corresponding PCR product by sequencing. Canine whole blood was incubated with colcemid in order to arrest the cultured cells in metaphases. The verified BAC DNA was digoxigenin labeled and used as a probe in fluorescence in situ hybridization (FISH. Ten well spread metaphases were examined indicating a signal on canine chromosome 10 on both chromatids. A detailed fine mapping was performed indicating the canine THADA gene locus on the q-arm of chromosome 10. Results The canine THADA gene locus was mapped on chromosome 10q25. Our mapping results obtained in this study following the previously described nomenclature for the canine karyotype. Conclusion We analysed whether the THADA gene locus is a hotspot of canine chromosomal rearrangements in canine neoplastic lesions of the thyroid and in addition might play a role as a candidate gene for a possible malignant transformation of canine thyroid adenomas. Although the available cytogenetic data of canine thyroid adenomas are still insufficient the chromosomal region to which the canine THADA has been mapped seems to be no

  14. Chromosomal breakpoints characterization of two supernumerary ring chromosomes 20.

    Science.gov (United States)

    Guediche, N; Brisset, S; Benichou, J-J; Guérin, N; Mabboux, P; Maurin, M-L; Bas, C; Laroudie, M; Picone, O; Goldszmidt, D; Prévot, S; Labrune, P; Tachdjian, G

    2010-02-01

    The occurrence of an additional ring chromosome 20 is a rare chromosome abnormality, and no common phenotype has been yet described. We report on two new patients presenting with a supernumerary ring chromosome 20 both prenatally diagnosed. The first presented with intrauterine growth retardation and some craniofacial dysmorphism, and the second case had a normal phenotype except for obesity. Conventional cytogenetic studies showed for each patient a small supernumerary marker chromosome (SMC). Using fluorescence in situ hybridization, these SMCs corresponded to ring chromosomes 20 including a part of short and long arms of chromosome 20. Detailed molecular cytogenetic characterization showed different breakpoints (20p11.23 and 20q11.23 for Patient 1 and 20p11.21 and 20q11.21 for Patient 2) and sizes of the two ring chromosomes 20 (13.6 Mb for case 1 and 4.8 Mb for case 2). Review of the 13 case reports of an extra r(20) ascertained postnatally (8 cases) and prenatally (5 cases) showed varying degrees of phenotypic abnormalities. We document a detailed molecular cytogenetic chromosomal breakpoints characterization of two cases of supernumerary ring chromosomes 20. These results emphasize the need to characterize precisely chromosomal breakpoints of supernumerary ring chromosomes 20 in order to establish genotype-phenotype correlation. This report may be helpful for prediction of natural history and outcome, particularly in prenatal diagnosis.

  15. Anticipatory Artificial Autopoiesis

    OpenAIRE

    DuBois, Daniel; Holmberg, Stig C.

    2010-01-01

    In examining relationships between autopoiesis and anticipation in artificial life (Alife) systems it is demonstrated that anticipation may increase efficiency and viability in artificial autopoietic living systems. This paper, firstly, gives a review of the Varela et al [1974] automata algorithm of an autopoietic living cell. Some problems in this algorithm must be corrected. Secondly, a new and original anticipatory artificial autopoiesis algorithm for automata is presented. ...

  16. Inteligencia artificial en vehiculo

    OpenAIRE

    Amador Díaz, Pedro

    2012-01-01

    Desarrollo de un robot seguidor de líneas, en el que se implementan diversas soluciones de las áreas de sistemas embebidos e inteligencia artificial. Desenvolupament d'un robot seguidor de línies, en el qual s'implementen diverses solucions de les àrees de sistemes encastats i intel·ligència artificial. Follower robot development of lines, in which various solutions are implemented in the areas of artificial intelligence embedded systems.

  17. Artificial cognition architectures

    CERN Document Server

    Crowder, James A; Friess, Shelli A

    2013-01-01

    The goal of this book is to establish the foundation, principles, theory, and concepts that are the backbone of real, autonomous Artificial Intelligence. Presented here are some basic human intelligence concepts framed for Artificial Intelligence systems. These include concepts like Metacognition and Metamemory, along with architectural constructs for Artificial Intelligence versions of human brain functions like the prefrontal cortex. Also presented are possible hardware and software architectures that lend themselves to learning, reasoning, and self-evolution

  18. Disruption of Netrin G1 by a balanced chromosome translocation in a girl with Rett syndrome

    DEFF Research Database (Denmark)

    Borg, Isabella; Freude, Kristine; Kübart, Sabine;

    2005-01-01

    We have identified a girl with characteristic features of Rett syndrome (RTT) who carries a de novo balanced translocation involving chromosomes 1 and 7. Both breakpoints were mapped by fluorescence in situ hybridization with selected genomic clones from the regions of interest. Southern blot...

  19. The Relationship Between Spontaneous Telomere Loss and Chromosome Instability in a Human Tumor Cell Line

    Directory of Open Access Journals (Sweden)

    Bijan Fouladi

    2000-01-01

    Full Text Available Chromosome instability plays an important role in cancer by promoting the alterations in the genome required for tumor cell progression. The loss of telomeres that protect the ends of chromosomes and prevent chromosome fusion has been proposed as one mechanism for chromosome instability in cancer cells, however, there is little direct evidence to support this hypothesis. To investigate the relationship between spontaneous telomere loss and chromosome instability in human cancer cells, clones of the EJ-30 tumor cell line were isolated in which a herpes simplex virus thymidine kinase (HSV-tk gene was integrated immediately adjacent to a telomere. Selection for HSV-tkdeficient cells with ganciclovir demonstrated a high rate of loss of the end these "marked" chromosomes (10-4 events/cell per generation. DNA sequence and cytogenetic analysis suggests that the loss of function of the HSV-tk gene most often involves telomere loss, sister chromatid fusion, and prolonged periods of chromosome instability. In some HSV-tk-deficient cells, telomeric repeat sequences were added on to the end of the truncated HSV-tk gene at a new location, whereas in others, no telomere was detected on the end of the marked chromosome. These results suggest that spontaneous telomere loss is a mechanism for chromosome instability in human cancer cells.

  20. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human

    Energy Technology Data Exchange (ETDEWEB)

    Blatt, C.; Eversole-Cire, P.; Cohn, V.H.; Zollman, S.; Fournier, R.E.K.; Mohandas, L.T.; Nesbitt, M.; Lugo, T.; Jones, D.T.; Reed, R.R.; Weiner, L.P.; Sparkes, R.S.; Simon, M.I. (Weizmann Institute, Rehovoth (Israel))

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding {alpha}-subunit proteins, two different {beta} subunits, and one {gamma} subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The {beta} subunits were also assigned-GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extend of the G{alpha} gene family and may help in attempts to correlate specific genetic diseases and with genes corresponding to G proteins.

  1. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    Science.gov (United States)

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  2. Familial complex chromosomal rearrangement resulting in a recombinant chromosome.

    Science.gov (United States)

    Berend, Sue Ann; Bodamer, Olaf A F; Shapira, Stuart K; Shaffer, Lisa G; Bacino, Carlos A

    2002-05-15

    Familial complex chromosomal rearrangements (CCRs) are rare and tend to involve fewer breakpoints and fewer chromosomes than CCRs that are de novo in origin. We report on a CCR identified in a child with congenital heart disease and dysmorphic features. Initially, the child's karyotype was thought to involve a straightforward three-way translocation between chromosomes 3, 8, and 16. However, after analyzing the mother's chromosomes, the mother was found to have a more complex rearrangement that resulted in a recombinant chromosome in the child. The mother's karyotype included an inverted chromosome 2 and multiple translocations involving chromosomes 3, 5, 8, and 16. No evidence of deletion or duplication that could account for the clinical findings in the child was identified.

  3. Physical mapping of the split hand/split foot (SHSF) locus on chromosome 7 reveals a relationship between SHSF and the syndromic ectrodactylies

    Energy Technology Data Exchange (ETDEWEB)

    Poorkaj, P.; Nunes, M.E.; Geshuri, D. [Univ. of Washington, Seattle, WA (United States)] [and others

    1994-09-01

    Split hand/split foot (also knows as ectrodactyly) is a human developmental malformation characterized by missing digits and claw-like extremities. An autosomal dominant form of this disorder has been mapped to 7q21.3-q22.1 on the basis of SHSF-associated chromosomal rearrangements: this locus has been designated SHFD1. We have constructed a physical map of the SHFD1 region that consists of contiguous yeast artificial chromosome clones and spans approximately 8 Mb. Somatic cell hybrid and fluorescent in situ hybridization analyses were used to define SHSF-associated chromosomal breakpoints in fourteen patients. A critical interval of about 1 Mb was established for SHFD1 by analysis of six patients with deletions. Translocation and inversion breakpoints in seven other patients were found to localize within a 500-700 kb interval within the critical region. Several candidate genes including DLX5 and DLX6 (members of the Drosophilia Distal-less homeobox-containing gene family) localize to this region. At least four of these genes are expressed in the developing mouse limb bud. Of particular interest is the observation that 8 of the 14 patients studied have syndromic ectrodactyly, which is characterized by the association of SHSF with a variety of other anomalies including cleft lip/palate, ectodermal dysplasia, and renal anomalies. Thus, these data implicate a single gene or cluster of genes at the SHFD1 locus in a wide range of developmental processes and serve to establish a molecular genetic relationship between simple SHSF and a broad group of human birth defects.

  4. Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

    International Nuclear Information System (INIS)

    Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector λpSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB+ clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA+ transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB+ and lysA+. The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes

  5. Duplication and loss of chromosome 21 in two children with Down Syndrome and acute leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Rogan, P.K.; Close, P.; Seip, J.R. [Pennsylvania State Univ. College of Medicine, Hershey, PA (United States)] [and others

    1994-09-01

    Acute leukemia in patients with Trisomy 21 (Down Syndrome; DS) may often result in additional karyotypic changes in the number or structure of chromosome 21. We present two DS patients whose immunoblast karyotypes were associated with changes in chromosome 21 ploidy. Patient L.E. developed acute lymphocytic leukemia concomitant with the loss of a single copy of chromosome 21. Trisomy 21 in this individual was due to maternal meiosis I nondisjunction. A recombination event resulted in reduction of maternal alleles to homozygosity distal to D21S167. Loss of the paternal chromosomes in the leukemia clone produced uniparental maternal disomy with isodisomy over a 25cM interval. This could, in theory, permit the unopposed expression of one or more homozygous recessive maternal tumor-associated genes, thus providing an explanation for leukemogenesis in this patient. Patient E.H. was diagnosed with acute monoblastic leukemia and consistently displayed tetrasomy 21 in the blast cell population. The DS karyotype probably arose from a mitotic error in which the paternal chromosome was duplicated. DNA polymorphism analysis indicated that the additional chromosome in the leukemia clone was of maternal origin. The presence of equal numbers of maternal and paternal chromosomes in the tetraploid blast clone would not appear to be consistent with the expression of a mutant tumor suppressor gene in this patient. Although tetrasomy 21 could be a non-specific karyotypic abnormality unrelated to leukemogenesis, it is possible that monoblastic leukemia may be a consequence of increased expression of one or more genes on this chromosome.

  6. More Genetic Engineering With Cloned Hemoglobin Genes

    Science.gov (United States)

    Bailey, James E.

    1992-01-01

    Cells modified to enhance growth and production of proteins. Method for enhancing both growth of micro-organisms in vitro and production of various proteins or metalbolites in these micro-organisms provides for incorporation of selected chromosomal or extrachormosomal deoxyribonucleic acid (DNA) sequences into micro-organisms from other cells or from artificial sources. Incorporated DNA includes parts encoding desired product(s) or characteristic(s) of cells and parts that control expression of productor characteristic-encoding parts in response to variations in environment. Extended method enables increased research into growth of organisms in oxygen-poor environments. Industrial applications found in enhancement of processing steps requiring oxygen in fermentation, enzymatic degradation, treatment of wastes containing toxic chemicals, brewing, and some oxidative chemical reactions.

  7. Ethical issues in animal cloning.

    Science.gov (United States)

    Fiester, Autumn

    2005-01-01

    The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.

  8. 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.

  9. Karyotyping of Chromosomes in Human Bronchial Epithelial Cells Transformed by High Energy Fe Ions

    Science.gov (United States)

    Yeshitla, Samrawit; Zhang, Ye; Park, Seongmi; Story, Michael D.; Wilson, Bobby; Wu, Honglu

    2015-01-01

    Lung cancer induced from exposures to space radiation is one of the most significant health risks for long-term space travels. Evidences show that low- and high- Linear energy transfer (LET)-induced transformation of normal human bronchial epithelial cells (HBEC) that are immortalized through the expression of Cdk4 and hTERT. The cells were exposed to gamma rays and high-energy Fe ions for the selection of transformed clones. Transformed HBEC are identified and analyzed chromosome aberrations (i.e. genomic instability) using the multi-color fluorescent in situ hybridization (mFISH), as well as the multi-banding in situ hybridization (mBAND) techniques. Our results show chromosomal translocations between different chromosomes and several of the breaks occurred in the q-arm of chromosome 3. We also identified copy number variations between the transformed and the parental HBEC regardless of the exposure conditions. We observed chromosomal aberrations in the lowand high-LET radiation-induced transformed clones and they are imperfectly different from clones obtain in spontaneous soft agar growth.

  10. Development of T. aestivum L.-H. californicum alien chromosome lines and assignment of homoeologous groups of Hordeum californicum chromosomes.

    Science.gov (United States)

    Fang, Yuhui; Yuan, Jingya; Wang, Zhangjun; Wang, Haiyan; Xiao, Jin; Yang, Zhixi; Zhang, Ruiqi; Qi, Zengjun; Xu, Weigang; Hu, Lin; Wang, Xiu-E

    2014-08-20

    Hordeum californicum (2n = 2x = 14, HH) is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring (CS)-H. californicum amphidiploid (2n = 6x = 56, AABBDDHH) was established. By genomic in situ hybridization (GISH) and multicolor fluorescent in situ hybridization (FISH) using repetitive DNA clones (pTa71, pTa794 and pSc119.2) as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheat-alien chromosome lines, including four disomic addition lines (DAH1, DAH3, DAH5 and DAH6), five telosomic addition lines (MtH7L, MtH1S, MtH1L, DtH6S and DtH6L), one multiple addition line involving H. californicum chromosome H2, one disomic substitution line (DSH4) and one translocation line (TH7S/1BL), were identified from the progenies derived from the crosses of CS-H. californicum amphidiploid with common wheat varieties. A total of 482 EST (expressed sequence tag) or SSR (simple sequence repeat) markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2, H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5H(c), 2H(c), 6H(c), 3H(c) and 1H(c), respectively. The chromosomes H1 and H6 were designated as 7H(c) and 4H(c), respectively, by referring to SSR markers located on rye chromosomes.

  11. A rapid molecular approach for chromosomal phasing.

    Directory of Open Access Journals (Sweden)

    John F Regan

    Full Text Available Determining the chromosomal phase of pairs of sequence variants - the arrangement of specific alleles as haplotypes - is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information, and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11-116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours, scalable (to hundreds of samples, and effective at long genomic distances (200 kb.

  12. Artificial life and life artificialization in Tron

    Directory of Open Access Journals (Sweden)

    Carolina Dantas Figueiredo

    2012-12-01

    Full Text Available Cinema constantly shows the struggle between the men and artificial intelligences. Fiction, and more specifically fiction films, lends itself to explore possibilities asking “what if?”. “What if”, in this case, is related to the eventual rebellion of artificial intelligences, theme explored in the movies Tron (1982 and Tron Legacy (2010 trat portray the conflict between programs and users. The present paper examines these films, observing particularly the possibility programs empowering. Finally, is briefly mentioned the concept of cyborg as a possibility of response to human concerns.

  13. Chromosomal evolution of the PKD1 gene family in primates

    Directory of Open Access Journals (Sweden)

    Krawczak Michael

    2008-09-01

    Full Text Available Abstract Background The autosomal dominant polycystic kidney disease (ADPKD is mostly caused by mutations in the PKD1 (polycystic kidney disease 1 gene located in 16p13.3. Moreover, there are six pseudogenes of PKD1 that are located proximal to the master gene in 16p13.1. In contrast, no pseudogene could be detected in the mouse genome, only a single copy gene on chromosome 17. The question arises how the human situation originated phylogenetically. To address this question we applied comparative FISH-mapping of a human PKD1-containing genomic BAC clone and a PKD1-cDNA clone to chromosomes of a variety of primate species and the dog as a non-primate outgroup species. Results Comparative FISH with the PKD1-cDNA clone clearly shows that in all primate species studied distinct single signals map in subtelomeric chromosomal positions orthologous to the short arm of human chromosome 16 harbouring the master PKD1 gene. Only in human and African great apes, but not in orangutan, FISH with both BAC and cDNA clones reveals additional signal clusters located proximal of and clearly separated from the PKD1 master genes indicating the chromosomal position of PKD1 pseudogenes in 16p of these species, respectively. Indeed, this is in accordance with sequencing data in human, chimpanzee and orangutan. Apart from the master PKD1 gene, six pseudogenes are identified in both, human and chimpanzee, while only a single-copy gene is present in the whole-genome sequence of orangutan. The phylogenetic reconstruction of the PKD1-tree reveals that all human pseudogenes are closely related to the human PKD1 gene, and all chimpanzee pseudogenes are closely related to the chimpanzee PKD1 gene. However, our statistical analyses provide strong indication that gene conversion events may have occurred within the PKD1 family members of human and chimpanzee, respectively. Conclusion PKD1 must have undergone amplification very recently in hominid evolution. Duplicative

  14. Recombinant protein expression by targeting pre-selected chromosomal loci

    Directory of Open Access Journals (Sweden)

    Krömer Wolfgang

    2009-12-01

    Full Text Available Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs were targeted by Flp recombinase mediated cassette exchange (RMCE. The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context

  15. DETECTION OF CROSS-PROJECT BENEFICIAL CLONES

    OpenAIRE

    Ms.Kavitha Esther Rajakumari; Dr.T.Jebarajan

    2014-01-01

    Duplicate codes are also known as code clones. They are considered as one of the main factors that deteriorate the quality of software. They are usually discarded by using automatic clone detection tools. In this paper the clones are detected using a data mining approach. The clones are well analyzed and the beneficial code clones are retained. These clones are maintained separately and are used in software maintenance. The beneficial clones will definitely help in reducing the overall time s...

  16. A note on clones with nullary operations

    OpenAIRE

    Behrisch, Mike

    2013-01-01

    This report discusses clones with nullary operations and the corresponding relational clones, both defined on arbitrary non-empty sets. The relationship between such clones and clones in the usual sense, i.e. without nullary operations, is investigated, and in particular the latter type of clones is located in the lattice of all clones. By means of two pairs of kernel and closure operators, a framework is developed that allows to transfer statements about usual clones to statements about clon...

  17. The HOX-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2.

    Science.gov (United States)

    Stubbs, L; Huxley, C; Hogan, B; Evans, T; Fried, M; Duboule, D; Lehrach, H

    1990-04-01

    Using an interspecies backcross, we have mapped the HOX-5 and surfeit (surf) gene clusters within the proximal portion of mouse chromosome 2. While the HOX-5 cluster of homeobox-containing genes has been localized to chromosome 2, bands C3-E1, by in situ hybridization, its more precise position relative to the genes and cloned markers of chromosome 2 was not known. Surfeit, a tight cluster of at least six highly conserved "housekeeping" genes, has not been previously mapped in mouse, but has been localized to human chromosome 9q, a region of the human genome with strong homology to proximal mouse chromosome 2. The data presented here place HOX-5 in the vicinity of the closely linked set of developmental mutations rachiterata, lethargic, and fidget and place surf close to the proto-oncogene Abl, near the centromere of chromosome 2.

  18. A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast

    Directory of Open Access Journals (Sweden)

    Barrett J Carl

    2003-04-01

    Full Text Available Abstract Background Transformation-associated recombination (TAR cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes. The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (~ 60 bp 5' and 3' gene targeting sequences (hooks. Result TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating sequence (ARS that can function as the origin of replication in yeast, which prevents wide application of the method. In this paper, we describe a novel TAR cloning system that allows isolation of genomic regions lacking yeast ARS-like sequences. ARS is inserted into the TAR vector along with URA3 as a counter-selectable marker. The hooks are placed between the TATA box and the transcription initiation site of URA3. Insertion of any sequence between hooks results in inactivation of URA3 expression. That inactivation confers resistance to 5-fluoroorotic acid, allowing selection of TAR cloning events against background vector recircularization events. Conclusion The new system greatly expands the area of application of TAR cloning by allowing isolation of any chromosomal region from eukaryotic and prokaryotic genomes regardless of the presence of autonomously replicating sequences.

  19. Cytogenetically Unrelated Clones in Acute Myeloid Leukemia Showing Different Responses to Chemotherapy

    Directory of Open Access Journals (Sweden)

    Kohei Kasahara

    2016-01-01

    Full Text Available We report a case of acute myeloid leukemia (AML with two cytogenetically unrelated clones. The patient was a 45-year-old male who was diagnosed with acute monoblastic leukemia (AMoL. Initial G-band analysis showed 51,XY,+6,+8,inv(9(p12q13c,+11,+13,+19[12]/52,idem,+Y[8], but G-band analysis after induction therapy showed 45,XY,-7,inv(9(p12q13c[19]/46,XY,inv(9(p12q13c[1]. Retrospective FISH analysis revealed a cryptic monosomy 7 clone in the initial AML sample. The clone with multiple trisomies was eliminated after induction therapy and never recurred, but a clone with monosomy 7 was still detected in myelodysplastic marrow with a normal blast percentage. Both clones were successfully eliminated after related peripheral blood stem cell transplantation, but the patient died of relapsed AML with monosomy 7. We concluded that one clone was de novo AMoL with chromosome 6, 8, 11, 13, and 19 trisomy and that the other was acute myeloid leukemia with myelodysplasia-related changes (AML-MRC with chromosome 7 monosomy showing different responses to chemotherapy. Simultaneous onset of cytogenetically unrelated hematological malignancies that each have a different disease status is a rare phenomenon but is important to diagnose for a correct understanding of the disease status and for establishing an appropriate treatment strategy.

  20. Therapeutic cloning in the mouse

    OpenAIRE

    Mombaerts, Peter

    2003-01-01

    Nuclear transfer technology can be applied to produce autologous differentiated cells for therapeutic purposes, a concept termed therapeutic cloning. Countless articles have been published on the ethics and politics of human therapeutic cloning, reflecting the high expectations from this new opportunity for rejuvenation of the aging or diseased body. Yet the research literature on therapeutic cloning, strictly speaking, is comprised of only four articles, all in the mouse. The efficiency of d...

  1. Artificial insemination in poultry

    Science.gov (United States)

    Artificial insemination is a relative simple yet powerful tool geneticists can employ for the propagation of economically important traits in livestock and poultry. In this chapter, we address the fundamental methods of the artificial insemination of poultry, including semen collection, semen evalu...

  2. Chromosome numbers and meiotic analysis in the pre-breeding of Brachiaria decumbens (Poaceae)

    Indian Academy of Sciences (India)

    Gléia Cristina Laverde Ricci; Alice Maria De Souza-Kaneshima; Mariana Ferrari Felismino; Andrea Beatriz Mendes-Bonato; Maria Suely Pagliarini; Cacilda Borges Do Valle

    2011-08-01

    A total of 44 accessions of Brachiaria decumbens were analysed for chromosome count and meiotic behaviour in order to identify potential progenitors for crosses. Among them, 15 accessions presented $2n = 18$; 27 accessions, $2n = 36$; and 2 accessions, $2n = 45$ chromosomes. Among the diploid accessions, the rate of meiotic abnormalities was low, ranging from 0.82% to 7.93%. In the 27 tetraploid accessions, the rate of meiotic abnormalities ranged from 18.41% to 65.83%. The most common meiotic abnormalities were related to irregular chromosome segregation, but chromosome stickiness and abnormal cytokinesis were observed in low frequency. All abnormalities can compromise pollen viability by generating unbalanced gametes. Based on the chromosome number and meiotic stability, the present study indicates the apomictic tetraploid accessions that can act as male genitor to produce interspecific hybrids with B. ruziziensis or intraspecific hybrids with recently artificially tetraploidized accessions.

  3. Assignment of Chinook salmon (Oncorhynchus tshawytscha) linkage groups to specific chromosomes reveals a karyotype with multiple rearrangements of the chromosome arms of rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Phillips, Ruth B; Park, Linda K; Naish, Kerry A

    2013-12-09

    The Chinook salmon genetic linkage groups have been assigned to specific chromosomes using fluorescence in situ hybridization with bacterial artificial chromosome probes containing genetic markers mapped to each linkage group in Chinook salmon and rainbow trout. Comparison of the Chinook salmon chromosome map with that of rainbow trout provides strong evidence for conservation of large syntenic blocks in these species, corresponding to entire chromosome arms in the rainbow trout as expected. In almost every case, the markers were found at approximately the same location on the chromosome arm in each species, suggesting conservation of marker order on the chromosome arms of the two species in most cases. Although theoretically a few centric fissions could convert the karyotype of rainbow trout (2N = 58-64) into that of Chinook salmon (2N = 68) or vice versa, our data suggest that chromosome arms underwent multiple centric fissions and subsequent new centric fusions to form the current karyotypes. The morphology of only approximately one-third of the chromosome pairs have been conserved between the two species.

  4. Therapeutic cloning: The ethical limits

    International Nuclear Information System (INIS)

    A brief outline of stem cells, stem cell therapy and therapeutic cloning is given. The position of therapeutic cloning with regard to other embryonic manipulations - IVF-based reproduction, embryonic stem formation from IVF embryos and reproductive cloning - is indicated. The main ethically challenging stages in therapeutic cloning are considered to be the nuclear transfer process including the source of eggs for this and the destruction of an embryo to provide stem cells for therapeutic use. The extremely polarised nature of the debate regarding the status of an early human embryo is noted, and some potential alternative strategies for preparing immunocompatible pluripotent stem cells are indicated

  5. Onion artificial muscles

    Science.gov (United States)

    Chen, Chien-Chun; Shih, Wen-Pin; Chang, Pei-Zen; Lai, Hsi-Mei; Chang, Shing-Yun; Huang, Pin-Chun; Jeng, Huai-An

    2015-05-01

    Artificial muscles are soft actuators with the capability of either bending or contraction/elongation subjected to external stimulation. However, there are currently no artificial muscles that can accomplish these actions simultaneously. We found that the single layered, latticed microstructure of onion epidermal cells after acid treatment became elastic and could simultaneously stretch and bend when an electric field was applied. By modulating the magnitude of the voltage, the artificial muscle made of onion epidermal cells would deflect in opposing directions while either contracting or elongating. At voltages of 0-50 V, the artificial muscle elongated and had a maximum deflection of -30 μm; at voltages of 50-1000 V, the artificial muscle contracted and deflected 1.0 mm. The maximum force response is 20 μN at 1000 V.

  6. Self-Cloning CRISPR.

    Science.gov (United States)

    Arbab, Mandana; Sherwood, Richard I

    2016-01-01

    CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR-based addition of short homology arms, we achieve efficient site-specific knock-in of transgenes such as GFP without traditional plasmid cloning or genome-integrated selection cassette (2% to 4% knock-in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc. PMID:27532819

  7. A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome-arm identification, integration of genetic linkage groups and analysis of major repeat family distribution.

    Science.gov (United States)

    Paesold, Susanne; Borchardt, Dietrich; Schmidt, Thomas; Dechyeva, Daryna

    2012-11-01

    We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North-South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome-specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S-5.8S-25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber-FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.

  8. A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome-arm identification, integration of genetic linkage groups and analysis of major repeat family distribution.

    Science.gov (United States)

    Paesold, Susanne; Borchardt, Dietrich; Schmidt, Thomas; Dechyeva, Daryna

    2012-11-01

    We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North-South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome-specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S-5.8S-25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber-FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA. PMID:22775355

  9. Microcell-mediated transfer of a single human chromosome complements xeroderma pigmentosum group A fibroblasts

    International Nuclear Information System (INIS)

    Chromosomes from an immortalized aneuploid human fibroblast cell line were randomly tagged with the selectable marker neo by transfection with the plasmid pSV2neo. Somatic cell fusions between transfected human cells and mouse A9 cells generated pools of G418-resistant human-mouse hybrid clones containing various numbers of human chromosomes. Microcell-mediated chromosome transfer from the hybrid pools to xeroderma pigmentosum complementation group A (XP-A) cells in culture and selection for G418-resistant colonies resulted in the identification of XP cells with enhanced resistance to ultraviolet radiation. Screening of subclones from selected pools of human-mouse hybrids facilitated the identification of hybrids containing a single neo-tagged human chromosome. Transfer of this chromosome to XP-A cells (but not to XP-F or XP-C cells) results in enhanced resistance to ultraviolet light and enhanced excision repair capacity. The identification of a single human chromosome that complements the phenotype of XP-A cells in culture provides the potential for genetic mapping of the complementing gene and for its isolation by molecular cloning

  10. A molecularly defined duplication set for the X chromosome of Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Venken, Koen J. T.; Popodi, Ellen; Holtzman, Stacy L.; Schulze, Karen L.; Park, Soo; Carlson, Joseph W.; Hoskins, Roger A.; Bellen, Hugo J.; Kaufman, Thomas C.

    2010-07-22

    We describe a molecularly defined duplication kit for the X chromosome of Drosophila melanogaster. A set of 408 overlapping P[acman] BAC clones was used to create small duplications (average length 88 kb) covering the 22-Mb sequenced portion of the chromosome. The BAC clones were inserted into an attP docking site on chromosome 3L using C31 integrase, allowing direct comparison of different transgenes. The insertions complement 92% of the essential and viable mutations and deletions tested, demonstrating that almost all Drosophila genes are compact and that the current annotations of the genome are reasonably accurate. Moreover, almost all genes are tolerated at twice the normal dosage. Finally, we more precisely mapped two regions at which duplications cause diplo-lethality in males. This collection comprises the first molecularly defined duplication set to cover a whole chromosome in a multicellular organism. The work presented removes a long-standing barrier to genetic analysis of the Drosophila X chromosome, will greatly facilitate functional assays of X-linked genes in vivo, and provides a model for functional analyses of entire chromosomes in other species.

  11. Complete Genomic Sequence and an Infectious BAC Clone of Feline Herpesvirus-1 (FHV-1)

    Science.gov (United States)

    Feline herpesvirus type 1 (FHV-1) is classified under the genus Varicellovirus within the Alphaherpesvirinae subfamily, and is a major cause of upper respiratory infection in cats. In this report, we present the first complete genomic sequence of FHV-1, as well as a bacterial artificial chromosome (...

  12. BURDEN OF ABNORMAL HEMATOPOIETIC CLONE IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To investigate the role of the burden of abnormal hematopoietic clone in the development of myelodys plastic syndromes (MDS).Methods The ratio of the bone marrow cells with abnormal chromosomes to the total counted bone marrow cells was regarded as the index of MDS clone burden. The disease severity related parameters including white blood cell count, hemoglobin, platelet count, lactate dehydrogenase level, bone marrow blast, myeloid differentiation index, micromegakaryocyte, transfusion, interleukin-2, tumor necrosis factor ( TNF), CD4 + and CD8 + T cells of MDS patients were assayed, and the correlations between those parameters and MDS clone burden were also analyzed.Results The clone burden of MDS patients was 67.4% ± 36. 2%. MDS clone burden positively correlated with bone marrow blasts (r=0.483, P<0.05), negatively with hemoglobin level (r=-0.445, P<0.05). The number of blasts, hemoglobin, and erythrocytes in high clone burden (>50%) and low clone burden (≤50%) groups were 7.78%±5.51% and 3.45%±3.34%, 56.06±14. 28 g/L and 76.40±24.44 g/L, (1.82±0.48)×1012/L and (2. 32±0.66)×1012/L, respectively (all P <0.05). CD4 + T lymphocytes of MDS patients and normal controls were (0. 274±0.719)×109/L and (0.455±0.206)×109/L, respectively (P<0.05). CD8 ± T lymphocytes of MDS patients and normal controls were (0.240±0.150)×109/L and (0.305 ±0.145)×109/L, respectively. The serum level of interleukin-2 of MDS patients (6.29±3.58 ng/mL) was significantly higher than normal control (3.11±1.40ng/mL, P<0.05). The serum level of TNF of MDS patients and normal control group were 2.42±1.79 ng/mL and 1.68 ±0.69 ng/mL, respectively. The ratio of CD4 to CD8 was higher in high clone burden MDS patients (1.90 ±0.52) than that in low clone burden patients (0.97±0.44, P<0.05).Conclusion The quantitive clonal karyotype abnormalities and deficient T cell immunity are important parameters for evaluating MDS severity and predicting its

  13. Artificial intelligence in medicine.

    Science.gov (United States)

    Ramesh, A. N.; Kambhampati, C.; Monson, J. R. T.; Drew, P. J.

    2004-01-01

    INTRODUCTION: Artificial intelligence is a branch of computer science capable of analysing complex medical data. Their potential to exploit meaningful relationship with in a data set can be used in the diagnosis, treatment and predicting outcome in many clinical scenarios. METHODS: Medline and internet searches were carried out using the keywords 'artificial intelligence' and 'neural networks (computer)'. Further references were obtained by cross-referencing from key articles. An overview of different artificial intelligent techniques is presented in this paper along with the review of important clinical applications. RESULTS: The proficiency of artificial intelligent techniques has been explored in almost every field of medicine. Artificial neural network was the most commonly used analytical tool whilst other artificial intelligent techniques such as fuzzy expert systems, evolutionary computation and hybrid intelligent systems have all been used in different clinical settings. DISCUSSION: Artificial intelligence techniques have the potential to be applied in almost every field of medicine. There is need for further clinical trials which are appropriately designed before these emergent techniques find application in the real clinical setting. PMID:15333167

  14. A YAC contig and an EST map in the pericentromeric region of chromosome 13 surrounding the loci for neurosensory nonsyndromic deafness (DFNB1 and DFNA3) and Limb-Girdle muscular dystrophy type 2C (LGMD2C)

    Energy Technology Data Exchange (ETDEWEB)

    Guilford, P.; Crozet, F.; Blanchard, S. [Institut Pasteur, Paris (France)] [and others

    1995-09-01

    Two forms of inherited childhood nonsyndromic deafness (DFNB1 and DFNA3) and a Duchenne-like form of progressive muscular dystrophy (LGMD2C) have been mapped to the pericentromeric region of chromosome 13. To clone the genes responsible for these diseases we constructed a yeast artificial chromosome (YAC) contig spanning an 8-cM region between the polymorphic markers D13S221. The contig comprises 24 sequence-tagged sites, among which 15 were newly obtained. This contig allowed us to order the polymorphic markers centromere- D13S175-D13S141-D13S143-D13S115-AFM128yc1-D13S292-D13S283-AFM323vh5-D13S221-telomere. Eight expressed sequence tags, previously assigned to 13q11-q12 (D13S182E, D13S183E, D13S502E, D13S504E, D13S505E, D13S837E, TUBA2, ATP1AL1), were localized on the YAC contig. YAC screening of a cDNA library derived from mouse cochlea allowed us to identify an {alpha}-tubulin gene (TUBA2) that was subsequently precisely mapped within the candidate region. 36 refs., 2 figs., 2 tabs.

  15. Intraspecific chromosome variability

    Directory of Open Access Journals (Sweden)

    N Dubinin

    2010-12-01

    Full Text Available (Editorial preface. The publication is presented in order to remind us of one of dramatic pages of the history of genetics. It re-opens for the contemporary reader a comprehensive work marking the priority change from plant cytogenetics to animal cytogenetics led by wide population studies which were conducted on Drosophila polytene chromosomes. The year of the publication (1937 became the point of irretrievable branching between the directions of Old World and New World genetics connected with the problems of chromosome variability and its significance for the evolution of the species. The famous book of T. Dobzhansky (1937 was published by Columbia University in the US under the title “Genetics and the origin of species”, and in the shadow of this American ‘skybuilding’ all other works grew dim. It is remarkable that both Dobzhansky and Dubinin come to similar conclusions about the role of chromosomes in speciation. This is not surprising given that they both might be considered as representatives of the Russian genetic school, by their birth and education. Interestingly, Dobzhansky had never referred to the full paper of Dubinin et al. (1937, though a previous short communication in Nature (1936 was included together with all former papers on the related subject. In full, the volume of the original publication printed in the Biological Journal in Moscow comprised 47 pages, in that number 41 pages of the Russian text accompanied by 16 Figs, a table and reference list, and, above all, 6 pages of the English summary. This final part in English is now reproduced in the authors’ version with the only addition being the reference list in the originally printed form.

  16. Chromosome assortment in Saccharum.

    Science.gov (United States)

    Al-Janabi, S M; Honeycutt, R J; Sobral, B W

    1994-12-01

    Recent work has revealed random chromosome pairing and assortment in Saccharum spontaneum L., the most widely distributed, and morphologically and cytologically variable of the species of Saccharum. This conclusion was based on the analysis of a segregating population from across between S. spontaneum 'SES 208' and a spontaneously-doubled haploid of itself, derived from anther culture. To determine whether polysomic inheritance is common in Saccharum and whether it is observed in a typical biparental cross, we studied chromosome pairing and assortment in 44 progeny of a cross between euploid, meiotically regular, 2n=80 forms of Saccharum officinarum 'LA Purple' and Saccharum robustum ' Mol 5829'. Papuan 2n=80 forms of S. robustum have been suggested as the immediate progenitor species for cultivated sugarcane (S. officinarum). A total of 738 loci in LA Purple and 720 loci in Mol 5829 were amplified and typed in the progeny by arbitrarily primed PCR using 45 primers. Fifty and 33 single-dose polymorphisms were identified in the S. officinarum and S. robustum genomes, respectively (χ 2 at 98%). Linkage analysis of single-dose polymorphisms in both genomes revealed linkages in repulsion and coupling phases. In the S. officinarum genome, a map hypothesis gave 7 linkage groups with 17 linked and 33 unlinked markers. Four of 13 pairwise linkages were in repulsion phase and 9 were in coupling phase. In the S. robustum genome, a map hypothesis gave 5 linkage groups, defined by 12 markers, with 21 markers unlinked, and 2 of 9 pairwise linkages were in repulsion phase. Therefore, complete polysomic inheritance was not observed in either species, suggesting that chromosomal behavior is different from that observed by linkage analysis of over 500 markers in the S. spontaneum map. Implications of this finding for evolution and breeding are discussed.

  17. CATO: The Clone Alignment Tool.

    Directory of Open Access Journals (Sweden)

    Peter V Henstock

    Full Text Available High-throughput cloning efforts produce large numbers of sequences that need to be aligned, edited, compared with reference sequences, and organized as files and selected clones. Different pieces of software are typically required to perform each of these tasks. We have designed a single piece of software, CATO, the Clone Alignment Tool, that allows a user to align, evaluate, edit, and select clone sequences based on comparisons to reference sequences. The input and output are designed to be compatible with standard data formats, and thus suitable for integration into a clone processing pipeline. CATO provides both sequence alignment and visualizations to facilitate the analysis of cloning experiments. The alignment algorithm matches each of the relevant candidate sequences against each reference sequence. The visualization portion displays three levels of matching: 1 a top-level summary of the top candidate sequences aligned to each reference sequence, 2 a focused alignment view with the nucleotides of matched sequences displayed against one reference sequence, and 3 a pair-wise alignment of a single reference and candidate sequence pair. Users can select the minimum matching criteria for valid clones, edit or swap reference sequences, and export the results to a summary file as part of the high-throughput cloning workflow.

  18. [The discrete horror of cloning].

    Science.gov (United States)

    Guibourg, Ricardo A

    2009-01-01

    The author raises the topic of cloning after the decision of the Argentine government, which concerned for the "dignity of the human person", passed a decree of need and urgency, No. 200/97 (Annex), prohibiting cloning experiments with human beings. Therefore, considering that the topic is so terribly urgent and necessary, the author feels it is timely to consider it.

  19. Sequencing and association analysis of the type 1 diabetes – linked region on chromosome 10p12-q11

    Directory of Open Access Journals (Sweden)

    Barratt Bryan J

    2007-05-01

    Full Text Available Abstract Background In an effort to locate susceptibility genes for type 1 diabetes (T1D several genome-wide linkage scans have been undertaken. A chromosomal region designated IDDM10 retained genome-wide significance in a combined analysis of the main linkage scans. Here, we studied sequence polymorphisms in 23 Mb on chromosome 10p12-q11, including the putative IDDM10 region, to identify genes associated with T1D. Results Initially, we resequenced the functional candidate genes, CREM and SDF1, located in this region, genotyped 13 tag single nucleotide polymorphisms (SNPs and found no association with T1D. We then undertook analysis of the whole 23 Mb region. We constructed and sequenced a contig tile path from two bacterial artificial clone libraries. By comparison with a clone library from an unrelated person used in the Human Genome Project, we identified 12,058 SNPs. We genotyped 303 SNPs and 25 polymorphic microsatellite markers in 765 multiplex T1D families and followed up 22 associated polymorphisms in up to 2,857 families. We found nominal evidence of association in six loci (P = 0.05 – 0.0026, located near the PAPD1 gene. Therefore, we resequenced 38.8 kb in this region, found 147 SNPs and genotyped 84 of them in the T1D families. We also tested 13 polymorphisms in the PAPD1 gene and in five other loci in 1,612 T1D patients and 1,828 controls from the UK. Overall, only the D10S193 microsatellite marker located 28 kb downstream of PAPD1 showed nominal evidence of association in both T1D families and in the case-control sample (P = 0.037 and 0.03, respectively. Conclusion We conclude that polymorphisms in the CREM and SDF1 genes have no major effect on T1D. The weak T1D association that we detected in the association scan near the PAPD1 gene may be either false or due to a small genuine effect, and cannot explain linkage at the IDDM10 region.

  20. Quantum probabilistically cloning and computation

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this article we make a review on the usefulness of probabilistically cloning and present examples of quantum computation tasks for which quantum cloning offers an advantage which cannot be matched by any approach that does not resort to it.In these quantum computations,one needs to distribute quantum information contained in states about which we have some partial information.To perform quantum computations,one uses state-dependent probabilistic quantum cloning procedure to distribute quantum information in the middle of a quantum computation.And we discuss the achievable efficiencies and the efficient quantum logic network for probabilistic cloning the quantum states used in implementing quantum computation tasks for which cloning provides enhancement in performance.

  1. Assignment of genetic linkage maps to diploid Solanum tuberosum pachytene chromosomes by BAC-FISH technology

    NARCIS (Netherlands)

    Tang, X.; Boer, de J.M.; Eck, van H.J.; Bachem, C.W.B.; Visser, R.G.F.; Jong, de J.H.

    2009-01-01

    A cytogenetic map has been developed for diploid potato (Solanum tuberosum), in which the arms of the 12 potato bivalents can be identified in pachytene complements using multicolor fluorescence in situ hybridization (FISH) with a set of 60 genetically anchored bacterial artificial chromosome (BAC)

  2. Engineering targeted chromosomal amplifications in human breast epithelial cells.

    Science.gov (United States)

    Springer, Simeon; Yi, Kyung H; Park, Jeenah; Rajpurohit, Anandita; Price, Amanda J; Lauring, Josh

    2015-07-01

    Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

  3. Molecular mapping of chromosomes 17 and X. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Barker, D.F.

    1989-12-31

    The basic aims of this project are the construction of high density genetic maps of chromosomes 17 and X and the utilization of these maps for the subsequent isolation of a set of physically overlapping DNA segment clones. The strategy depends on the utilization of chromosome specific libraries of small (1--15 kb) segments from each of the two chromosomes. Since the time of submission of our previous progress report, we have refined the genetic map of markers which we had previously isolated for chromosome 17. We have completed our genetic mapping in CEPH reference and NF1 families of 15 markers in the pericentric region of chromosome 17. Physical mapping results with three probes, were shown be in very close genetic proximity to the NF1 gene, with respect to two translocation breakpoints which disrupt the activity of the gene. All three of the probes were found to lie between the centromere and the most proximal translocation breakpoint, providing important genetic markers proximal to the NF1 gene. Our primary focus has shifted to the X chromosome. We have isolated an additional 30 polymorphic markers, bringing the total number we have isolated to over 80. We have invested substantial effort in characterizing the polymorphisms at each of these loci and constructed plasmid subclones which reveal the polymorphisms for nearly all of the loci. These subclones are of practical value in that they produce simpler and stronger patterns on human genomic Southern blots, thus improving the efficiency of the genetic mapping experiments. These subclones may also be of value for deriving DNA sequence information at each locus, necessary for establishing polymerase chain reaction primers specific for each locus. Such information would allow the use of each locus as a sequence tagged site.

  4. Chromosomal anomalies in radiation-induced fibrosis in the pig

    International Nuclear Information System (INIS)

    R-banded karyotypes were established on fibroblasts from fibrotic tissues derived from experimental fibrosis induced in pigs, either surgically or by 64 Gy of γ-rays from iridium-192. No chromosome aberrations were observed in the surgical fibrosis. In radiation-induced fibrosis, the high frequency of abnormal karyotypes and frequent complexity of the chromosomal rearrangements suggest that the fibroblasts originated either from the 64-Gy area, or from the penumbra, but certainly not from non-irradiated areas. At early passages in vitro, almost all karyotypes were different, demonstrating a multiclonal origin of fibrotic tissue. At late passages (above 24), the situation was quite different, with the persistence of one or two clones only, demonstrating a strong selective pressure occurring in vitro. (author). 23 refs., 2 figs., 2 tabs

  5. Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells.

    Science.gov (United States)

    Yamano, Noriko; Takahashi, Mai; Ali Haghparast, Seyed Mohammad; Onitsuka, Masayoshi; Kumamoto, Toshitaka; Frank, Jana; Omasa, Takeshi

    2016-08-01

    Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production.

  6. Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells.

    Science.gov (United States)

    Yamano, Noriko; Takahashi, Mai; Ali Haghparast, Seyed Mohammad; Onitsuka, Masayoshi; Kumamoto, Toshitaka; Frank, Jana; Omasa, Takeshi

    2016-08-01

    Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production. PMID:26850366

  7. Transformation with DNA from 5-azacytidine-reactivated X chromosomes.

    OpenAIRE

    Venolia, L; Gartler, S M; Wassman, E R; Yen, P.; Mohandas, T; Shapiro, L J

    1982-01-01

    It has been shown that 5-azacytidine (5-Aza-Cyd) can reactivate genes on the inactive human X chromosome. It is assumed that the 5-Aza-Cyd acts by causing demethylation of the DNA at specific sites, but this cannot be demonstrated directly without a cloned probe. Instead, we have utilized the technique of DNA-mediated transformation to show that the 5-Aza-Cyd-induced reactivation occurs at the DNA level. DNAs from various mouse-human or hamster-human hybrid cell lines, deficient for mouse or ...

  8. X chromosome inactivation: Activation of Silencing

    NARCIS (Netherlands)

    I.H. Jonkers (Iris)

    2009-01-01

    textabstractX chromosome inactivation is a process that ensures equal expression of the X chromosomes between males, which have one X and one Y chromosome, and females, which have two X chromosomes, in mammals. Females initiate inactivation of one of their two X chromosomes early during embryogenesi

  9. Chromosome Connections: Compelling Clues to Common Ancestry

    Science.gov (United States)

    Flammer, Larry

    2013-01-01

    Students compare banding patterns on hominid chromosomes and see striking evidence of their common ancestry. To test this, human chromosome no. 2 is matched with two shorter chimpanzee chromosomes, leading to the hypothesis that human chromosome 2 resulted from the fusion of the two shorter chromosomes. Students test that hypothesis by looking for…

  10. X-chromosome workshop.

    Science.gov (United States)

    Paterson, A D

    1998-01-01

    Researchers presented results of ongoing research to the X-chromosome workshop of the Fifth World Congress on Psychiatric Genetics, covering a wide range of disorders: X-linked infantile spasms; a complex phenotype associated with deletions of Xp11; male homosexuality; degree of handedness; bipolar affective disorder; schizophrenia; childhood onset psychosis; and autism. This report summarizes the presentations, as well as reviewing previous studies. The focus of this report is on linkage findings for schizophrenia and bipolar disorder from a number of groups. For schizophrenia, low positive lod scores were obtained for markers DXS991 and DXS993 from two studies, although the sharing of alleles was greatest from brother-brother pairs in one study, and sister-sister in the other. Data from the Irish schizophrenia study was also submitted, with no strong evidence for linkage on the X chromosome. For bipolar disease, following the report of a Finnish family linked to Xq24-q27, the Columbia group reported some positive results for this region from 57 families, however, another group found no evidence for linkage to this region. Of interest, is the clustering of low positive linkage results that point to regions for possible further study. PMID:9686435

  11. Principles of artificial intelligence

    CERN Document Server

    Nilsson, Nils J

    1980-01-01

    A classic introduction to artificial intelligence intended to bridge the gap between theory and practice, Principles of Artificial Intelligence describes fundamental AI ideas that underlie applications such as natural language processing, automatic programming, robotics, machine vision, automatic theorem proving, and intelligent data retrieval. Rather than focusing on the subject matter of the applications, the book is organized around general computational concepts involving the kinds of data structures used, the types of operations performed on the data structures, and the properties of th

  12. The Artificial Anal Sphincter

    OpenAIRE

    Christiansen, John

    2000-01-01

    The artificial anal sphincter as treatment for end stage anal incontinence was first described in 1987. Published series concern a total of 42 patients, with a success rate of approximately 80%. Infection has been the most serious complication, but a number of technical complications related to the device have also occurred and required revisional procedures in 40% to 60% of the patients. The artificial anal sphincter may be used for the same indications as dynamic graciloplasty except in pat...

  13. Clone Networks, Clone Extensions and Biregularizations of Varieties of Algebras

    Institute of Scientific and Technical Information of China (English)

    J. Plonka

    2001-01-01

    We consider algebras of type τ- without nullary operations. An identity ψ≈ψ of type τ is clone compatible if ψ and ψ are the same variable or the sets of fundamental operation symbols in ψ and ψ are non-empty and identical. For a variety V, we denote by Vc the variety defined by all clone compatible identities from Id(V). In this paper, we give a construction of algebras called a clone network. Under some assumptions, we describe algebras from Vc by means of this construction. We find some properties of Vc and applications.

  14. Genes on chromosomes 4, 9, and 19 involved in 11q23 abnormalities in acute leukemia share sequence homology and/or common motifs.

    OpenAIRE

    Nakamura, T.(International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan); Alder, H; Y. Gu; R. Prasad; Canaani, O; Kamada, N; Gale, R P; Lange, B; Crist, W M; Nowell, P C

    1993-01-01

    Chromosome translocations involving band 11q23 are associated with human acute leukemias. These translocations fuse the ALL-1 gene, homolog of Drosophila trithorax and located at chromosome band 11q23, to genes from a variety of chromosomes. We cloned and sequenced cDNAs derived from transcripts of the AF-4 and AF-9 genes involved in the most common chromosome abnormalities, t(4:11)(q21:q23) and t(9:11)(p22:q23), respectively. Sequence analysis indicates high homology between the AF-9 gene pr...

  15. Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Redkar Rajendra

    2004-02-01

    Full Text Available Abstract Background Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH was used to identify specific chromosomal sequences unique to B. anthracis. Results Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. Conclusions Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

  16. Characterization of the OmyY1 region on the rainbow trout Y chromosome

    Science.gov (United States)

    Phillips, Ruth B.; DeKoning, Jenefer J.; Brunelli, Joseph P.; Faber-Hammond, Joshua J.; Hansen, John D.; Christensen, Kris A.; Renn, Suzy C.P.; Thorgaard, Gary H.

    2013-01-01

    We characterized the male-specific region on the Y chromosome of rainbow trout, which contains both sdY (the sex-determining gene) and the male-specific genetic marker, OmyY1. Several clones containing the OmyY1 marker were screened from a BAC library from a YY clonal line and found to be part of an 800 kb BAC contig. Using fluorescence in situ hybridization (FISH), these clones were localized to the end of the short arm of the Y chromosome in rainbow trout, with an additional signal on the end of the X chromosome in many cells. We sequenced a minimum tiling path of these clones using Illumina and 454 pyrosequencing. The region is rich in transposons and rDNA, but also appears to contain several single-copy protein-coding genes. Most of these genes are also found on the X chromosome; and in several cases sex-specific SNPs in these genes were identified between the male (YY) and female (XX) homozygous clonal lines. Additional genes were identified by hybridization of the BACs to the cGRASP salmonid 4x44K oligo microarray. By BLASTn evaluations using hypothetical transcripts of OmyY1-linked candidate genes as query against several EST databases, we conclude at least 12 of these candidate genes are likely functional, and expressed.

  17. YAC contigs of the Rab1 and wobbler (wr) spinal muscular atrophy gene region on proximal mouse chromosome 11 and of the homologous region on human chromosome 2p

    Energy Technology Data Exchange (ETDEWEB)

    Wedemeyer, N.; Lengeling, A.; Ronsiek, M. [Univ. of Bielefeld (Germany)] [and others

    1996-03-05

    Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr2p). We have localized the wobbler spinal atrophy gene wr to proximal mouse Chr 11, tightly linked to Rab1, a gene coding for a small GTP-binding protein, and Glns-ps1, an intronless pseudogene of the glutamine synthetase gene. We have not used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of the Rab1 region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescence in situ hybridization (FISH), and sequence-tagged site (STS) isolation and mapping. Rab1 and Glns-ps1 were found to be only 200 kb apart. A potential CpG island near a methylated NarI site and a trapped exon, ETG1.1, were found over 250 kb from Rab1. Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising the RAB1 locus, AHY1.1, and the human homologue of ETG1.1, indicating a high degree of conservation of this region in the two species. We mapped AHY1.1 and thus human RAB1 on Chr 2p13.4-p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the gene LMGMD2B for a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13-p16. The conservation between the mouse Rab1 and human RAB1 regions will be helpful in identifying candidate genes for the wobbler spinal muscular atrophy and in clarifying a possible relationship between wr and LMGMD2B. 33 refs., 7 figs., 3 tabs.

  18. Construction and analysis of an hn-cDNA library derived from the p-arm of pig chromosome 12.

    Science.gov (United States)

    Anderson Dear, D V; Miller, J R

    1996-09-01

    Our aim is to find unidentified genes on specific pig chromosomes or chromosome fragments. Our approach has involved the construction of a heterogeneous nuclear complementary (hn-c) DNA library of the p-arm of pig Chromosome (Chr) 12, the only pig chromosome present in the pig x hamster hybrid cell line 8990. Total RNA was extracted from the cells and first-strand synthesis of hn-cDNA carried out with random and oligo dT primers. Pig hn-cDNA was isolated by amplification of first-strand synthesized hn-cDNA with primers specific for Short Interspersed Repeat Elements (SINEs) via the polymerase chain reaction (PCR). Hn-cDNAs were size selected and cloned in E. coli XL-1 blue cells with PCR-Script as the vector. The library consisted of 6000 clones. Clone inserts were amplified by PCR with vector-specific primers, and randomly picked inserts greater than 600 bp were sequenced. Homology searches were carried out with the FASTA search program on the GenEmbl database. Thirty clones were sequenced, and of these three showed strong homologies to GenEmbl sequences: (1) to sheep, mouse, human, and rat mammary gland factor (MGF); (2) to MLN-50, a gene that is amplified in human familial breast cancer and is present on human Chr 17; the latter is homologous to pig chromosome 12; (3) to a family of unassigned overlapping human ESTs. Of the other sequenced clones, seven were over 80% homologous with pig SINE sequences; three were over 75% homologous to human LINE sequences; six displayed open reading frames over a mean distance equivalent to 50 amino acids, although these showed no significant similarities with sequences in the databases. Using this approach, we have been able to identify several new genes on the p-arm of pig Chr 12. This is the first report of gene isolation from a library derived from a pig chromosome fragment. PMID:8703117

  19. Integration of 28 STSs into the physical map of human chromosome 18

    Energy Technology Data Exchange (ETDEWEB)

    Gerken, S.; White, R.; Bradley, P. [Univ. of Utah, Salt Lake City, UT (United States)] [and others

    1994-12-01

    Genes on human chromosome 18 are associated with familial glucocorticoid deficiency (MC2R), pemphigus vulgaris (DSG3) and foliaceus (DSG1), familial amyloidosis (TTR), colorectal carcinoma (DCC), erythropoietic protoporphyria (FECH), follicular lymphoma (BCL2, FVT1), and congenital methemoglobinemia (CYB5). As the resolution of human genetic maps improves, linkage between other diseases and specific regions of chromosome 18 will occur. A physical map of human chromosome 18 will prove useful in identifying candidate genes that are associated with these disorders. Using various physical and genetic mapping techniques, over 35 genes and 19 expressed sequence tags (ESTs) are assigned to human chromosome 18. Most of these genes and several of the ESTs were sublocalized using a well-defined panel of somatic cell hybrids that contain different segments of human chromosome 18. Despite recent efforts, progress in mapping human chromosome 18 has lagged behind that achieved for other chromosomes. Thus, the purpose of this study was to integrate 9 new transcriptional tags [8 brain ESTs (8) and the melanocortin 4 receptor (MC4R) (3)] and 19 simple sequence repeats (SSRs) into the physical map of human chromosome 18. The SSRs were isolated by screening genomic DNA libraries constructed in M13mp18 vectors with oligonucleotide probes that detected dinucleotide d(CA)- and tetranucleotide-repeat motifs. DNA sequences of clones that contained microsatellite repeats were obtained by thermal-cycle sequencing, and STSs were developed from clones that contained numerous repeats. STSs that identified highly polymorphic loci in eight unrelated CEPH parents were used for genotyping. Results of linkage analyses and estimates of heterozygosity for these markers will be reported. 9 refs., 1 fig., 1 tab.

  20. Causes of oncogenic chromosomal translocation

    OpenAIRE

    Aplan, Peter D.

    2005-01-01

    Non-random chromosomal translocations are frequently associated with a variety of cancers, especially hematologic malignancies and childhood sarcomas In addition to their diagnostic utility, chromosomal translocations are increasingly being used in the clinic to guide therapeutic decisions. However, the mechanisms which cause these translocations remain poorly understood. Illegit...