WorldWideScience

Sample records for human chromosomal dna

  1. [DNA image-fluorimetry of individual human chromosomes].

    Science.gov (United States)

    Agafonova, N A; Sakuta, G A; Rozanov, Iu M; Shteĭn, G I; Kudriavtsev, B N

    2013-01-01

    Mucrofluorimetric method for the determination of DNA content in individual human chromosomes has been developed. The method is based on a preliminary identification of chromosomes with Hoechst 33258, followed by staining of the chromosomes with Feulgen reaction using Schiffs reagent type ethidium bromide-SO2, then measuring the fluorescence intensity of the chromosomes using an image analyzer. The method allows to determine the DNA content of individual chromosomes with accuracy up to 4.5 fg. DNA content of individual human chromosomes, their p-and q-arms as well as homologous chromosomes were measured using the developed method. It has been shown that the DNA content in the chromosomes of normal human karyotype is unstable. Fluctuations in the DNA content in some chromosomes can vary 35-40 fg.

  2. Isolation and characterization of DNA probes for human chromosome 21.

    Science.gov (United States)

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  3. The origin of human chromosome 2 analyzed by comparative chromosome mapping with a DNA microlibrary

    OpenAIRE

    Wienberg, Johannes; Jauch, Anna; Lüdecke, H J; Senger, G.; Horsthemke, B; Claussen, U.; Cremer, Thomas; Arnold, N; Lengauer, Christoph

    1994-01-01

    Fluorescencein situ hybridization (FISH) of microlibraries established from distinct chromosome subregions can test the evolutionary conservation of chromosome bands as well as chromosomal rearrangements that occurred during primate evolution and will help to clarify phylogenetic relationships. We used a DNA library established by microdissection and microcloning from the entire long arm of human chromosome 2 for fluorescencein situ hybridization and comparative mapping of the chromosomes of ...

  4. Evolutionarily different alphoid repeat DNA on homologous chromosomes in human and chimpanzee.

    OpenAIRE

    Jørgensen, A L; Laursen, H B; Jones, C; Bak, A L

    1992-01-01

    Centromeric alphoid DNA in primates represents a class of evolving repeat DNA. In humans, chromosomes 13 and 21 share one subfamily of alphoid DNA while chromosomes 14 and 22 share another subfamily. We show that similar pairwise homogenizations occur in the chimpanzee (Pan troglodytes), where chromosomes 14 and 22, homologous to human chromosomes 13 and 21, share one partially homogenized alphoid DNA subfamily and chromosomes 15 and 23, homologous to human chromosomes 14 and 22, share anothe...

  5. DNA methylation profiling of human chromosomes 6, 20 and 22

    OpenAIRE

    Eckhardt, Florian; Lewin, Joern; Cortese, Rene; Rakyan, Vardhman K.; Attwood, John; Burger, Matthias; Burton, John; Cox, Tony V.; Davies, Rob; Down, Thomas A; Haefliger, Carolina; Horton, Roger; Howe, Kevin; Jackson, David K.; Kunde, Jan

    2006-01-01

    DNA methylation constitutes the most stable type of epigenetic modifications modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation reference profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of 6 annotation categories, revealed evolutionary conserved regions to be the predominant sites for differential DNA methyl...

  6. The DNA sequence of the human X chromosome.

    Science.gov (United States)

    Ross, Mark T; Grafham, Darren V; Coffey, Alison J; Scherer, Steven; McLay, Kirsten; Muzny, Donna; Platzer, Matthias; Howell, Gareth R; Burrows, Christine; Bird, Christine P; Frankish, Adam; Lovell, Frances L; Howe, Kevin L; Ashurst, Jennifer L; Fulton, Robert S; Sudbrak, Ralf; Wen, Gaiping; Jones, Matthew C; Hurles, Matthew E; Andrews, T Daniel; Scott, Carol E; Searle, Stephen; Ramser, Juliane; Whittaker, Adam; Deadman, Rebecca; Carter, Nigel P; Hunt, Sarah E; Chen, Rui; Cree, Andrew; Gunaratne, Preethi; Havlak, Paul; Hodgson, Anne; Metzker, Michael L; Richards, Stephen; Scott, Graham; Steffen, David; Sodergren, Erica; Wheeler, David A; Worley, Kim C; Ainscough, Rachael; Ambrose, Kerrie D; Ansari-Lari, M Ali; Aradhya, Swaroop; Ashwell, Robert I S; Babbage, Anne K; Bagguley, Claire L; Ballabio, Andrea; Banerjee, Ruby; Barker, Gary E; Barlow, Karen F; Barrett, Ian P; Bates, Karen N; Beare, David M; Beasley, Helen; Beasley, Oliver; Beck, Alfred; Bethel, Graeme; Blechschmidt, Karin; Brady, Nicola; Bray-Allen, Sarah; Bridgeman, Anne M; Brown, Andrew J; Brown, Mary J; Bonnin, David; Bruford, Elspeth A; Buhay, Christian; Burch, Paula; Burford, Deborah; Burgess, Joanne; Burrill, Wayne; Burton, John; Bye, Jackie M; Carder, Carol; Carrel, Laura; Chako, Joseph; Chapman, Joanne C; Chavez, Dean; Chen, Ellson; Chen, Guan; Chen, Yuan; Chen, Zhijian; Chinault, Craig; Ciccodicola, Alfredo; Clark, Sue Y; Clarke, Graham; Clee, Chris M; Clegg, Sheila; Clerc-Blankenburg, Kerstin; Clifford, Karen; Cobley, Vicky; Cole, Charlotte G; Conquer, Jen S; Corby, Nicole; Connor, Richard E; David, Robert; Davies, Joy; Davis, Clay; Davis, John; Delgado, Oliver; Deshazo, Denise; Dhami, Pawandeep; Ding, Yan; Dinh, Huyen; Dodsworth, Steve; Draper, Heather; Dugan-Rocha, Shannon; Dunham, Andrew; Dunn, Matthew; Durbin, K James; Dutta, Ireena; Eades, Tamsin; Ellwood, Matthew; Emery-Cohen, Alexandra; Errington, Helen; Evans, Kathryn L; Faulkner, Louisa; Francis, Fiona; Frankland, John; Fraser, Audrey E; Galgoczy, Petra; Gilbert, James; Gill, Rachel; Glöckner, Gernot; Gregory, Simon G; Gribble, Susan; Griffiths, Coline; Grocock, Russell; Gu, Yanghong; Gwilliam, Rhian; Hamilton, Cerissa; Hart, Elizabeth A; Hawes, Alicia; Heath, Paul D; Heitmann, Katja; Hennig, Steffen; Hernandez, Judith; Hinzmann, Bernd; Ho, Sarah; Hoffs, Michael; Howden, Phillip J; Huckle, Elizabeth J; Hume, Jennifer; Hunt, Paul J; Hunt, Adrienne R; Isherwood, Judith; Jacob, Leni; Johnson, David; Jones, Sally; de Jong, Pieter J; Joseph, Shirin S; Keenan, Stephen; Kelly, Susan; Kershaw, Joanne K; Khan, Ziad; Kioschis, Petra; Klages, Sven; Knights, Andrew J; Kosiura, Anna; Kovar-Smith, Christie; Laird, Gavin K; Langford, Cordelia; Lawlor, Stephanie; Leversha, Margaret; Lewis, Lora; Liu, Wen; Lloyd, Christine; Lloyd, David M; Loulseged, Hermela; Loveland, Jane E; Lovell, Jamieson D; Lozado, Ryan; Lu, Jing; Lyne, Rachael; Ma, Jie; Maheshwari, Manjula; Matthews, Lucy H; McDowall, Jennifer; McLaren, Stuart; McMurray, Amanda; Meidl, Patrick; Meitinger, Thomas; Milne, Sarah; Miner, George; Mistry, Shailesh L; Morgan, Margaret; Morris, Sidney; Müller, Ines; Mullikin, James C; Nguyen, Ngoc; Nordsiek, Gabriele; Nyakatura, Gerald; O'Dell, Christopher N; Okwuonu, Geoffery; Palmer, Sophie; Pandian, Richard; Parker, David; Parrish, Julia; Pasternak, Shiran; Patel, Dina; Pearce, Alex V; Pearson, Danita M; Pelan, Sarah E; Perez, Lesette; Porter, Keith M; Ramsey, Yvonne; Reichwald, Kathrin; Rhodes, Susan; Ridler, Kerry A; Schlessinger, David; Schueler, Mary G; Sehra, Harminder K; Shaw-Smith, Charles; Shen, Hua; Sheridan, Elizabeth M; Shownkeen, Ratna; Skuce, Carl D; Smith, Michelle L; Sotheran, Elizabeth C; Steingruber, Helen E; Steward, Charles A; Storey, Roy; Swann, R Mark; Swarbreck, David; Tabor, Paul E; Taudien, Stefan; Taylor, Tineace; Teague, Brian; Thomas, Karen; Thorpe, Andrea; Timms, Kirsten; Tracey, Alan; Trevanion, Steve; Tromans, Anthony C; d'Urso, Michele; Verduzco, Daniel; Villasana, Donna; Waldron, Lenee; Wall, Melanie; Wang, Qiaoyan; Warren, James; Warry, Georgina L; Wei, Xuehong; West, Anthony; Whitehead, Siobhan L; Whiteley, Mathew N; Wilkinson, Jane E; Willey, David L; Williams, Gabrielle; Williams, Leanne; Williamson, Angela; Williamson, Helen; Wilming, Laurens; Woodmansey, Rebecca L; Wray, Paul W; Yen, Jennifer; Zhang, Jingkun; Zhou, Jianling; Zoghbi, Huda; Zorilla, Sara; Buck, David; Reinhardt, Richard; Poustka, Annemarie; Rosenthal, André; Lehrach, Hans; Meindl, Alfons; Minx, Patrick J; Hillier, Ladeana W; Willard, Huntington F; Wilson, Richard K; Waterston, Robert H; Rice, Catherine M; Vaudin, Mark; Coulson, Alan; Nelson, David L; Weinstock, George; Sulston, John E; Durbin, Richard; Hubbard, Tim; Gibbs, Richard A; Beck, Stephan; Rogers, Jane; Bentley, David R

    2005-03-17

    The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

  7. The DNA sequence and biology of human chromosome 19.

    Science.gov (United States)

    Grimwood, Jane; Gordon, Laurie A; Olsen, Anne; Terry, Astrid; Schmutz, Jeremy; Lamerdin, Jane; Hellsten, Uffe; Goodstein, David; Couronne, Olivier; Tran-Gyamfi, Mary; Aerts, Andrea; Altherr, Michael; Ashworth, Linda; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caenepeel, Sean; Carrano, Anthony; Caoile, Chenier; Chan, Yee Man; Christensen, Mari; Cleland, Catherine A; Copeland, Alex; Dalin, Eileen; Dehal, Paramvir; Denys, Mirian; Detter, John C; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Garcia, Carmen; Georgescu, Anca M; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Ho, Isaac; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Larionov, Vladimer; Leem, Sun-Hee; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Malfatti, Stephanie; Martinez, Diego; McCready, Paula; Medina, Catherine; Morgan, Jenna; Nelson, Kathryn; Nolan, Matt; Ovcharenko, Ivan; Pitluck, Sam; Pollard, Martin; Popkie, Anthony P; Predki, Paul; Quan, Glenda; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanine; Salamov, Asaf; Salazar, Angelica; She, Xinwei; Smith, Doug; Slezak, Tom; Solovyev, Victor; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wagner, Mark; Wheeler, Jeremy; Wu, Kevin; Xie, Gary; Yang, Joan; Dubchak, Inna; Furey, Terrence S; DeJong, Pieter; Dickson, Mark; Gordon, David; Eichler, Evan E; Pennacchio, Len A; Richardson, Paul; Stubbs, Lisa; Rokhsar, Daniel S; Myers, Richard M; Rubin, Edward M; Lucas, Susan M

    2004-04-01

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

  8. The DNA sequence and biology of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Grimwood, J; Gordon, L A; Olsen, A; Terry, A; Schmutz, J; Lamerdin, J; Hellsten, U; Goodstein, D; Couronne, O; Tran-Gyamfi, M

    2004-04-06

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high GC content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in Mendelian disorders, including familial hypercholesterolemia and insulin-resistant diabetes. Nearly one quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

  9. The DNA sequence and biology of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Grimwood, Jane; Gordon, Laurie A.; Olsen, Anne; Terry, Astrid; Schmutz, Jeremy; Lamerdin, Jane; Hellsten, Uffe; Goodstein, David; Couronne, Olivier; Tran-Gyamfi, Mary; Aerts, Andrea; Altherr, Michael; Ashworth, Linda; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caenepeel, Sean; Carrano, Anthony; Caoile, Chenier; Chan, Yee Man; Christensen, Mari; Cleland, Catherine A.; Copeland, Alex; Dalin, Eileen; Dehal, Paramvir; Denys, Mirian; Detter, John C.; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Garcia, Carmen; Georgescu, Anca M.; Glavina, Tijana; Gomez, Maria; Gonzales, Eldelyn; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Ho, Issac; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Larionov, Vladimer; Leem, Sun-Hee; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Malfatti, Stephanie; Martinez, Diego; McCready, Paula; Medina, Catherine; Morgan, Jenna; Nelson, Kathryn; Nolan, Matt; Ovcharenko, Ivan; Pitluck, Sam; Pollard, Martin; Popkie, Anthony P.; Predki, Paul; Quan, Glenda; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanine; Salamov, Asaf; Salazar, Angelica; She, Xinwei; Smith, Doug; Slezak, Tom; Solovyev, Victor; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wagner, Mark; Wheeler, Jeremy; Wu, Kevin; Xie, Gary; Yang, Joan; Dubchak, Inna; Furey, Terrence S.; DeJong, Pieter; Dickson, Mark; Gordon, David; Eichler, Evan E.; Pennacchio, Len A.; Richardson, Paul; Stubbs, Lisa; Rokhsar, Daniel S.; Myers, Richard M.; Rubin, Edward M.; Lucas, Susan M.

    2003-09-15

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G1C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9 percent of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25 percent of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, a nd segments of coding and non-coding conservation with the distant fish species Takifugu.

  10. The DNA sequence, annotation and analysis of human chromosome 3

    DEFF Research Database (Denmark)

    Muzny, Donna M; Scherer, Steven E; Kaul, Rajinder

    2006-01-01

    After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chr...

  11. The DNA sequence of the human X chromosome

    OpenAIRE

    Ross, Mark T.; Grafham, Darren V.; Coffey, Alison J; Scherer, Steven; McLay, Kirsten; Muzny, Donna; Platzer, Matthias; Howell, Gareth R.; Burrows, Christine; Bird, Christine P.; Frankish, Adam; Lovell, Frances L.; Howe, Kevin L; Jennifer L Ashurst; Fulton, Robert S.

    2005-01-01

    The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a...

  12. DNA Catenation Maintains Structure of Human Metaphase Chromosomes

    DEFF Research Database (Denmark)

    L. V. Bauer, David; Marie, Rodolphe; Rasmussen, Kristian Hagsted

    2012-01-01

    Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip...

  13. The finished DNA sequence of human chromosome 12.

    Science.gov (United States)

    Scherer, Steven E; Muzny, Donna M; Buhay, Christian J; Chen, Rui; Cree, Andrew; Ding, Yan; Dugan-Rocha, Shannon; Gill, Rachel; Gunaratne, Preethi; Harris, R Alan; Hawes, Alicia C; Hernandez, Judith; Hodgson, Anne V; Hume, Jennifer; Jackson, Andrew; Khan, Ziad Mohid; Kovar-Smith, Christie; Lewis, Lora R; Lozado, Ryan J; Metzker, Michael L; Milosavljevic, Aleksandar; Miner, George R; Montgomery, Kate T; Morgan, Margaret B; Nazareth, Lynne V; Scott, Graham; Sodergren, Erica; Song, Xing-Zhi; Steffen, David; Lovering, Ruth C; Wheeler, David A; Worley, Kim C; Yuan, Yi; Zhang, Zhengdong; Adams, Charles Q; Ansari-Lari, M Ali; Ayele, Mulu; Brown, Mary J; Chen, Guan; Chen, Zhijian; Clerc-Blankenburg, Kerstin P; Davis, Clay; Delgado, Oliver; Dinh, Huyen H; Draper, Heather; Gonzalez-Garay, Manuel L; Havlak, Paul; Jackson, Laronda R; Jacob, Leni S; Kelly, Susan H; Li, Li; Li, Zhangwan; Liu, Jing; Liu, Wen; Lu, Jing; Maheshwari, Manjula; Nguyen, Bao-Viet; Okwuonu, Geoffrey O; Pasternak, Shiran; Perez, Lesette M; Plopper, Farah J H; Santibanez, Jireh; Shen, Hua; Tabor, Paul E; Verduzco, Daniel; Waldron, Lenee; Wang, Qiaoyan; Williams, Gabrielle A; Zhang, Jingkun; Zhou, Jianling; Allen, Carlana C; Amin, Anita G; Anyalebechi, Vivian; Bailey, Michael; Barbaria, Joseph A; Bimage, Kesha E; Bryant, Nathaniel P; Burch, Paula E; Burkett, Carrie E; Burrell, Kevin L; Calderon, Eliana; Cardenas, Veronica; Carter, Kelvin; Casias, Kristal; Cavazos, Iracema; Cavazos, Sandra R; Ceasar, Heather; Chacko, Joseph; Chan, Sheryl N; Chavez, Dean; Christopoulos, Constantine; Chu, Joseph; Cockrell, Raynard; Cox, Caroline D; Dang, Michelle; Dathorne, Stephanie R; David, Robert; Davis, Candi Mon'Et; Davy-Carroll, Latarsha; Deshazo, Denise R; Donlin, Jeremy E; D'Souza, Lisa; Eaves, Kristy A; Egan, Amy; Emery-Cohen, Alexandra J; Escotto, Michael; Flagg, Nicole; Forbes, Lisa D; Gabisi, Abdul M; Garza, Melissa; Hamilton, Cerissa; Henderson, Nicholas; Hernandez, Omar; Hines, Sandra; Hogues, Marilyn E; Huang, Mei; Idlebird, DeVincent G; Johnson, Rudy; Jolivet, Angela; Jones, Sally; Kagan, Ryan; King, Laquisha M; Leal, Belita; Lebow, Heather; Lee, Sandra; LeVan, Jaclyn M; Lewis, Lakeshia C; London, Pamela; Lorensuhewa, Lorna M; Loulseged, Hermela; Lovett, Demetria A; Lucier, Alice; Lucier, Raymond L; Ma, Jie; Madu, Renita C; Mapua, Patricia; Martindale, Ashley D; Martinez, Evangelina; Massey, Elizabeth; Mawhiney, Samantha; Meador, Michael G; Mendez, Sylvia; Mercado, Christian; Mercado, Iracema C; Merritt, Christina E; Miner, Zachary L; Minja, Emmanuel; Mitchell, Teresa; Mohabbat, Farida; Mohabbat, Khatera; Montgomery, Baize; Moore, Niki; Morris, Sidney; Munidasa, Mala; Ngo, Robin N; Nguyen, Ngoc B; Nickerson, Elizabeth; Nwaokelemeh, Ogechi O; Nwokenkwo, Stanley; Obregon, Melissa; Oguh, Maryann; Oragunye, Njideka; Oviedo, Rodolfo J; Parish, Bridgette J; Parker, David N; Parrish, Julia; Parks, Kenya L; Paul, Heidie A; Payton, Brett A; Perez, Agapito; Perrin, William; Pickens, Adam; Primus, Eltrick L; Pu, Ling-Ling; Puazo, Maria; Quiles, Miyo M; Quiroz, Juana B; Rabata, Dina; Reeves, Kacy; Ruiz, San Juana; Shao, Hongmei; Sisson, Ida; Sonaike, Titilola; Sorelle, Richard P; Sutton, Angelica E; Svatek, Amanda F; Svetz, Leah Anne; Tamerisa, Kavitha S; Taylor, Tineace R; Teague, Brian; Thomas, Nicole; Thorn, Rachel D; Trejos, Zulma Y; Trevino, Brenda K; Ukegbu, Ogechi N; Urban, Jeremy B; Vasquez, Lydia I; Vera, Virginia A; Villasana, Donna M; Wang, Ling; Ward-Moore, Stephanie; Warren, James T; Wei, Xuehong; White, Flower; Williamson, Angela L; Wleczyk, Regina; Wooden, Hailey S; Wooden, Steven H; Yen, Jennifer; Yoon, Lillienne; Yoon, Vivienne; Zorrilla, Sara E; Nelson, David; Kucherlapati, Raju; Weinstock, George; Gibbs, Richard A

    2006-03-16

    Human chromosome 12 contains more than 1,400 coding genes and 487 loci that have been directly implicated in human disease. The q arm of chromosome 12 contains one of the largest blocks of linkage disequilibrium found in the human genome. Here we present the finished sequence of human chromosome 12, which has been finished to high quality and spans approximately 132 megabases, representing approximately 4.5% of the human genome. Alignment of the human chromosome 12 sequence across vertebrates reveals the origin of individual segments in chicken, and a unique history of rearrangement through rodent and primate lineages. The rate of base substitutions in recent evolutionary history shows an overall slowing in hominids compared with primates and rodents.

  14. Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

    Science.gov (United States)

    Baldini, A; Archidiacono, N; Carbone, R; Bolino, A; Shridhar, V; Miller, O J; Miller, D A; Ward, D C; Rocchi, M

    1992-01-01

    We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.

  15. The DNA Sequence And Comparative Analysis Of Human Chromosome5

    Energy Technology Data Exchange (ETDEWEB)

    Schmutz, Jeremy; Martin, Joel; Terry, Astrid; Couronne, Olivier; Grimwood, Jane; Lowry, Steve; Gordon, Laurie A.; Scott, Duncan; Xie,Gary; Huang, Wayne; Hellsten, Uffe; Tran-Gyamfi, Mary; She, Xinwei; Prabhakar, Shyam; Aerts, Andrea; Altherr, Michael; Bajorek, Eva; Black,Stacey; Branscomb, Elbert; Caoile, Chenier; Challacombe, Jean F.; Chan,Yee Man; Denys, Mirian; Detter, John C.; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Goodstein, David; Grigoriev, Igor; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Israni, Sanjay; Jett, Jamie; Kadner,Kristen; Kimball, Heather; Kobayashi, Arthur; Lopez, Frederick; Lou,Yunian; Martinez, Diego; Medina, Catherine; Morgan, Jenna; Nandkeshwar,Richard; Noonan, James P.; Pitluck, Sam; Pollard, Martin; Predki, Paul; Priest, James; Ramirez, Lucia; Retterer, James; Rodriguez, Alex; Rogers,Stephanie; Salamov, Asaf; Salazar, Angelica; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wheeler, Jeremy; Wu, Kevin; Yang,Joan; Dickson, Mark; Cheng, Jan-Fang; Eichler, Evan E.; Olsen, Anne; Pennacchio, Len A.; Rokhsar, Daniel S.; Richardson, Paul; Lucas, SusanM.; Myers, Richard M.; Rubin, Edward M.

    2004-08-01

    Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.

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

    Science.gov (United States)

    D'Aiuto, L; Antonacci, R; Marzella, R; Archidiacono, N; Rocchi, M

    1993-11-01

    We 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.

  17. 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.

  18. Comparative mapping of a gorilla-derived alpha satellite DNA clone on great ape and human chromosomes.

    Science.gov (United States)

    Baldini, A; Miller, D A; Shridhar, V; Rocchi, M; Miller, O J; Ward, D C

    1991-11-01

    We have isolated an alpha satellite DNA clone, pG3.9, from gorilla DNA. Fluorescence in situ hybridization on banded chromosomes under high stringency conditions revealed that pG3.9 identifies homologous sequences at the centromeric region of ten gorilla chromosomes, and, with few exceptions, also recognizes the homologous chromosomes in human. A pG3.9-like alphoid DNA is present on a larger number of orangutan chromosomes, but, in contrast, is present on only two chromosomes in the chimpanzee. These results show that the chromosomal subsets of related alpha satellite DNA sequences may undergo different patterns of evolution.

  19. Chromosomal mapping of 18S-28S rRNA genes and 10 cDNA clones of human chromosome 1 in the musk shrew (Suncus murinus).

    Science.gov (United States)

    Kuroiwa, A; Matsubara, K; Nagase, T; Nomura, N; Seong, J K; Ishikawa, A; Anunciado, R V; Tanaka, K; Yamagata, T; Masangkay, J S; Dang, V B; Namikawa, T; Matsuda, Y

    2001-01-01

    The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.

  20. A 6. 5-Mb yeast artificial chromosome contig incorporating 33 DNA markers on the human X chromosome at Xq22

    Energy Technology Data Exchange (ETDEWEB)

    Vetrie, D.; Kendall, E.; Coffey, A.; Hassock, S.; Collins, J.; Todd, C.; Bobrow, M.; Bentley, D.R. (Paediatric Research Unit, London (United Kingdom)); Lehrach, H. (Imperial Cancer Research Fund, London (United Kingdom)); Harris, A. (John Radcliffe Hospital, Oxford (United Kingdom))

    1994-01-01

    The Xq22 region of the human X chromosome contains genes for a number of inherited disorders. Sixty-nine yeast artificial chromosome clones have been isolated and assembled into a 6.5-Mb contig that contains 33 DNA markers localized to this region. This contig extends distally from DXS366 to beyond DXS87 and includes the genes involved in X-linked agammaglobulinemia (btk), Fabry disease (GLA), and Pelizaeus-Merzbacher disease (PLP). The order of markers in this contig is consistent with the known genetic and physical mapping information of Xq22. This cloned material provides a source from which to isolate other genes located in this part of the X chromosome. 45 refs., 2 figs., 2 tabs.

  1. 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.

  2. Universal spectrum for DNA base C+G frequency distribution in Human chromosomes 1 to 24

    CERN Document Server

    Selvam, A M

    2007-01-01

    Power spectra of human DNA base C+G frequency distribution in all available contiguous sections exhibit the universal inverse power law form of the statistical normal distribution for the 24 chromosomes. Inverse power law form for power spectra of space-time fluctuations is generic to dynamical systems in nature and indicate long-range space-time correlations. A recently developed general systems theory predicts the observed non-local connections as intrinsic to quantumlike chaos governing space-time fluctuations of dynamical systems. The model predicts the following. (1) The quasiperiodic Penrose tiling pattern for the nested coiled structure of the DNA molecule in the chromosome resulting in maximum packing efficiency. (2) The DNA molecule functions as a unified whole fuzzy logic network with ordered two-way signal transmission between the coding and non-coding regions. Recent studies indicate influence of non-coding regions on functions of coding regions in the DNA molecule.

  3. Large-scale oscillation of structure-related DNA sequence features in human chromosome 21

    Science.gov (United States)

    Li, Wentian; Miramontes, Pedro

    2006-08-01

    Human chromosome 21 is the only chromosome in the human genome that exhibits oscillation of the (G+C) content of a cycle length of hundreds kilobases (kb) ( 500kb near the right telomere). We aim at establishing the existence of a similar periodicity in structure-related sequence features in order to relate this (G+C)% oscillation to other biological phenomena. The following quantities are shown to oscillate with the same 500kb periodicity in human chromosome 21: binding energy calculated by two sets of dinucleotide-based thermodynamic parameters, AA/TT and AAA/TTT bi- and tri-nucleotide density, 5'-TA-3' dinucleotide density, and signal for 10- or 11-base periodicity of AA/TT or AAA/TTT. These intrinsic quantities are related to structural features of the double helix of DNA molecules, such as base-pair binding, untwisting or unwinding, stiffness, and a putative tendency for nucleosome formation.

  4. Isolation, Mapping, DNA Sequence and RFLPs Studies of Random Single-Copy DNA Segments on Human X Chromosome

    Institute of Scientific and Technical Information of China (English)

    谭骏; 邱信芳; 薛京伦; 朱锡华; 纪贤文; 张冬梅; 秦世真

    1994-01-01

    Using the total human/mouse DNA as the probe, screening has been carried out three times with in situ plaque hybridization to obtain the single-copy DNA sequence from the human X chromosome genomic library. The effective rate of screening is 1. 45%. DNAs from clones containing single-copy inserts have been analyzed by a panel of hybrid cells with or without human X chromosome. Three segments, designated by DXFD52,73,75, are mapped to the X chromosome. DXFD52 has been precisely localized on Xq12-q13 with in situ chromosomal hybridization. DXFD52 has been partially sequenced. The results indicate that DXFD52 is a new isolated single-copy segment on the X chromosome. Great progress in the RFLPs study with DXFD52 has been achieved in the population of Chongqing, Sichuan Province. The results show that the DXFD52 can be used to detect the RFLP with Hind Ⅲ, Bgl Ⅱ, and Hinf Ⅰ. DXFD52 will be a potential "landmark" for the construction of the complete linkage map of human genome and the analysis of genomic s

  5. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

    Directory of Open Access Journals (Sweden)

    Lifeng Liang

    Full Text Available A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH to evaluate accuracy of the results. We found that most (58.1% of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s, partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal

  6. Genetic linkage analysis of familial amyotrophic lateral sclerosis using human chromosome 21 microsatellite DNA markers

    Energy Technology Data Exchange (ETDEWEB)

    Rosen, D.R.; Sapp, P.; O`Regan, J.; McKenna-Yasek, D.; Schlumpf, K.S.; Haines, J.L.; Gusella, J.F.; Horvitz, H.R.; Brown, R.H. Jr. [Massachusetts Institute of Technology, Cambridge, MA (United States)

    1994-05-15

    Amyotrophic lateral sclerosis (ALS; Lou Gehrig`s Disease) is a lethal neurodegenerative disease of upper and lower motorneurons in the brain and spinal cord. We previously reported linkage of a gene for familial ALS (FALS) to human chromosome 21 using 4 restriction fragment length polymorphism DNA markers and identified disease-associated mutations in the superoxide dismutase (SOD)-1 gene in some ALS families. We report here the genetic linkage data that led us to examine the SOD-1 gene for mutations. We also report a new microsatellite DNA marker for D21S63, derived from the cosmid PW517. Ten microsatellite DNA markers, including the new marker D21S63, were used to reinvestigate linkage of FALS to chromosome 21. Genetic linkage analysis performed with 13 ALS familes for these 10 DNA markers confirmed the presence of a FALS gene on chromosome 21. The highest total 2-point LOD score for all families was 4.33, obtained at a distance of 10 cM from the marker D21S223. For 5 ALS families linked to chromosome 21, a peak 2-point LOD score of 5.94 was obtained at the DNA marker D21S223. A multipoint score of 6.50 was obtained with the markers D21S213, D21S223, D21S167, and FALS for 5 chromosome 21-linked ALS families. The haplotypes of these families for the 10 DNA markers reveal recombination events that further refined the location of the FALS gene to a segment of approximately 5 megabases (Mb) between D21S213 and D21S219. The only characterized gene within this segment was SOD-1, the structural gene for Cu, Zn SOD. 30 refs., 4 figs., 4 tabs.

  7. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  8. Chromosome loss caused by DNA fragmentation induced in main nuclei and micronuclei of human lymphoblastoid cells treated with colcemid.

    Science.gov (United States)

    Yamamoto, Mika; Wakata, Akihiro; Aoki, Yoshinobu; Miyamae, Yoichi; Kodama, Seiji

    2014-04-01

    Aneuploidy, a change in the number of chromosomes, plays an essential role in tumorigenesis. Our previous study demonstrated that a loss of a whole chromosome is induced in human lymphocytes by colcemid, a well-known aneugen. Here, to clarify the mechanism for colcemid-induced chromosome loss, we investigated the relationship between chromosome loss and DNA fragmentation in human lymphoblastoid cells treated with colcemid (an aneugen) compared with methyl methanesulfonate (MMS; a clastogen). We analyzed the number of fluorescence in situ hybridization (FISH) signals targeted for a whole chromosome 2 in cytokinesis-blocked binucleated TK6 cells and WTK-1 cells treated with colcemid and MMS, and concurrently detected DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results revealed that DNA fragmentation occurred in 60% of all binucleated TK6 cells harboring colcemid-induced chromosome loss (30% of micronuclei and 30% of main nuclei). DNA fragmentation was observed in colcemid-induced micronuclei containing a whole chromosome but not in MMS-induced micronuclei containing chromosome fragments. In contrast, colcemid-induced nondisjunction had no effect on induction of DNA fragmentation, suggesting that DNA fragmentation was triggered by micronuclei containing a whole chromosome but not by micronuclei containing chromosome fragments or nondisjunction. In addition, the frequency of binucleated cells harboring chromosome loss with DNA fragmentation in micronuclei or main nuclei was higher in wild-type p53 TK6 cells than in mutated-p53 WTK-1 cells treated with colcemid. Taken together, these present and previous results suggest that colcemid-induced chromosome loss is caused by DNA fragmentation, which is triggered by a micronucleus with a whole chromosome and controlled by the p53-dependent pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. A device for extraction, manipulation and stretching of DNA from single human chromosomes

    DEFF Research Database (Denmark)

    Rasmussen, Kristian Hagsted; Marie, Rodolphe; Moresco, Jacob Lange

    2011-01-01

    by time-lapse imaging; pressure-driven flow was then used to shunt the chromosomal DNA package into a nanoslit. A long linear DNA strand (>1.3 Mbp) was seen to stretch out from the DNA package and along the length of the nanoslit. Delivery of DNA in its native metaphase chromosome package as well...... as the microfluidic environment prevented DNA from shearing and will be important for preparing ultra-long lengths of DNA for nanofluidic analysis....

  10. Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes.

    Science.gov (United States)

    Shimizu, Nobuyoshi; Maekawa, Masahiko; Asai, Satoko; Shimizu, Yoshiko

    2015-12-01

    We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.

  11. Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Rossner, Pavel, E-mail: prossner@biomed.cas.cz; Rossnerova, Andrea; Beskid, Olena; Tabashidze, Nana; Libalova, Helena; Uhlirova, Katerina; Topinka, Jan; Sram, Radim J.

    2014-05-15

    Highlights: • We analyzed the effect of air pollutants on NHEJ and chromosome aberrations. • In HEL12469 cells B[a]P and extractable organic matter induced DSBs. • The compounds induced XRCC4 expression and a weak Ku70/80 response. • We found increased frequency of aberrations of chromosomes 1, 2, 4, 5, 7 and 17. • The tested compounds preferentially affected chromosome 7. - Abstract: In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 μm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24 h with the following concentrations of tested chemicals: B[a]P: 1 μM, 10 μM, 25 μM; EOMs: 1 μg/ml, 10 μg/ml, 25 μg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25 μM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and

  12. Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

    Science.gov (United States)

    Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

    2011-01-01

    Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

  13. In situ amplification of DNA fragments specific for human Y chromosome in cellular nuclei by PCR

    Institute of Scientific and Technical Information of China (English)

    张锡元; 姜海波; 李立家; 马琦; 杨建琪; 刘汀

    1996-01-01

    Using single primer pairs Y3 and Y4, in siru polymerase chain reaction (in situ PCR) was successfully performed on the specimen slides of peripheral leukocytes. By both of the direct digpxiginin-11-dUTP incorporation into PCR products with in situ PCR (direct in situ PCR) and in situ PCR followed by detection of in situ hybridization (indirect in siru PCR), DNA fragments specific for human Y chromosome were obviously amplified in cellular nuclei of specimens on the slides. The results were verified by Southern analysis. The methodology of in situ PCR and its application were discussed.

  14. Chromosomal assignment of human DNA fingerprint sequences by simultaneous hybridization to arbitrarily primed PCR products from human/rodent monochromosome cell hybrids

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Jun; Sekiya, Takao [National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Navarro, J.M. [Burnham Institute, La Jolla, CA (United States)] [and others

    1996-05-15

    We have developed a technique for the simultaneous chromosomal assignment of multiple human DNA sequences from DNA fingerprints obtained by the arbitrarily primed polymerase chain reaction (AP-PCR). Radioactively labeled human AP-PCR products are hybridized to DNA fingerprints generated with the same arbitrary primer from human/rodent monochromosome cell hybrids after electroblotting to a nylong membrane. Human-specific hybridization bands in the human/rodent fingerprints unambiguously determine their chromosome of origin. We named this method simultaneous hybridization of arbitrarily primed PCR DNA fingerprinting products (SHARP). Using this approach, we determined the chromosomal origins of most major bands of human AP-PCR fingerprints obtained with two arbitrary primers. Altogether, the chromosomal localization of near 50 DNA fragments, comprehensive of all human chromosomes except chromosomes 21 and Y, was achieved in this simple manner. Chromosome assignment of fingerprint bands is essential for molecular karyotyping of cancer by AP-PCR DNA fingerprinting. The SHARP method provides a convenient and powerful tool for this purpose. 23 refs., 3 figs., 2 tabs.

  15. Chromosomal aberrations and DNA damage in human populations exposed to the processing of electronics waste.

    Science.gov (United States)

    Liu, Qiang; Cao, Jia; Li, Ke Qiu; Miao, Xu Hong; Li, Guang; Fan, Fei Yue; Zhao, Yong Cheng

    2009-05-01

    It has been known that the pollutants of electronic wastes (E-wastes) can lead to severe pollution to the environment. It has been reported that about 50% to 80% of E-wastes from developed countries are exported to Asia and Africa. It has become a major global environmental problem to deal with 'E-wastes'. E-waste recycling has remained primitive in Jinghai, China. This not only produces enormous environmental pollution but also can bring about toxic or genotoxic effects on the human body, threatening the health of both current residents and future generations living in the local environment. The concentration of lead in the blood of children in the E-waste polluted area in China is higher than that of the control area. But little is known about the cytogenetic effect to human beings caused by the pollution of E-wastes. In the present study, experiments have been performed to investigate the genetics of permanent residents of three villages with numerous E-waste disposal sites and to analyze the harmful effects of exposure to E-wastes. In total, 171 villagers (exposed group) were randomly selected from permanent residents of three villages located in Jinghai County of Tianjin, China, where there has been massive disposal of E-wastes. Thirty villagers were selected from the neighboring towns without E-waste disposal sites to serve as controls. Chromosomal aberrations and cytokinesis blocking micronucleus were performed to detect the cytogenetic effect, dic + r (dicentric and ring chromosome), monomer, fragments (acentric fragments, minute chromosomes, and acentric rings), translocation, satellite, quadriradial, total aberrations, and micronuclear rate were scored for each subject. DNA damage was detected using comet assay; the DNA percentage in the comet tail (TDNA%), tail moment (TM), and Olive tail moment (OTM) were recorded to describe DNA damage to lymphocytes. The total chromosome aberration rates (5.50%) and micronuclear rates (16.99%) of the exposure group

  16. 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 ...

  17. A neocentromere on human chromosome 3 without detectable alpha-satellite DNA forms morphologically normal kinetochores

    DEFF Research Database (Denmark)

    Wandall, A; Tranebjaerg, L; Tommerup, Niels

    1998-01-01

    A neocentromere at 3q26 was observed in a father and his daughter on a chromosome 3 with deleted centromeric region. No alpha-satellite DNA was detectable at the 3q26 neocentromere, but it was weakly positive with anticentromere (CREST) antibodies. Electron microscopy showed that the neocentromere...... formed microtubule-associated kinetochores with normal morphology and of the same size as the kinetochores of other large chromosomes. The deleted centromere formed a small linear marker chromosome that reacted strongly with anticentromere antibodies, but showed reduced kinetochore size. The 3q26...

  18. Tissue-specific variation in DNA methylation levels along human chromosome 1

    Directory of Open Access Journals (Sweden)

    De Bustos Cecilia

    2009-06-01

    Full Text Available Abstract Background DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. Results Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined. Conclusion The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.

  19. Studies on the integration of hepatitis B virusDNA sequence in human sperm chromosomes

    Institute of Scientific and Technical Information of China (English)

    Jian-MinHUANG; Tian-HuaHUANG

    2002-01-01

    Aim:To study the integration of hepatitis Bvirus(HBV)DNAinto sperm chromosomes in hepatitsBpatients and the features of its integration.Methods:Sperm chromosomes of 14subjects(5healthy controls and9HBpatients,including1acute hepatitis B,2chronic active hepatitisB,4chronic persistent hepatitsB,2HBsAg chronic carriers with no clinical symptoms)were prepared using imterspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa.Fluosescence in situ hybridization(FISH)to sperm chromosome spreads was carried out with biotin-labeled full length HBVDNAprobe to detect the specificHBVDNA sequences in the sperm chromosomes.Results:Specific fluorescent signal spots for HBVDNAwere seen iv sperm chromosomes of one patient with chronic persistent hepatitisB.In9(9/42)sperm chromosome complements containing fluorescent signal spots,one presented5obvious FISHspots and the others2to4signals.The fluorescence intensity showed significant difference among the signal spots.The distribution of signal sites among chromosomes seems to be random.Con clusion:HBV could integrate into human sperm chromosomes.Results suggest that the possibility of vertical transmission of HBVvia the germ line tothe next generation is present.

  20. Intergenic DNA sequences from the human X chromosome reveal high rates of global gene flow

    Directory of Open Access Journals (Sweden)

    Wall Jeffrey D

    2008-11-01

    Full Text Available Abstract Background Despite intensive efforts devoted to collecting human polymorphism data, little is known about the role of gene flow in the ancestry of human populations. This is partly because most analyses have applied one of two simple models of population structure, the island model or the splitting model, which make unrealistic biological assumptions. Results Here, we analyze 98-kb of DNA sequence from 20 independently evolving intergenic regions on the X chromosome in a sample of 90 humans from six globally diverse populations. We employ an isolation-with-migration (IM model, which assumes that populations split and subsequently exchange migrants, to independently estimate effective population sizes and migration rates. While the maximum effective size of modern humans is estimated at ~10,000, individual populations vary substantially in size, with African populations tending to be larger (2,300–9,000 than non-African populations (300–3,300. We estimate mean rates of bidirectional gene flow at 4.8 × 10-4/generation. Bidirectional migration rates are ~5-fold higher among non-African populations (1.5 × 10-3 than among African populations (2.7 × 10-4. Interestingly, because effective sizes and migration rates are inversely related in African and non-African populations, population migration rates are similar within Africa and Eurasia (e.g., global mean Nm = 2.4. Conclusion We conclude that gene flow has played an important role in structuring global human populations and that migration rates should be incorporated as critical parameters in models of human demography.

  1. 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.

  2. Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

    Science.gov (United States)

    Waye, J S; Willard, H F

    1986-09-01

    The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.

  3. DNA methylation signature in peripheral blood reveals distinct characteristics of human X chromosome numerical aberrations

    OpenAIRE

    2015-01-01

    Background Abnormal sex chromosome numbers in humans are observed in Turner (45,X) and Klinefelter (47,XXY) syndromes. Both syndromes are associated with several clinical phenotypes, whose molecular mechanisms are obscure, and show a range of inter-individual penetrance. In order to understand the effect of abnormal numbers of X chromosome on the methylome and its correlation to the variable clinical phenotype, we performed a genome-wide methylation analysis using MeDIP and Illumina’s Infiniu...

  4. Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States); Kaur, G.P. [Temple Univ. Medical School, Philadelphia, PA (United States)] [and others

    1997-05-01

    We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as {open_quotes}ST1C1{close_quotes} - although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated {open_quotes}SULT.{close_quotes} The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5{prime}-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2. 14 refs., 2 figs.

  5. New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes for radiation molecular cytogenetics

    Directory of Open Access Journals (Sweden)

    Repin Mikhail V

    2009-06-01

    Full Text Available Abstract Background The objective of this work is to obtain the correct relative DNA contents of chromosomes in the normal male and female human diploid genomes for the use at FISH analysis of radiation-induced chromosome aberrations. Results The relative DNA contents of chromosomes in the male and female human diploid genomes have been calculated from the publicly available international Human Genome Project data. New sequence-based data on the relative DNA contents of human chromosomes were compared with the data recommended by the International Atomic Energy Agency in 2001. The differences in the values of the relative DNA contents of chromosomes obtained by using different approaches for 15 human chromosomes, mainly for large chromosomes, were below 2%. For the chromosomes 13, 17, 20 and 22 the differences were above 5%. Conclusion New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes were obtained. This approach, based on the genome sequence, can be recommended for the use in radiation molecular cytogenetics.

  6. Chromosomal aneuploidies and DNA fragmentation of human spermatozoa from patients exposed to perfluorinated compounds.

    Science.gov (United States)

    Governini, L; Guerranti, C; De Leo, V; Boschi, L; Luddi, A; Gori, M; Orvieto, R; Piomboni, P

    2015-11-01

    This study investigated chromosomal aneuploidies and DNA damage in spermatozoa from male patients contaminated by perfluorinated compounds (PFCs) in whole blood and seminal plasma. Sperm aneuploidy and diploidy rate for chromosomes 18, X and Y were evaluated by FISH; sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling technique coupled to flow cytometry. Our results indicated that PFC contamination was present in 58% of subjects included in the study. A significant increase in alterations of sperm parameters was observed in PFC-positive subjects compared to PFC-negative subjects. As regards the sperm aneuploidy, both disomy and diploidy rates resulted significantly increased in subjects positive for PFC contamination compared to PFC-negative samples. In addition, sperm DNA fragmentation index resulted significantly increased in PFC-contaminated subjects compared to PFC-non-contaminated subjects, with a significant increased level of dimmer DNA fragmentation index. Our results clearly indicate that PFC contamination may detrimentally affect spermatogenesis, disturbing both meiotic segregation and DNA integrity. We could therefore suggest cautions to reduce or eliminate any contact with these compounds because the long-term effects of PFC accumulation in the body are not predictable. © 2014 Blackwell Verlag GmbH.

  7. Sequence information encoded in DNA that may influence long-range chromatin structure correlates with human chromosome functions.

    Directory of Open Access Journals (Sweden)

    Taichi E Takasuka

    Full Text Available Little is known about the possible function of the bulk of the human genome. We have recently shown that long-range regular oscillation in the motif non-T, A/T, G (VWG existing at ten-nucleotide multiples influences large-scale nucleosome array formation. In this work, we have determined the locations of all 100 kb regions that are predicted to form distinctive chromatin structures throughout each human chromosome (except Y. Using these data, we found that a significantly greater fraction of 300 kb sequences lacked annotated transcripts in genomic DNA regions > or = 300 kb that contained nearly continuous chromatin organizing signals than in control regions. We also found a relationship between the meiotic recombination frequency and the presence of strong VWG chromatin organizing signals. Large (> or = 300 kb genomic DNA regions having low average recombination frequency are enriched in chromatin organizing signals. As additional controls, we show using chromosome 1 that the VWG motif signals are not enriched in randomly selected DNA regions having the mean size of the recombination coldspots, and that non-VWG motif sets do not generate signals that are enriched in recombination coldspots. We also show that tandemly repeated alpha satellite DNA contains strong VWG signals for the formation of distinctive nucleosome arrays, consistent with the low recombination activity of centromeres. Our correlations cannot be explained simply by variations in the GC content. Our findings suggest that a specific set of periodic DNA motifs encoded in genomic DNA, which provide signals for chromatin organization, influence human chromosome function.

  8. Analysis of repetitive DNA in chromosomes by flow cytometry.

    Science.gov (United States)

    Brind'Amour, Julie; Lansdorp, Peter M

    2011-06-01

    We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.

  9. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Gyoyeon [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of); Lee, Hansol [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Jiyeon, E-mail: jylee@kist.re.kr [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of)

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

  10. Micro RNAs and DNA methylation are regulatory players in human cells with altered X chromosome to autosome balance

    Science.gov (United States)

    Rajpathak, Shriram N.; Deobagkar, Deepti D.

    2017-01-01

    The gene balance hypothesis predicts that an imbalance in the dosage sensitive genes affects the cascade of gene networks that may influence the fitness of individuals. The phenotypes associated with chromosomal aneuploidies demonstrate the importance of gene dosage balance. We have employed untransformed human fibroblast cells with different number of X chromosomes to assess the expression of miRNAs and autosomal genes in addition to the DNA methylation status. High throughput NGS analysis using illumina Next seq500 has detected several autosomal as well as X linked miRNAs as differentially expressed in X monosomy and trisomy cells. Two of these miRNAs (hsa-miR-125a-5p and 335-5p) are likely to be involved in regulation of the autosomal gene expression. Additionally, our data demonstrates altered expression and DNA methylation signatures of autosomal genes in X monosomy and trisomy cells. In addition to miRNAs, expression of DNMT1 which is an important epigenetic player involved in many processes including cancer, is seen to be altered. Overall, present study provides a proof for regulatory roles of micro RNAs and DNA methylation in human X aneuploidy cells opening up possible new ways for designing therapeutic strategies. PMID:28233878

  11. Localization of the human fibromodulin gene (FMOD) to chromosome 1q32 and completion of the cDNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Sztrolovics, R.; Grover, J.; Roughley, P.J. [McGill Univ., Montreal (Canada)] [and others

    1994-10-01

    This report describes the cloning of the 3{prime}-untranslated region of the human fibromodulin cDNA and its use to map the gene. For somatic cell hybrids, the generation of the PCR product was concordant with the presence of chromosome 1 and discordant with the presence of all other chromosomes, confirming that the fibromodulin gene is located within region q32 of chromosome 1. The physical mapping of genes is a critical step in the process of identifying which genes may be responsible for various inherited disorders. Specifically, the mapping of the fibromodulin gene now provides the information necessary to evaluate its potential role in genetic disorders of connective tissues. The analysis of previously reported diseases mapped to chromosome 1 reveals two genes located in the proximity of the fibromodulin locus. These are Usher syndrome type II, a recessive disorder characterized by hearing loss and retinitis pigmentosa, and Van der Woude syndrome, a dominant condition associated with abnormalities such as cleft lip and palate and hyperdontia. The genes for both of these disorders have been projected to be localized to 1q32 of a physical map that integrates available genetic linkage and physical data. However, it seems improbable that either of these disorders, exhibiting restricted tissue involvement, could be linked to the fibromodulin gene, given the wide tissue distribution of the encoded proteoglycan, although it remains possible that the relative importance of the quantity and function of the proteoglycan may avry between tissues. 11 refs., 1 fig.

  12. 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.

  13. 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.

  14. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  15. Structural chromosome abnormalities, increased DNA strand breaks and DNA strand break repair deficiency in dermal fibroblasts from old female human donors.

    Science.gov (United States)

    Kalfalah, Faiza; Seggewiß, Sabine; Walter, Regina; Tigges, Julia; Moreno-Villanueva, María; Bürkle, Alexander; Ohse, Sebastian; Busch, Hauke; Boerries, Melanie; Hildebrandt, Barbara; Royer-Pokora, Brigitte; Boege, Fritz

    2015-02-01

    Dermal fibroblasts provide a paradigmatic model of cellular adaptation to long-term exogenous stress and ageing processes driven thereby. Here we addressed whether fibroblast ageing analysedex vivo entails genome instability. Dermal fibroblasts from human female donors aged 20-67 years were studied in primary culture at low population doubling. Under these conditions, the incidence of replicative senescence and rates of age-correlated telomere shortening were insignificant. Genome-wide gene expression analysis revealed age-related impairment of mitosis, telomere and chromosome maintenance and induction of genes associated with DNA repair and non-homologous end-joining, most notably XRCC4 and ligase 4. We observed an age-correlated drop in proliferative capacity and age-correlated increases in heterochromatin marks, structural chromosome abnormalities (deletions, translocations and chromatid breaks), DNA strand breaks and histone H2AX-phosphorylation. In a third of the cells from old and middle-aged donors repair of X-ray induced DNA strand breaks was impaired despite up-regulation of DNA repair genes. The distinct phenotype of genome instability, increased heterochromatinisation and (in 30% of the cases futile) up-regulation of DNA repair genes was stably maintained over several cell passages indicating that it represents a feature of geroconversion that is distinct from cellular senescence, as it does not encompass a block of proliferation.

  16. DNA double-strand breaks coupled with PARP1 and HNRNPA2B1 binding sites flank coordinately expressed domains in human chromosomes.

    Directory of Open Access Journals (Sweden)

    Nickolai A Tchurikov

    2013-04-01

    Full Text Available Genome instability plays a key role in multiple biological processes and diseases, including cancer. Genome-wide mapping of DNA double-strand breaks (DSBs is important for understanding both chromosomal architecture and specific chromosomal regions at DSBs. We developed a method for precise genome-wide mapping of blunt-ended DSBs in human chromosomes, and observed non-random fragmentation and DSB hot spots. These hot spots are scattered along chromosomes and delimit protected 50-250 kb DNA domains. We found that about 30% of the domains (denoted forum domains possess coordinately expressed genes and that PARP1 and HNRNPA2B1 specifically bind DNA sequences at the forum domain termini. Thus, our data suggest a novel type of gene regulation: a coordinated transcription or silencing of gene clusters delimited by DSB hot spots as well as PARP1 and HNRNPa2B1 binding sites.

  17. Intermittency as a universal characteristic of the complete chromosome DNA sequences of eukaryotes: From protozoa to human genomes

    Science.gov (United States)

    Rybalko, S.; Larionov, S.; Poptsova, M.; Loskutov, A.

    2011-10-01

    Large-scale dynamical properties of complete chromosome DNA sequences of eukaryotes are considered. Using the proposed deterministic models with intermittency and symbolic dynamics we describe a wide spectrum of large-scale patterns inherent in these sequences, such as segmental duplications, tandem repeats, and other complex sequence structures. It is shown that the recently discovered gene number balance on the strands is not of a random nature, and certain subsystems of a complete chromosome DNA sequence exhibit the properties of deterministic chaos.

  18. Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

    Science.gov (United States)

    Montiel, J F; Norbury, C J; Tuite, M F; Dobson, M J; Mills, J S; Kingsman, A J; Kingsman, S M

    1984-01-01

    We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence. PMID:6320114

  19. Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

    OpenAIRE

    Wood, S.; Starr, T V; Shukin, R J

    1986-01-01

    We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes dete...

  20. Assignment of heregulin (HGL) to human chromosome 8p22-p11 by PCR analysis of somatic cell hybrid DNA

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J.; Wood, W.I. (Genentech, Inc., South San Francisco, CA (United States))

    1993-06-01

    Heregulin is a recently described ligand and specific activator of the receptor tyrosine kinase designated HER2 (also called erbB2 or neu). Heregulin is a secreted 45-kDa protein that contains a region with amino acid sequence similarity to epidermal growth factor (EGF). The receptors for heregulin and EGF are encoded by related protooncogenes that can produce cellular transformation and are associated with human malignancies. The overexpression of HER2 correlates with a poor prognosis in breast, ovarian, and other cancers. Heregulin was purified from the conditioned medium of a human breast cancer cell line. The isolation of cDNA clones encoding heregulin show that several alternatively spliced variants of the protein are produced. The mature protein is derived from the N-terminal half of a 640-amino-acid precursor that includes a hydrophobic domain that may anchor the precursor in the cell membrane. Recombinantly produced heregulin stimulates the tyrosine phosphorylation of HER2 in a number of cell lines and augments the proliferation of the HER2-overexpressing cell line, SK-BR-3. Heregulin mRNA is expressed in many human tissues including breast, brain, ovary, testis, small intestine, and others. The rat homologue of heregulin has also been isolated, and cDNA clones encoding this protein have been described. The rat protein has been termed Neu differentiation factor based on its ability to induce the differentiation of a mammary tumor cell line. The gene for heregulin was mapped to human chromosome 8 by PCR amplification using primers specific for human but not rodent DNA in somatic cell hybrids.

  1. Forensic use of Y-chromosome DNA: a general overview

    NARCIS (Netherlands)

    M.H. Kayser (Manfred)

    2017-01-01

    textabstractThe male-specific part of the human Y chromosome is widely used in forensic DNA analysis, particularly in cases where standard autosomal DNA profiling is not informative. A Y-chromosomal gene fragment is applied for inferring the biological sex of a crime scene trace donor. Haplotypes co

  2. Unrepaired DNA damage facilitates elimination of uniparental chromosomes in interspecific hybrid cells.

    Science.gov (United States)

    Wang, Zheng; Yin, Hao; Lv, Lei; Feng, Yingying; Chen, Shaopeng; Liang, Junting; Huang, Yun; Jiang, Xiaohua; Jiang, Hanwei; Bukhari, Ihtisham; Wu, Lijun; Cooke, Howard J; Shi, Qinghua

    2014-01-01

    Elimination of uniparental chromosomes occurs frequently in interspecific hybrid cells. For example, human chromosomes are always eliminated during clone formation when human cells are fused with mouse cells. However, the underlying mechanisms are still elusive. Here, we show that the elimination of human chromosomes in human-mouse hybrid cells is accompanied by continued cell division at the presence of DNA damage on human chromosomes. Deficiency in DNA damage repair on human chromosomes occurs after cell fusion. Furthermore, increasing the level of DNA damage on human chromosomes by irradiation accelerates human chromosome loss in hybrid cells. Our results indicate that the elimination of human chromosomes in human-mouse hybrid cells results from unrepaired DNA damage on human chromosomes. We therefore provide a novel mechanism underlying chromosome instability which may facilitate the understanding of carcinogenesis.

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

    Institute of Scientific and Technical Information of China (English)

    赵勇[1; 余龙[2; 高洁[3; 付强[4; 华益民[5; 张宏来[6; 赵寿元[7

    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

  4. 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.

  5. Z-DNA immunoreactivity in fixed metaphase chromosomes of primates.

    Science.gov (United States)

    Viegas-Péquignot, E; Derbin, C; Malfoy, B; Taillandier, E; Leng, M; Dutrillaux, B

    1983-10-01

    Antibodies against Z-DNA bind to fixed metaphase chromosomes of man and Cebus albifrons (Platyrrhini, Primate). By indirect immunofluorescence and indirect immunoperoxidase techniques, a heavy staining is detected in some segments of chromosomes of C. albifrons. These segments correspond to R-band-positive heterochromatin, which has a high G + C-base content. Euchromatin of human and Cebus chromosomes show a weak and heterogeneous staining that consistently reproduces an R- and T-banding pattern in both species. Because chromosome homologies previously were demonstrated between these distantly related species by chromosome banding, our results suggest that Z-DNA has been conserved during the course of primate evolution.

  6. Evolutionarily conserved sequences on human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Frazer, Kelly A.; Sheehan, John B.; Stokowski, Renee P.; Chen, Xiyin; Hosseini, Roya; Cheng, Jan-Fang; Fodor, Stephen P.A.; Cox, David R.; Patil, Nila

    2001-09-01

    Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarily conserved elements are present in regions on chromosome 21 that do not encode known genes.

  7. Characterization of a new aberration of the human Y chromosome by banding methods and DNA restriction endonuclease analysis.

    Science.gov (United States)

    Schmid, M; Gall, H; Schempp, W; Weber, L; Schmidtke, J

    1981-01-01

    Comparative cytogenetic analyses were performed with ten different banding methods on a previously undescribed, inherited structural aberration of a Y chromosome, and the results compared with those of normal Y chromosomes occurring in the same family. The value of the individual staining techniques in investigations of Y chromosomal aberrations is emphasized. The aberrant Y chromosome analyzed can be formally derived from an isodicentric Y chromosome for the short arm with a very terminal long-arm breakpoint, in which the centromere, an entire short arm, and the proximal region on one long arm was lost. This interpretation was confirmed by determining the amount of the two Y-specific DNA sequences (2.1 and 3.4 kb in length) by means of Hae III restriction endonuclease analysis. The karyotype-phenotype correlations in the men with this aberrant Y chromosome, especially the fertility dysfunctions (oligoasthenoteratozoospermia, cryptozoospermia), are discussed. The possibility of the existence of fertility factors involved in the control of spermatogenesis within the quinacrine-bright heterochromatic region of the Y long arm is presented.

  8. Universal spectrum for DNA base C+G concentration variability in Human chromosome Y

    CERN Document Server

    Selvam, A M

    2004-01-01

    The spatial distribution of DNA base sequence A, C, G and T exhibit selfsimilar fractal fluctuations and the corresponding power spectra follow inverse power law form, which implies the following: (1) A scale invariant eddy continuum, namely, the amplitudes of component eddies are related to each other by a scale factor alone. In general, the scale factor is different for different scale ranges and indicates a multifractal structure for the spatial distribution of DNA base sequence. (2) Long-range spatial correlations of the eddy fluctuations. Multifractal structure to space-time fluctuations and the associated inverse power law form for power spectra is generic to spatially extended dynamical systems in nature and is a signature of self-organized criticality. The exact physical mechanism for the observed self-organized criticality is not yet identified. The author has developed a general systems theory where quantum mechanical laws emerge as self-consistent explanations for the observed long-range space-time...

  9. Z-DNA immunoreactivity in fixed metaphase chromosomes of primates.

    OpenAIRE

    Viegas-Péquignot, E; Derbin, C; Malfoy, B; Taillandier, E; Leng, M; Dutrillaux, B

    1983-01-01

    Antibodies against Z-DNA bind to fixed metaphase chromosomes of man and Cebus albifrons (Platyrrhini, Primate). By indirect immunofluorescence and indirect immunoperoxidase techniques, a heavy staining is detected in some segments of chromosomes of C. albifrons. These segments correspond to R-band-positive heterochromatin, which has a high G + C-base content. Euchromatin of human and Cebus chromosomes show a weak and heterogeneous staining that consistently reproduces an R- and T-banding patt...

  10. 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

    An extensive study on spontaneous and 5-Fluorodeoxyuridine induced fragile sites identified Xq31 in cattle (Bos taurus) and (Xq24, Xq26) in sheep (Ovis aries) in addition to several autosomal fragile sites (under publication). A ZOO-FISH study using three cloned human fragile-X probes with CCG....../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...... showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X....

  11. 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

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

  12. A highly conserved pericentromeric domain in human and gorilla chromosomes.

    Science.gov (United States)

    Pita, M; Gosálvez, J; Gosálvez, A; Nieddu, M; López-Fernández, C; Mezzanotte, R

    2009-01-01

    Significant similarity between human and gorilla genomes has been found in all chromosome arms, but not in centromeres, using whole-comparative genomic hybridization (W-CGH). In human chromosomes, centromeric regions, generally containing highly repetitive DNAs, are characterized by the presence of specific human DNA sequences and an absence of homology with gorilla DNA sequences. The only exception is the pericentromeric area of human chromosome 9, which, in addition to a large block of human DNA, also contains a region of homology with gorilla DNA sequences; the localization of these sequences coincides with that of human satellite III. Since highly repetitive DNAs are known for their high mutation frequency, we hypothesized that the chromosome 9 pericentromeric DNA conserved in human chromosomes and deriving from the gorilla genome may thus play some important functional role.

  13. Physical mapping of human chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, G.R.

    1992-01-01

    Project aims for the past year have been to refine the cytogenetic based physical map of human chromosome 16. This has been achieved by extending the panel of mouse/human hybrids of chromosome 16 to over sixty hybrids and mapping approximately 250 DNA makers. The high resolution of this physical map, with an average distance between breakpoints of less than 1.6 Mb, and the availability of at least one STS in the majority of these intervals, will be the basis for constructing extensive contigs of cloned DNA.

  14. Molecular and evolutionary characteristics of the fraction of human alpha satellite DNA associated with CENP-A at the centromeres of chromosomes 1, 5, 19, and 21

    Directory of Open Access Journals (Sweden)

    Roizès Gérard

    2010-03-01

    Full Text Available Abstract Background The mode of evolution of the highly homogeneous Higher-Order-Repeat-containing alpha satellite arrays is still subject to discussion. This is also true of the CENP-A associated repeats where the centromere is formed. Results In this paper, we show that the molecular mechanisms by which these arrays evolve are identical in multiple chromosomes: i accumulation of crossovers that homogenise and expand the arrays into different domains and subdomains that are mostly unshared between homologues and ii sporadic mutations and conversion events that simultaneously differentiate them from one another. Individual arrays are affected by these mechanisms to different extents that presumably increase with time. Repeats associated with CENP-A, where the centromere is formed, are subjected to the same evolutionary mechanisms, but constitute minor subsets that exhibit subtle sequence differences from those of the bulk repeats. While the DNA sequence per se is not essential for centromere localisation along an array, it appears that certain sequences can be selected against. On chromosomes 1 and 19, which are more affected by the above evolutionary mechanisms than are chromosomes 21 and 5, CENP-A associated repeats were also recovered from a second homogeneous array present on each chromosome. This could be a way for chromosomes to sustain mitosis and meiosis when the normal centromere locus is ineluctably undermined by the above mechanisms. Conclusion We discuss, in light of these observations, possible scenarios for the normal evolutionary fates of human centromeric regions.

  15. Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the [alpha]1 (XVIII) collagen gene to mouse chromosome 10 and human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Oh, S.P.; Warman, M.L.; Timmons, S.; Olsen, B.R.; Knoll, J.H.M. (Harvard Medical School, Boston, MA (United States)); Seldin, M.F. (Duke Univ. Medical Center, Durham, NC (United States)); Cheng, Sou-De (Children' s Hospital/Harvard Medical School, Boston, MA (United States))

    1994-02-01

    Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which the authors have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, they report the isolation of human cDNAs and genomic DNAs encoding the [alpha]1 (XVIII) collagen chain. Utilizing a genomic clone as probe, they have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, they have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2. 16 refs., 5 figs.

  16. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

    Science.gov (United States)

    Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

  17. Micromechanical study of protein-DNA interactions and chromosomes

    Science.gov (United States)

    Marko, John

    I will discuss micromechanics experiments that our group has used to analyze protein-DNA interactions and chromosome organization. In single-DNA experiments we have found that a feature of protein-DNA complexes is that their dissociation rates can depend strikingly on bulk solution concentrations of other proteins and DNA segments; I will describe experiments which demonstrate this effect, which can involve tens-fold changes in off-rates with submicromolar changes in solution concentrations. Second, I will discuss experiments aimed at analyzing large-scale human chromosome structure; we isolate metaphase chromosomes, which in their native form behave as remarkably elastic networks of chromatin. Exposure to DNA-cutting restriction enzymes completely eliminates this elasticity, indicating that there is not a mechanically contiguous protein ''scaffold'' from which the chromosome gains its stability. I will show results of siRNA experiments indicating that depletion of condensin proteins leads to destabilization of chromosome mechanics, indicating condensin's role as the major chromatin ''cross-linker'' in metaphase chromosomes. Finally I will discuss similar experiments on human G1 nuclei, where we use genetic and chemical modifications to separate the contributions of the nuclear lamina and chromatin to the mechanical stiffness of the nucleus as a whole. Supported by the NSF (DMR-1206868, MCB-1022117) and the NIH (GM105847, CA193419).

  18. Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition.

    Science.gov (United States)

    Uchiyama, Susumu; Kawahara, Kazuki; Hosokawa, Yuki; Fukakusa, Shunsuke; Oki, Hiroya; Nakamura, Shota; Kojima, Yukiko; Noda, Masanori; Takino, Rie; Miyahara, Yuya; Maruno, Takahiro; Kobayashi, Yuji; Ohkubo, Tadayasu; Fukui, Kiichi

    2015-12-01

    Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ~30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition.

  19. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: Chromosomal assignment of the gene in the human, mouse, and rat genomes

    Energy Technology Data Exchange (ETDEWEB)

    Pausova, Z.; Bourdon, J.; Clayton, D.; Janicic, N.; Goltzman, D.; Hendy, G.N. (McGill Univ. and Royal Victoria Hospital, Montreal Quebec (Canada)); Mattei, M.G. (INSERM, Marseille (France)); Seldin, M.F. (Duke Univ. Medical Center, Durham, NC (United States)); Riviere, M.; Szpirer, J. (Universite Libre de Bruxelles, Rhode-St-Genese (Belgium)) (and others)

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acelpeptide hydrolase gene (APEH). These results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes. 34 refs., 7 figs. 1 tab.

  20. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: chromosomal assignment of the gene in the human, mouse, and rat genomes.

    Science.gov (United States)

    Pausova, Z; Bourdon, J; Clayton, D; Mattei, M G; Seldin, M F; Janicic, N; Rivière, M; Szpirer, J; Levan, G; Szpirer, C

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acylpeptide hydrolase gene (APEH). Our results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes.

  1. Engineered human dicentric chromosomes show centromere plasticity.

    Science.gov (United States)

    Higgins, Anne W; Gustashaw, Karen M; Willard, Huntington F

    2005-01-01

    The centromere is essential for the faithful distribution of a cell's genetic material to subsequent generations. Despite intense scrutiny, the precise genetic and epigenetic basis for centromere function is still unknown. Here, we have used engineered dicentric human chromosomes to investigate mammalian centromere structure and function. We describe three classes of dicentric chromosomes isolated in different cell lines: functionally monocentric chromosomes, in which one of the two genetically identical centromeres is consistently inactivated; functionally dicentric chromosomes, in which both centromeres are consistently active; and dicentric chromosomes heterogeneous with respect to centromere activity. A study of serial single cell clones from heterogeneous cell lines revealed that while centromere activity is usually clonal, the centromere state (i.e. functionally monocentric or dicentric) in some lines can switch within a growing population of cells. Because pulsed field gel analysis indicated that the DNA at the centromeres of these chromosomes did not change detectably, this switching of the centromere state is most likely due to epigenetic changes. Inactivation of one of the two active centromeres in a functionally dicentric chromosome was observed in a percentage of cells after treatment with Trichostatin A, an inhibitor of histone deacetylation. This study provides evidence that the activity of human centromeres, while largely stable, can be subject to dynamic change, most likely due to epigenetic modification.

  2. The nuclear position of pericentromeric DNA of chromosome 11 appears to be random in G0 and non-random in G1 human lymphocytes.

    Science.gov (United States)

    Hulspas, R; Houtsmuller, A B; Krijtenburg, P J; Bauman, J G; Nanninga, N

    1994-07-01

    The nuclear topography of pericentromeric DNA of chromosome 11 was analyzed in G0 (nonstimulated) and G1 [phytohemagglutinin (PHA) stimulated] human lymphocytes by confocal microscopy. In addition to the nuclear center, the centrosome was used as a second point of reference in the three-dimensional (3D) analysis. Pericentromeric DNA of chromosome 11 and the centrosome were labeled using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence. To preserve the 3D morphology of the cells, these techniques were performed on whole cells in suspension. Three-dimensional images of the cells were analyzed with a recently developed 3D software program (Interactive Measurement of Axes and Positioning in 3 Dimensions). The distribution of the chromosome 11 centromeres appeared to be random during the G0 stage but clearly non-random during the G1 stage, when the nuclear center was used as a reference point. Further statistical analysis of the G1 cells revealed that the centromeres were randomly distributed in a shell underlying the nuclear membrane. A topographical relationship between the centrosome and the centromeres appeared to be absent during the G0 and G1 stages of the cell cycle.

  3. Molecular genetic evidence for the human settlement of the Pacific: analysis of mitochondrial DNA, Y chromosome and HLA markers.

    Science.gov (United States)

    Hagelberg, E; Kayser, M; Nagy, M; Roewer, L; Zimdahl, H; Krawczak, M; Lió, P; Schiefenhövel, W

    1999-01-29

    Present-day Pacific islanders are thought to be the descendants of Neolithic agriculturalists who expanded from island South-east Asia several thousand years ago. They speak languages belonging to the Austronesian language family, spoken today in an area spanning half of the circumference of the world, from Madagascar to Easter Island, and from Taiwan to New Zealand. To investigate the genetic affinities of the Austronesian-speaking peoples, we analysed mitochondrial DNA, HLA and Y-chromosome polymorphisms in individuals from eight geographical locations in Asia and the Pacific (China, Taiwan, Java, New Guinea highlands, New Guinea coast, Trobriand Islands, New Britain and Western Samoa). Our results show that the demographic expansion of the Austronesians has left a genetic footprint. However, there is no simple correlation between languages and genes in the Pacific.

  4. Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen

    Energy Technology Data Exchange (ETDEWEB)

    Federsppiel, B.; Melhado, I.G.; Delaney, A.; Clark-Lewis, I. (Univ. of British Columbia, Vancouver (Canada)); Duncan, A.M.V. (Queens Univ., Kinston, Ontario (Canada)); Jirik, F.R. (Hospital for Sick Children, Toronto, Ontario (Canada))

    1993-06-01

    A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E, was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin. 32 refs., 5 figs.

  5. 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...... 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...

  6. Selection and fine mapping of chromosome-specific cDNAs: application to human chromosome 1.

    Science.gov (United States)

    Mancini, M; Sala, C; Rivella, S; Toniolo, D

    1996-12-01

    We have developed a methodology for identification and fine mapping of chromosome-specific transcripts. Combining digestion of DNA with different restriction enzymes, ligation to "bubble" linkers, and PCR amplification from Alu and "bubble" primers, we have synthesized human chromosome 1-specific sequences from DNA of a somatic cell hybrid, A9Neol. After hybridization to human fetal brain cDNA, we could efficiently capture chromosome 1-specific cDNAs. The cDNAs were sequenced and used as probes in hybridizations to high-density filters containing the arrayed CEPH Mega-YAC library and to the arrayed cDNA library from infant brain made by B. Soares, which has been extensively sequenced. By this approach we have been able to select chromosome 1-specific cDNAs, to map them to chromosome 1 YAC contigs, and to identify and map corresponding longer cDNAs and ESTs.

  7. 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.

  8. Associated chromosomal DNA changes in polyploids.

    Science.gov (United States)

    Raina, S N; Parida, A; Koul, K K; Salimath, S S; Bisht, M S; Raja, V; Khoshoo, T N

    1994-08-01

    The 2C and 4C nuclear DNA amounts were estimated in eight diploid species, belonging to three diverse genera (Vicia, Tephrosia, and Phlox) and their corresponding colchitetraploids. In P. drummondii, T. purpurea, and T. oxygona tetraploids the deviation from the expectation was highly significant. The DNA in P. drummondii was further discarded in subsequent (C1, C2) generations, thus attaining an overall reduction of about 25%. The DNA content in the subsequent generations was the same as that of C2. It is concluded that rapid DNA loss in the first and subsequent generations was not only associated with the substantial increase (30-66%) in the seed set, but it also helped in the establishment and stabilization of the tetraploid. The possible relationship between such a nucleotypic change and success of polyploids is discussed. The DNA change from the expected value in the P. drummondii tetraploid was achieved by equal decrement to each chromosome independent of size, i.e., small chromosomes loose the same amount of DNA as the large chromosomes.

  9. Effects of hepatitis B virus infection on human sperm chromosomes

    Institute of Scientific and Technical Information of China (English)

    Jian-Min Huang; Tian-Hua Huang; Huan-Ying Qiu; Xiao-Wu Fang; Tian-Gang Zhuang; Hong-Xi Liu; Yong-Hua Wang; Li-Zhi Deng; Jie-Wen Qiu

    2003-01-01

    AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and

  10. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  11. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  12. Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities.

    Science.gov (United States)

    Norkin, L C; Steinberg, V I; Kosz-Vnenchak, M

    1985-02-01

    A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.

  13. Numerically abnormal chromosome constitutions in humans

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1993-12-31

    Chapter 24, discusses numerically abnormal chromosome constitutions in humans. This involves abnormalities of human chromosome number, including polyploidy (when the number of sets of chromosomes increases) and aneuploidy (when the number of individual normal chromosomes changes). Chapter sections discuss the following chromosomal abnormalities: human triploids, imprinting and uniparental disomy, human tetraploids, hydatidiform moles, anomalies caused by chromosomal imbalance, 13 trisomy (D{sub 1} trisomy, Patau syndrome), 21 trisomy (Down syndrome), 18 trisomy syndrome (Edwards syndrome), other autosomal aneuploidy syndromes, and spontaneous abortions. The chapter concludes with remarks on the nonrandom participation of chromosomes in trisomy. 69 refs., 3 figs., 4 tabs.

  14. Homologies between human and marmoset (Callithrix jacchus) chromosomes revealed by comparative chromosome painting

    Energy Technology Data Exchange (ETDEWEB)

    Sherlock, J.K.; Griffin, D.K.; Delhanty, J.D.A.; Parrington, J.M. [Univ. College London (United Kingdom)

    1996-04-15

    Regions of DNA homology between human and marmoset (Callithrix jacchus) chromosomes have been demonstrated using fluorescence in situ hybridization. All 24 chromosome paints and two centromere repeat sequences from Homo sapiens (HSA) have been annealed to previously G-banded metaphase spreads of Callithrix jacchus. All human paint probes, except Y, successfully hybridized to marmoset chromosomes. Fifteen of them hybridized to one region only, seven to two regions, and paint 1 to three regions. Homologies proposed from previous banding comparisons have been confirmed for HSA 2, 4-6, 10-12, 18, 19, 21, and X and partially confirmed for HSA 1 and 3, but were not in agreement for HSA 14 and 17. Human centromere repeat sequences for X and 18 did not hybridize to marmoset chromosomes. Because, at present, there is the confusing situation of several different numbering systems for marmoset chromosomes, we propose a new simpler nomenclature based on descending order of chromosome size. 25 refs., 3 figs.

  15. Human chromosomes: Structure, behavior, and effects

    Energy Technology Data Exchange (ETDEWEB)

    Therman, E.; Susman, M.

    1993-12-31

    The book `Human Chromosomes: Structure, Behavior, and Effects` covers the most important topics regarding human chromosomes and current research in cytogenetics. Attention is given both to structure and function of autosomes and sex chromosomes, as well as definitions and causes of chromosomal aberrations. This often involves discussion about various aspects of the cell cycle (both mitosis and meiosis). Methods and techniques involved in researching and mapping human chromosomes are also discussed.

  16. Development of specific chromosomal DNA pool for rice field eel and their application to gene mapping

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The chromosomes 1, 3, 5, 6, 7, 10 and 12 of rice field eel (Monopterus albus Zuiew) have been microdissected successfully from meiosis I diakinesis spreads by using glass microneedle under an inverted microscope. And the DOP-PCR products of the single chromosome dotted on the nylon membrane as "specific chromosomal DNA pool", have been hybridized with 6 probes to map these genes. The mapping results show that Zfa has been mapped to chromosome 1, rDNA to chromosomes 3 and 7, both Gh and Pdeg to chromosome 10, Hsl to chromosome 5 and Hox genes have been detected on chromosomes 1, 3, 6 and 10 meantime. It has initiatively been suggested that chromosome 10 of rice field eel might possess the commom conserved synteny to that on chromosome 17 of human, chromosome 11 of mouse,chromosome 12 of pig and chromosome 19 of bovine. And so chromosome 3 of rice field eel might also contain the commom conserved synteny to that on chromosome 2 of zebrafish. Our study is an attempt to establish a new and feasible method to advance the study of gene mapping and chromosome evolution in fish, and also to provide a new idea to distinguish each chromosome on the base of molecular markers for fish.

  17. Construction and characterization of genomic libraries from specific human chromosomes.

    Science.gov (United States)

    Krumlauf, R; Jeanpierre, M; Young, B D

    1982-05-01

    Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.

  18. Flow karyotyping and sorting of human chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Gray, J.W.; Lucas, J.; Peters, D.; Pinkel, D.; Trask, B.; van den Engh, G.; Van Dilla, M.A.

    1986-07-16

    Flow cytometry and sorting are becoming increasingly useful as tools for chromosome classfication and for the detection of numerical and structural chromosome aberrations. Chromosomes of a single type can be purified with these tools to facilitate gene mapping or production of chromosome specific recombinant DNA libraries. For analysis of chromosomes with flow cytometry, the chromosomes are extracted from mitotic cells, stained with one or more fluorescent dyes and classified one-by-one according to their dye content(s). Thus, the flow approach is fundamentally different than conventional karyotyping where chromosomes are classified within the context of a metaphase spread. Flow sorting allows purification of chromosomes that can be distinguished flow cytometrically. The authors describe the basic principles of flow cytometric chromosome classification i.e. flow karyotyping, and chromosome sorting and describe several applications. 30 refs., 8 figs.

  19. 45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp.

    Directory of Open Access Journals (Sweden)

    Jing Huang

    Full Text Available BACKGROUND: In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported. METHODS AND RESULTS: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region. CONCLUSIONS: The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

  20. Fluorescence imaging of chromosomal DNA using click chemistry

    Science.gov (United States)

    Ishizuka, Takumi; Liu, Hong Shan; Ito, Kenichiro; Xu, Yan

    2016-09-01

    Chromosome visualization is essential for chromosome analysis and genetic diagnostics. Here, we developed a click chemistry approach for multicolor imaging of chromosomal DNA instead of the traditional dye method. We first demonstrated that the commercially available reagents allow for the multicolor staining of chromosomes. We then prepared two pro-fluorophore moieties that served as light-up reporters to stain chromosomal DNA based on click reaction and visualized the clear chromosomes in multicolor. We applied this strategy in fluorescence in situ hybridization (FISH) and identified, with high sensitivity and specificity, telomere DNA at the end of the chromosome. We further extended this approach to observe several basic stages of cell division. We found that the click reaction enables direct visualization of the chromosome behavior in cell division. These results suggest that the technique can be broadly used for imaging chromosomes and may serve as a new approach for chromosome analysis and genetic diagnostics.

  1. Fluorescence imaging of chromosomal DNA using click chemistry

    Science.gov (United States)

    Ishizuka, Takumi; Liu, Hong Shan; Ito, Kenichiro; Xu, Yan

    2016-01-01

    Chromosome visualization is essential for chromosome analysis and genetic diagnostics. Here, we developed a click chemistry approach for multicolor imaging of chromosomal DNA instead of the traditional dye method. We first demonstrated that the commercially available reagents allow for the multicolor staining of chromosomes. We then prepared two pro-fluorophore moieties that served as light-up reporters to stain chromosomal DNA based on click reaction and visualized the clear chromosomes in multicolor. We applied this strategy in fluorescence in situ hybridization (FISH) and identified, with high sensitivity and specificity, telomere DNA at the end of the chromosome. We further extended this approach to observe several basic stages of cell division. We found that the click reaction enables direct visualization of the chromosome behavior in cell division. These results suggest that the technique can be broadly used for imaging chromosomes and may serve as a new approach for chromosome analysis and genetic diagnostics. PMID:27620982

  2. Chromosome breakages associated with 45S ribosomal DNA sequences in spotted snakehead fish Channa punctatus.

    Science.gov (United States)

    Singh, Mamta; Barman, Anindya Sundar

    2013-01-01

    It is well known that transcriptionally inactive rRNA genes are correlated with DNA hyper-methylation and histone hypo-methylation and there is clear evidence in humans that DNA and histone modification which alter chromatin structure are related to chromosome fragility. Very little is known about the biological cause of 45S rDNA fragility. In this report we characterized the chromosome breakage or gap associated with 45S rDNA in a fish species Channa punctatus. The rDNA mapping in C. punctatus, showed many chromosome breakages or gap formations, and all occurred exclusively in the 45S rDNA sites in anterior kidney cells. We observed that the number of chromosomes plus chromosome fragments was often more than the expected 32 in most cells. Total 67 % metaphase spread showed the expected or normal 32 chromosomes, while 33 % metaphase spread showed 33 and/or 34 chromosomes and/or chromosome fragments. The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are also discussed in present report.

  3. Simulation of DNA Damage in Human Cells from Space Radiation Using a Physical Model of Stochastic Particle Tracks and Chromosomes

    Science.gov (United States)

    Ponomarev, Artem; Plante, Ianik; Hada, Megumi; George, Kerry; Wu, Honglu

    2015-01-01

    The formation of double-strand breaks (DSBs) and chromosomal aberrations (CAs) is of great importance in radiation research and, specifically, in space applications. We are presenting a recently developed model, in which chromosomes simulated by NASARTI (NASA Radiation Tracks Image) is combined with nanoscopic dose calculations performed with the Monte-Carlo simulation by RITRACKS (Relativistic Ion Tracks) in a voxelized space. The model produces the number of DSBs, as a function of dose for high-energy iron, oxygen, and carbon ions, and He ions. The combined model calculates yields of radiation-induced CAs and unrejoined chromosome breaks in normal and repair deficient cells. The merged computational model is calibrated using the relative frequencies and distributions of chromosomal aberrations reported in the literature. The model considers fractionated deposition of energy to approximate dose rates of the space flight environment. The merged model also predicts of the yields and sizes of translocations, dicentrics, rings, and more complex-type aberrations formed in the G0/G1 cell cycle phase during the first cell division after irradiation.

  4. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    Science.gov (United States)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  5. The gene for human glutaredoxin (GLRX) is localized to human chromosome 5q14

    Energy Technology Data Exchange (ETDEWEB)

    Padilla, C.A.; Holmgren, A. [Karolinska Inst., Stockholm (Sweden); Bajalica, S.; Lagercrantz, J. [Karolinska Hospital, Stockholm (Sweden)

    1996-03-05

    Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescence in situ hybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human-hamster and a human-mouse hybrid panel and using a human glutaredoxin cDNA as a probe. 13 refs., 2 figs.

  6. Small supernumerary marker chromosomes (sSMC in humans; are there B chromosomes hidden among them

    Directory of Open Access Journals (Sweden)

    Ogilvie Caroline

    2008-06-01

    Full Text Available Abstract Background Small supernumerary marker chromosomes (sSMC and B-chromosomes represent a heterogeneous collection of chromosomes added to the typical karyotype, and which are both small in size. They may consist of heterochromatic and/or euchromatic material. Also a predominance of maternal transmission was reported for both groups. Even though sSMC and B-chromosomes show some similarity it is still an open question if B-chromosomes are present among the heterogeneous group of sSMC. According to current theories, sSMC would need drive, drift or beneficial effects to increase in frequency in order to become B chromosome. However, up to now no B-chromosomes were described in human. Results Here we provide first evidence and discuss, that among sSMC B-chromosomes might be hidden. We present two potential candidates which may already be, or may in future evolve into B chromosomes in human: (i sSMC cases where the marker is stainable only by DNA derived from itself; and (ii acrocentric-derived inverted duplication sSMC without associated clinical phenotype. Here we report on the second sSMC stainable exclusively by its own DNA and show that for acrocentric derived sSMC 3.9× more are familial cases than reported for other sSMC. Conclusion The majority of sSMC are not to be considered as B-chromosomes. Nonetheless, a minority of sSMC show similarities to B-chromosomes. Further studies are necessary to come to final conclusions for that problem.

  7. Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

    Science.gov (United States)

    Moguilevsky, N; Varsalona, F; Noyer, M; Gillard, M; Guillaume, J P; Garcia, L; Szpirer, C; Szpirer, J; Bollen, A

    1994-09-01

    A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

  8. Correlation of physical and genetic maps of human chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, G.R.

    1991-01-01

    This project aimed to divide chromosome 16 into approximately 50 intervals of {approximately}2Mb in size by constructing a series of mouse/human somatic cell hybrids each containing a rearranged chromosome 16. Using these hybrids, DNA probes would be regionally mapped by Southern blot or PCR analysis. Preference would be given to mapping probes which demonstrated polymorphisms for which the CEPH panel of families had been typed. This would allow a correlation of the physical and linkage maps of this chromosome. The aims have been substantially achieved. 49 somatic cell hybrids have been constructed which have allowed definition of 46, and potentially 57, different physical intervals on the chromosome. 164 loci have been fully mapped into these intervals. A correlation of the physical and genetic maps of the chromosome is in an advanced stage of preparation. The somatic cell hybrids constructed have been widely distributed to groups working on chromosome 16 and other genome projects.

  9. Correlation of chromosome patterns in human leukemic cells with exposure to chemicals and/or radiation. Progress report, July 1992--August 1993

    Energy Technology Data Exchange (ETDEWEB)

    Rowley, J.D.

    1993-09-01

    Progress in identification of chromosomal transformations associated with leukemogenesis is described. In particular progress in DNA cloning of chromosomal break points in human cancer patients is described.

  10. 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.

  11. Physical mapping of human chromosome 16. Annual progress report

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, G.R.

    1992-08-01

    Project aims for the past year have been to refine the cytogenetic based physical map of human chromosome 16. This has been achieved by extending the panel of mouse/human hybrids of chromosome 16 to over sixty hybrids and mapping approximately 250 DNA makers. The high resolution of this physical map, with an average distance between breakpoints of less than 1.6 Mb, and the availability of at least one STS in the majority of these intervals, will be the basis for constructing extensive contigs of cloned DNA.

  12. Hierarchical radial and polar organisation of chromosomes in human sperm.

    Science.gov (United States)

    Millan, N M; Lau, P; Hann, M; Ioannou, D; Hoffman, D; Barrionuevo, M; Maxson, W; Ory, S; Tempest, H G

    2012-10-01

    It is well established that chromosomes occupy distinct positions within the interphase nuclei, conferring a potential functional implication to the genome. In addition, alterations in the nuclear organisation patterns have been associated with disease phenotypes (e.g. cancer or laminopathies). The human sperm is the smallest cell in the body with specific DNA packaging and the mission of delivering the paternal genome to the oocyte during fertilisation. Studies of nuclear organisation in the sperm have postulated nonrandom chromosome position and have proposed a chromocentre model with the centromeres facing toward the interior and the telomeres toward the periphery of the nucleus. Most studies have assessed the nuclear address in the sperm longitudinally predominantly using centromeric or telomeric probes and to a lesser extent with whole chromosome paints. To date, studies investigating the radial organisation of human sperm have been limited. The purpose of this study was to utilise whole chromosome paints for six clinically important chromosomes (18, 19, 21, 22, X, and Y) to investigate nuclear address by assessing their radial and longitudinal nuclear organisation. A total of 10,800 sperm were analysed in nine normozoospermic individuals. The results have shown nonrandom chromosome position for all chromosomes using both methods of analysis. We present novel radial and polar analysis of chromosome territory localization within the human sperm nucleus. Specifically, a hierarchical organisation was observed radially with chromosomes organised from the interior to the periphery (chromosomes 22, 21, Y, X, 19, and 18 respectively) and polar organisation from the sperm head to tail (chromosomes X, 19, Y, 22, 21, and 18, respectively). We provide evidence of defined nuclear organisation in the human sperm and discuss the function of organisation and potential possible clinical ramifications of these results in regards to male infertility and early human development.

  13. Chromosome Variations And Human Behavior

    Science.gov (United States)

    Soudek, D.

    1974-01-01

    Article focused on the science of cytogenetics, which studied the transmission of the units of heredity called chromosomes, and considered the advantage of proper diagnosis of genetic diseases, treated on the chromosomal level. (Author/RK)

  14. Chromosomal localization of mitochondrial transcription factor A (TCF6), single-stranded DNA-binding protein (SSBP), and endonuclease G (ENDOG), three human housekeeping genes involving in mitochondrial biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tiranti, V.; Rossi, G.; DiDonato, S. [Istituto Nazionale Neurologico, Carlo Besta (Italy)] [and others

    1995-01-20

    By using a PCR-based screening of a somatic cell hybrid panel and FISH, we have assigned the loci of mitochondrial single-stranded DNA-binding protein (SSBP), mitochondrial transcription factor A (TCF6), and mitochondrial endonuclease G (ENDOG) genes to human chromosomes 7q34, 10q21, and 9q34.1, respectively. The products of these three genes are involved in fundamental aspects of mitochondrial biogenesis, such as replication and transcription of the mitochondrial genome. The chromosomal localization of these genes is important to testing whether the corresponding proteins may play a role in the etiopathogenesis of human disorders associated with qualitative or quantitative abnormalities of mitochondrial DNA. 20 refs., 1 fig., 2 tabs.

  15. Physical mapping of human chromosome 16. Annual progress report

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, G.R.

    1993-08-01

    We aim to isolate cDNAs mapping to human chromosome 16 and localise such cDNAs on the high resolution physical map. In collaboration with LANL, PCR primers will be synthesised from cDNA sequences mapped to chromosome 16 and used as ESTs in the generation of mega-YAC contigs for this chromosome. Probing of high density cosmid grids will enable integration of the ESTs into cosmid contigs and location of the cosmid contigs on the YAC contig. A hn-cDNA library has been constructed from the hybrid CY18 which contains chromosome 16 as the only human chromosome. A modified screening protocol has been successfully developed and 15 hn-cDNA clones have been sequenced and localised on the hybrid map. Sequence analysis of four of these revealed that they were known cDNAs, which are now mapped to chromosome 16. Development of techniques to allow the isolation of longer cDNAs from the identified exons is in progress. This will depend on PCR amplification of cDNAs from a total human CDNA library.

  16. Characters of DNA Constitution in the Rye B Chromosome

    Institute of Scientific and Technical Information of China (English)

    Hong Long; Zhong-Xia Qi; Xiao-Ming Sun; Cheng-Bin Chen; Xiu-Lan Li; Wen-Qin Song; Rui-Yang Chen

    2008-01-01

    We have used chromosome microdissection and microcloning to construct a DNA library of the entire B chromosome (B) of rye. New rye B-specific sequences have been screened from this pool, blasted with other sequences and analyzed to elucidate the characters of DNA constitution and the possible pathway of the origin of the rye B chromosome. We report the discovery of a new sequence that is specific to the rye B centromere.

  17. The human chromosome. Electron microscopic observations on chromatin fiber organization.

    Science.gov (United States)

    Abuelo, J G; Moore, D E

    1969-04-01

    Human lymphocytes were grown in short-term tissue culture and were arrested in metaphase with Colcemid. Their chromosomes were prepared by the Langmuir trough-critical point drying technique and were examined under the electron microscope. In addition, some chromosomes were digested with trypsin, Pronase, or DNase. The chromosomes consist entirely of tightly packed, 240 +/- 50-A chromatin fibers. Trypsin and Pronase treatments induce relaxation of fiber packing and reveal certain underlying fiber arrangements. Furthermore, trypsin treatment demonstrates that the chromatin fiber has a 25-50 A trypsin-resistant core surrounded by a trypsin-sensitive sheath. DNase digestion suggests that this core contains DNA.

  18. Human Structural Variation: Mechanisms of Chromosome Rearrangements.

    Science.gov (United States)

    Weckselblatt, Brooke; Rudd, M Katharine

    2015-10-01

    Chromosome structural variation (SV) is a normal part of variation in the human genome, but some classes of SV can cause neurodevelopmental disorders. Analysis of the DNA sequence at SV breakpoints can reveal mutational mechanisms and risk factors for chromosome rearrangement. Large-scale SV breakpoint studies have become possible recently owing to advances in next-generation sequencing (NGS) including whole-genome sequencing (WGS). These findings have shed light on complex forms of SV such as triplications, inverted duplications, insertional translocations, and chromothripsis. Sequence-level breakpoint data resolve SV structure and determine how genes are disrupted, fused, and/or misregulated by breakpoints. Recent improvements in breakpoint sequencing have also revealed non-allelic homologous recombination (NAHR) between paralogous long interspersed nuclear element (LINE) or human endogenous retrovirus (HERV) repeats as a cause of deletions, duplications, and translocations. This review covers the genomic organization of simple and complex constitutional SVs, as well as the molecular mechanisms of their formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  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. Research on automatic human chromosome image analysis

    Science.gov (United States)

    Ming, Delie; Tian, Jinwen; Liu, Jian

    2007-11-01

    Human chromosome karyotyping is one of the essential tasks in cytogenetics, especially in genetic syndrome diagnoses. In this thesis, an automatic procedure is introduced for human chromosome image analysis. According to different status of touching and overlapping chromosomes, several segmentation methods are proposed to achieve the best results. Medial axis is extracted by the middle point algorithm. Chromosome band is enhanced by the algorithm based on multiscale B-spline wavelets, extracted by average gray profile, gradient profile and shape profile, and calculated by the WDD (Weighted Density Distribution) descriptors. The multilayer classifier is used in classification. Experiment results demonstrate that the algorithms perform well.

  1. A Plain English Map of the Human Chromosomes.

    Science.gov (United States)

    Offner, Susan

    1992-01-01

    Presents a chromosome map for 19 known chromosomes in human genetics. Describes the characteristics attributed to the genetic codes for each of the chromosomes and discusses the teaching applications of the chromosome map. (MDH)

  2. Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

    Directory of Open Access Journals (Sweden)

    Kaitlin M Stimpson

    Full Text Available The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA. The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2 causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

  3. Gene for non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young subtype) is linked to DNA polymorphism on human chromosome 20q

    Energy Technology Data Exchange (ETDEWEB)

    Bell, G.I.; Xiang, Kunsan; Newman, M.V.; Wu, Songhua; Cox, N.J. (Univ. of Chicago, IL (United States)); Wright, L.G.; Spielman, R.S. (Univ. of Pennsylvania, Philadelphia (United States)); Fajans, S.S. (Univ. of Michigan, Ann Arbor (United States))

    1991-02-15

    Maturity-onset diabetes of the young (MODY) is a form of non-insulin-dependent diabetes mellitus characterized by an early age of onset, usually before 25 years of age, and an autosomal dominant mode of inheritance. The largest and best-studies MODY pedigree is the TW family. The majority of the diabetic subjects in this pedigree has a reduced and delayed insulin-secretory response to glucose, and it has been proposed that this abnormal response is the manifestation of the basic genetic defect that leads to diabetes. Using DNA from members of the TW family, the authors tested more than 75 DNA markers for linkage with MODY. A DNA polymorphism in the adenosine deaminase gene (ADA) on the long arm of chromosome 20 was found to cosegregate with MODY. The maximum logarithm of adds (lod score) for linkage between MODY and ADA was 5.25 at a recombination fraction of 0.00. These results indicate that the odds are {gt}178,000:1 that the gene responsible for MODY is this family is tightly linked to the ADA gene on chromosome 20q.

  4. Extremely underwound chromosomal DNA in nucleoids of mouse sarcoma cells.

    Science.gov (United States)

    Hartwig, M; Matthes, E; Arnold, W

    1981-07-01

    The superhelical properties of chromosomal DNA from cells of a mouse sarcoma were investigated in neutral sucrose gradients containing ethidium bromide. Removal of negative supercoiling from the DNA of the sarcoma cells required a substantially higher dye concentration than was necessary in the case of DNA from cultured mouse fibroblasts. The calculated value of the mean superhelical density in malignant cells (sigma = -0.14) appears abnormally high compared with the value (sigma = -0.09) obtained for DNA of mouse fibroblasts. Chromosomal DNA from mouse sarcoma cells is therefore concluded to be highly deficient in helical turns.

  5. Paradigm Lost: The Human Chromosome Story.

    Science.gov (United States)

    Unger, Lawrence; Blystone, Robert V.

    1996-01-01

    Discusses whether the discovery in 1956 that humans have a chromosome number of 46, as opposed to 47 or 48 as previously thought, fits into a paradigm shift of the Kuhnian type. Concludes that Kuhn probably would not have considered the chromosome number shift to be large enough to be a focus for one of his paradigms. (AIM)

  6. 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.

  7. Meiotic chromosome abnormalities in human spermatogenesis.

    Science.gov (United States)

    Martin, Renée H

    2006-08-01

    The last few years have witnessed an explosion in the information about chromosome abnormalities in human sperm and the meiotic events that predispose to these abnormalities. We have determined that all chromosomes are susceptible to nondisjunction, but chromosomes 21 and 22 and, especially, the sex chromosomes have an increased frequency of aneuploidy. Studies are just beginning on the effects of potential mutagens on the chromosomal constitution of human sperm. The effects of pesticides and cancer therapeutic agents have been reviewed. In the last decade, there has been a great impetus to study chromosome abnormalities in sperm from infertile men because the advent of intracytoplasmic sperm injection (ICSI) made it possible for these men to father pregnancies. A large number of studies have demonstrated that infertile men have an increased frequency of chromosomally abnormal sperm and children, even when they have a normal somatic karyotype. Meiotic studies on the pachytene stage of spermatogenesis have demonstrated that infertile men have impaired chromosome synapsis, a significantly decreased frequency of recombination, and an increased frequency of chromosomes completely lacking a recombination site. Such errors make these cells susceptible to meiotic arrest and the production of aneuploid gametes.

  8. Polymorphic distribution of Y-chromosome haplotype and mitochondrial DNA in the Bouyei people in China

    Institute of Scientific and Technical Information of China (English)

    李永念; 左丽; 文波; 柯越海; 黄薇; 金力

    2004-01-01

    @@ In the evolution of humans, many kinds of mutations in the human genome have been accumulated, providing credible genetic evidence for the study of human origins and migrations. The "out-of-Africa" hypothesis of modern human evolution and the genetic origin of the Japanese has come about by studying mitochondrial DNA.l,2 Recently, researchers have recognized the power of Y-chromosome markers in resolving migratory patterns of modern humans as more and more Y-chromosome single nucleotide polymorphism markers have been found. The markers on the nonrecombinant part of the Y-chromosome allows for the reconstruction of intact haplotypes which are probably the best genetic tools to study human migrations. We can analyze the paternal history of some people in different areas by Y-chromosome haplotypes.

  9. A Revised Map of the Human Chromosomes.

    Science.gov (United States)

    Offner, Susan

    1993-01-01

    Presents an updated map of the human chromosomes, building on a "plain English map" that was previously published. A brief summary of genes research is included in the gene explanations accompanying the map. (PR)

  10. The Divergence of Neandertal and Modern Human Y Chromosomes.

    Science.gov (United States)

    Mendez, Fernando L; Poznik, G David; Castellano, Sergi; Bustamante, Carlos D

    2016-04-07

    Sequencing the genomes of extinct hominids has reshaped our understanding of modern human origins. Here, we analyze ∼120 kb of exome-captured Y-chromosome DNA from a Neandertal individual from El Sidrón, Spain. We investigate its divergence from orthologous chimpanzee and modern human sequences and find strong support for a model that places the Neandertal lineage as an outgroup to modern human Y chromosomes-including A00, the highly divergent basal haplogroup. We estimate that the time to the most recent common ancestor (TMRCA) of Neandertal and modern human Y chromosomes is ∼588 thousand years ago (kya) (95% confidence interval [CI]: 447-806 kya). This is ∼2.1 (95% CI: 1.7-2.9) times longer than the TMRCA of A00 and other extant modern human Y-chromosome lineages. This estimate suggests that the Y-chromosome divergence mirrors the population divergence of Neandertals and modern human ancestors, and it refutes alternative scenarios of a relatively recent or super-archaic origin of Neandertal Y chromosomes. The fact that the Neandertal Y we describe has never been observed in modern humans suggests that the lineage is most likely extinct. We identify protein-coding differences between Neandertal and modern human Y chromosomes, including potentially damaging changes to PCDH11Y, TMSB4Y, USP9Y, and KDM5D. Three of these changes are missense mutations in genes that produce male-specific minor histocompatibility (H-Y) antigens. Antigens derived from KDM5D, for example, are thought to elicit a maternal immune response during gestation. It is possible that incompatibilities at one or more of these genes played a role in the reproductive isolation of the two groups.

  11. 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.

  12. ASAR15, A cis-acting locus that controls chromosome-wide replication timing and stability of human chromosome 15.

    Directory of Open Access Journals (Sweden)

    Nathan Donley

    2015-01-01

    Full Text Available DNA replication initiates at multiple sites along each mammalian chromosome at different times during each S phase, following a temporal replication program. We have used a Cre/loxP-based strategy to identify cis-acting elements that control this replication-timing program on individual human chromosomes. In this report, we show that rearrangements at a complex locus at chromosome 15q24.3 result in delayed replication and structural instability of human chromosome 15. Characterization of this locus identified long, RNA transcripts that are retained in the nucleus and form a "cloud" on one homolog of chromosome 15. We also found that this locus displays asynchronous replication that is coordinated with other random monoallelic genes on chromosome 15. We have named this locus ASynchronous replication and Autosomal RNA on chromosome 15, or ASAR15. Previously, we found that disruption of the ASAR6 lincRNA gene results in delayed replication, delayed mitotic condensation and structural instability of human chromosome 6. Previous studies in the mouse found that deletion of the Xist gene, from the X chromosome in adult somatic cells, results in a delayed replication and instability phenotype that is indistinguishable from the phenotype caused by disruption of either ASAR6 or ASAR15. In addition, delayed replication and chromosome instability were detected following structural rearrangement of many different human or mouse chromosomes. These observations suggest that all mammalian chromosomes contain similar cis-acting loci. Thus, under this scenario, all mammalian chromosomes contain four distinct types of essential cis-acting elements: origins, telomeres, centromeres and "inactivation/stability centers", all functioning to promote proper replication, segregation and structural stability of each chromosome.

  13. Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits.

    Science.gov (United States)

    Piazza, Ilaria; Rutkowska, Anna; Ori, Alessandro; Walczak, Marta; Metz, Jutta; Pelechano, Vicent; Beck, Martin; Haering, Christian H

    2014-06-01

    Condensin complexes have central roles in the three-dimensional organization of chromosomes during cell divisions, but how they interact with chromatin to promote chromosome segregation is largely unknown. Previous work has suggested that condensin, in addition to encircling chromatin fibers topologically within the ring-shaped structure formed by its SMC and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA formed by the two HEAT-repeat subunits of the Saccharomyces cerevisiae condensin complex. From detailed mapping data of the interfaces between the HEAT-repeat and kleisin subunits, we generated condensin complexes that lack one of the HEAT-repeat subunits and consequently fail to associate with chromosomes in yeast and human cells. The finding that DNA binding by condensin's HEAT-repeat subunits stimulates the SMC ATPase activity suggests a multistep mechanism for the loading of condensin onto chromosomes.

  14. Microhomology directs diverse DNA break repair pathways and chromosomal translocations.

    Directory of Open Access Journals (Sweden)

    Diana D Villarreal

    Full Text Available Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR events after a DNA double-strand break (DSB in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.

  15. Hidden chromosome symmetry: in silico transformation reveals symmetry in 2D DNA walk trajectories of 671 chromosomes.

    Directory of Open Access Journals (Sweden)

    Maria S Poptsova

    Full Text Available Maps of 2D DNA walk of 671 examined chromosomes show composition complexity change from symmetrical half-turn in bacteria to pseudo-random trajectories in archaea, fungi and humans. In silico transformation of gene order and strand position returns most of the analyzed chromosomes to a symmetrical bacterial-like state with one transition point. The transformed chromosomal sequences also reveal remarkable segmental compositional symmetry between regions from different strands located equidistantly from the transition point. Despite extensive chromosome rearrangement the relation of gene numbers on opposite strands for chromosomes of different taxa varies in narrow limits around unity with Pearson coefficient r = 0.98. Similar relation is observed for total genes' length (r = 0.86 and cumulative GC (r = 0.95 and AT (r = 0.97 skews. This is also true for human coding sequences (CDS, which comprise only several percent of the entire chromosome length. We found that frequency distributions of the length of gene clusters, continuously located on the same strand, have close values for both strands. Eukaryotic gene distribution is believed to be non-random. Contribution of different subsystems to the noted symmetries and distributions, and evolutionary aspects of symmetry are discussed.

  16. Altered chromosome 6 in immortal human fibroblasts.

    Science.gov (United States)

    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

  17. Altered chromosome 6 in immortal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hubbard-Smith, K.; Pardinas, J.R.; Jha, K.K.; Ozer, H.L. (New Jersey Medical School, Newark, NJ (United States)); Patsalis, P.; Henderson, A.S. (City Univ. of New York, NY (United States))

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene. 66 refs., 6 figs., 2 tabs.

  18. [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.

  19. (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.

  20. [Fluorescence in situ hybridization with DNA probes derived from individual chromosomes and chromosome regions].

    Science.gov (United States)

    Bogomolov, A G; Karamysheva, T V; Rubtsov, N B

    2014-01-01

    A significant part of the eukaryotic genomes consists of repetitive DNA, which can form large clusters or distributed along euchromatic chromosome regions. Repeats located in chromosomal regions make a problem in analysis and identification of the chromosomal material with fluorescence in situ hybridization (FISH). In most cases, the identification of chromosome regions using FISH requires detection of the signal produced with unique sequences. The feasibility, advantages and disadvantages of traditional methods of suppression of repetitive DNA hybridization, methods of repeats-free probe construction and methods of chromosome-specific DNA sequences visualization using image processing of multicolor FISH results are considered in the paper. The efficiency of different techniques for DNA probe generation, different FISH protocols, and image processing of obtained microscopic images depends on the genomic size and structure of analyzing species. This problem was discussed and different approaches were considered for the analysis of the species with very large genome, rare species and species which specimens are too small in size to obtain the amount of genomic and Cot-1 DNA required for suppression of repetitive DNA hybridization.

  1. The Divergence of Neandertal and Modern Human Y Chromosomes

    Science.gov (United States)

    Mendez, Fernando L.; Poznik, G. David; Castellano, Sergi; Bustamante, Carlos D.

    2016-01-01

    Sequencing the genomes of extinct hominids has reshaped our understanding of modern human origins. Here, we analyze ∼120 kb of exome-captured Y-chromosome DNA from a Neandertal individual from El Sidrón, Spain. We investigate its divergence from orthologous chimpanzee and modern human sequences and find strong support for a model that places the Neandertal lineage as an outgroup to modern human Y chromosomes—including A00, the highly divergent basal haplogroup. We estimate that the time to the most recent common ancestor (TMRCA) of Neandertal and modern human Y chromosomes is ∼588 thousand years ago (kya) (95% confidence interval [CI]: 447–806 kya). This is ∼2.1 (95% CI: 1.7–2.9) times longer than the TMRCA of A00 and other extant modern human Y-chromosome lineages. This estimate suggests that the Y-chromosome divergence mirrors the population divergence of Neandertals and modern human ancestors, and it refutes alternative scenarios of a relatively recent or super-archaic origin of Neandertal Y chromosomes. The fact that the Neandertal Y we describe has never been observed in modern humans suggests that the lineage is most likely extinct. We identify protein-coding differences between Neandertal and modern human Y chromosomes, including potentially damaging changes to PCDH11Y, TMSB4Y, USP9Y, and KDM5D. Three of these changes are missense mutations in genes that produce male-specific minor histocompatibility (H-Y) antigens. Antigens derived from KDM5D, for example, are thought to elicit a maternal immune response during gestation. It is possible that incompatibilities at one or more of these genes played a role in the reproductive isolation of the two groups. PMID:27058445

  2. REVIEW ARTICLE: DNA protein interactions and bacterial chromosome architecture

    Science.gov (United States)

    Stavans, Joel; Oppenheim, Amos

    2006-12-01

    Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

  3. 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.

  4. Mammalian DNA ligase III: Molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jingwen; Danehower, S.; Besterman, J.M.; Husain, I. [Glaxo Research Inst., Research Triangle Park, NC (United States)] [and others

    1995-10-01

    Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating supermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replications. In contrast, elevated levels of DNA ligase III mRNA were observed in primary supermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells. 62 refs., 7 figs.

  5. Impact of chromosomal rearrangement upon DNA methylation patterns in leukemia

    Directory of Open Access Journals (Sweden)

    Byun Hyang-Min

    2017-05-01

    Full Text Available Genomic instability, including genetic mutations and chromosomal rearrangements, can lead to cancer development. Aberrant DNA methylation occurs commonly in cancer cells. The aim of this study is to determine the effects of a specific chromosomal lesion the BCR-ABL translocation t(9:22, in establishing DNA methylation profiles in cancer. Materials and methods We compared DNA methylation of 1,505 selected promoter CpGs in chronic myelogenous leukemia (CML, acute lymphoblastic leukemia (ALL with and without the Philadelphia chromosome t(9:22, CD34+ hematopoietic stem cells transfected with BCR-ABL, and other tumors without BCR-ABL (acute promyelocytic leukemia (APL and gastrointestinal stromal tumors (GIST. In this study, the DNA methylation profile of CML was more closely related to APL, another myeloid leukemia, than Ph+ ALL. Although DNA methylation profiles were consistent within a specific tumor type, overall DNA methylation profiles were no influenced by BCR-ABL gene translocation in the cancers and tissues studied. We conclude that DNA methylation profiles may reflect the cell of origin in cancers rather than the chromosomal lesions involved in leukemogenesis.

  6. Chromosomal Aberrations in Humans Induced by Urban Air Pollution

    DEFF Research Database (Denmark)

    Knudsen, Lisbeth E.; Norppa, Hannu; Gamborg, Michael O.

    1999-01-01

    We have studied the influence of individual susceptibility factors on the genotoxic effects of urban air pollution in 106 nonsmoking bus drivers and 101 postal workers in the Copenhagen metropolitan area. We used the frequency of chromosomal aberrations in peripheral blood lymphocytes...... that long-term exposure to urban air pollution (with traffic as the main contributor) induces chromosome damage in human somatic cells. Low DNA repair capacity and GSTM1 and NAT2 variants associated with reduced detoxification ability increase susceptibility to such damage. The effect of the GSTM1 genotype......, which was observed only in the bus drivers, appears to be associated with air pollution, whereas the NAT2 genotype effect, which affected all subjects, may influence the individual response to some other common exposure or the baseline level of chromosomal aberrations....

  7. Staining and embedding of human chromosomes for 3-d serial block-face scanning electron microscopy.

    Science.gov (United States)

    Yusuf, Mohammed; Chen, Bo; Hashimoto, Teruo; Estandarte, Ana Katrina; Thompson, George; Robinson, Ian

    2014-12-01

    The high-order structure of human chromosomes is an important biological question that is still under investigation. Studies have been done on imaging human mitotic chromosomes using mostly 2-D microscopy methods. To image micron-sized human chromosomes in 3-D, we developed a procedure for preparing samples for serial block-face scanning electron microscopy (SBFSEM). Polyamine chromosomes are first separated using a simple filtration method and then stained with heavy metal. We show that the DNA-specific platinum blue provides higher contrast than osmium tetroxide. A two-step procedure for embedding chromosomes in resin is then used to concentrate the chromosome samples. After stacking the SBFSEM images, a familiar X-shaped chromosome was observed in 3-D.

  8. A high-resolution interval map of the q21 region of the human X chromosome

    Energy Technology Data Exchange (ETDEWEB)

    Philippe, C.; Monaco, A.P. [ICRF Laboratories, Oxford (United Kingdom)] [and others; Arnould, C. [Laboratoire de Genetique Humaine, Vandoeuvre-les-Nancy (France)] [and others

    1995-06-10

    In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome. Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region. 26 refs., 1 fig., 1 tab.

  9. Small Supernumerary Marker Chromosomes in Human Infertility.

    Science.gov (United States)

    Armanet, Narjes; Tosca, Lucie; Brisset, Sophie; Liehr, Thomas; Tachdjian, Gérard

    2015-01-01

    Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be unambiguously identified by banding cytogenetics. The objective of this study was to provide an overview of sSMC frequency and characterization in a context of infertility and to review the literature describing sSMC in relation with male and female infertility. Therefore, a systematic literature review on sSMC associated with infertility was conducted by means of a PubMed literature and a sSMC database (http://ssmc-tl.com/sSMC.html) search. A total of 234 patients with infertility were identified as carriers of sSMC. All chromosomes, except chromosomes 10, 19 and the X, were involved in sSMC, and in 72% the sSMC originated from acrocentric chromosomes. Euchromatic imbalances were caused by the presence of sSMC in 30% of the cases. Putative genes have been identified in only 1.2% of sSMC associated with infertility. The implication of sSMC in infertility could be due to a partial trisomy of some genes but also to mechanical effects perturbing meiosis. Further precise molecular and interphase-architecture studies on sSMC are needed in the future to characterize the relationship between this chromosomal anomaly and human infertility.

  10. Guarding chromosomes from oxidative DNA damage to the very end

    Institute of Scientific and Technical Information of China (English)

    Rong Tan; Li Lan

    2016-01-01

    The ends of each chromosome are capped by the telomere assembly to protect chromosomal integrity from telomere attrition and DNA damage.In response to DNA damage,DNA repair factors are enriched at damage sites by a sophisticated signaling and recruitment cascade.However,DNA damage response at telomeres is different from non-telomeric region of genomic DNA due to specialized sequences and structures of the telomeres.In the course of normal DNA replication or DNA damage repair,both the telomere shelterin protein complex and the condensed telomeric chromatin structure in mammalian cells are modified to protect telomeres from exposing free DNA ends which are subject to both telemere shortening and chromosome end fusion.Initiation of either homologous recombination or non-homologous end joint repair at telomeres requires disassembling andaor post-translational modifications of the shelterin complex and telomeric chromatin.In addition,cancer cells utilize distinct mechanisms to maintain telomere length and cell survival upon damage.In this review,we summarize current studies that focus on telomere end protection and telomere DNA repair using different methodologies to model telomere DNA damage and disruption.These include genetic ablation of sheltering proteins,targeting endonuclease to telomeres,and delivering oxidative damage directly.These different approaches,when combined,offer better understanding of the mechanistic differences in DNA damage response between telomeric and genomic DNA,which will provide new hope to identify potential cancer therapeutic targets to curtail cancer cell proliferation via induction of telomere dysfunctions.

  11. Cell-free DNA screening and sex chromosome aneuploidies.

    Science.gov (United States)

    Mennuti, Michael T; Chandrasekaran, Suchitra; Khalek, Nahla; Dugoff, Lorraine

    2015-10-01

    Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed.

  12. Oxidative DNA damage in mouse sperm chromosomes: Size matters.

    Science.gov (United States)

    Kocer, Ayhan; Henry-Berger, Joelle; Noblanc, Anais; Champroux, Alexandre; Pogorelcnik, Romain; Guiton, Rachel; Janny, Laurent; Pons-Rejraji, Hanae; Saez, Fabrice; Johnson, Graham D; Krawetz, Stephen A; Alvarez, Juan G; Aitken, R John; Drevet, Joël R

    2015-12-01

    Normal embryo and foetal development as well as the health of the progeny are mostly dependent on gamete nuclear integrity. In the present study, in order to characterize more precisely oxidative DNA damage in mouse sperm we used two mouse models that display high levels of sperm oxidative DNA damage, a common alteration encountered both in in vivo and in vitro reproduction. Immunoprecipitation of oxidized sperm DNA coupled to deep sequencing showed that mouse chromosomes may be largely affected by oxidative alterations. We show that the vulnerability of chromosomes to oxidative attack inversely correlated with their size and was not linked to their GC richness. It was neither correlated with the chromosome content in persisting nucleosomes nor associated with methylated sequences. A strong correlation was found between oxidized sequences and sequences rich in short interspersed repeat elements (SINEs). Chromosome position in the sperm nucleus as revealed by fluorescent in situ hybridization appears to be a confounder. These data map for the first time fragile mouse sperm chromosomal regions when facing oxidative damage that may challenge the repair mechanisms of the oocyte post-fertilization.

  13. Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B

    Institute of Scientific and Technical Information of China (English)

    Gerald C Kimbi; Anna Kramvis; Michael C Kew

    2005-01-01

    AIM: To examine the serum from black African patients with acute hepatitis B to ascertain if integrants of viral DNA can be detected in fragments of cellular DNA leaking from damaged hepatocytes into the circulation.METHODS: DNA was extracted from the sera of five patients with uncomplicated acute hepatitis B and one with fulminant disease. Two subgenomic PCRs designed to amplify the complete genome of HBV were used and the resulting amplicons were cloned and sequenced.RESULTS: HBV and chromosomal DNA were amplified from the sera of all the patients. In one patient with uncomplicated disease, HBV DNA was integrated into host chromosome 7 q11.23 in the WBSCR1 gene. The viral DNA comprised 200 nucleotides covering the S and X genes in opposite orientation, with a 1 169 nudeotide deletion. The right virus/host junction was situated at nucleotide 1774 in the cohesive overlap region of the viral genome, at a preferred topoisomerase I cleavage motif. The chromosomal DNA was not rearranged.The patient made a full recovery and seroconverted to anti-HBs- and anti-HBe-positivity. Neither HBV nor chromosomal DNA could be amplified from his serum at that time.CONCLUSION: Integration of viral DNA into chromosomal DNA may occur rarely during acute hepatitis B and, with clonal propagation of the integrant, might play a role in hepatocarcinogenesis.

  14. Human male meiotic sex chromosome inactivation.

    Science.gov (United States)

    de Vries, Marieke; Vosters, Sanne; Merkx, Gerard; D'Hauwers, Kathleen; Wansink, Derick G; Ramos, Liliana; de Boer, Peter

    2012-01-01

    In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI), which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.

  15. Human male meiotic sex chromosome inactivation.

    Directory of Open Access Journals (Sweden)

    Marieke de Vries

    Full Text Available In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI, which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.

  16. Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae).

    Science.gov (United States)

    Zadesenets, Kira S; Karamysheva, Tatyana V; Katokhin, Alexei V; Mordvinov, Viatcheslav A; Rubtsov, Nikolay B

    2012-03-01

    Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.

  17. Dynamic organization of chromosomal DNA in Escherichia coli.

    Science.gov (United States)

    Niki, H; Yamaichi, Y; Hiraga, S

    2000-01-15

    We have revealed the subcellular localization of different DNA segments that are located at approximately 230-kb intervals on the Escherichia coli chromosome using fluorescence in situ hybridization (FISH). The series of chromosome segments is localized within the cell in the same order as the chromosome map. The large chromosome region including oriC shows similar localization patterns, which we call the Ori domain. In addition, the localization pattern of the large segment including dif is characteristic of the replication terminus region. The segment also shows similar localization patterns, which we call the Ter domain. In newborn cells, Ori and Ter domains of the chromosome are differentially localized near opposite cell poles. Subsequently, in the B period, the Ori domain moves toward mid-cell before the initiation of replication, and the Ter domain tends to relocate at mid-cell. An inversion mutant, in which the Ter domain is located close to oriC, shows abnormal subcellular localization of ori and dif segments, resulting in frequent production of anucleate cells. These studies thus suggest that the E. coli chromosome is organized to form a compacted ring structure with the Ori and Ter domains; these domains participate in the cell cycle-dependent localization of the chromosome.

  18. Chromosome protein framework from proteome analysis of isolated human metaphase chromosomes.

    Science.gov (United States)

    Fukui, Kiichi; Uchiyama, Susumu

    2007-01-01

    We have presented a structural model of the chromosome based on its constituent proteins. Development of a method of mass isolation for intact human metaphase chromosomes and proteome analysis by mass spectrometry of the isolated chromosomal proteins enabled us to develop a four-layer structural model of human metaphase chromosomes. The model consists of four layers, each with different chromosomal protein sets, i.e., chromosome coating proteins (CCPs), chromosome peripheral proteins (CPPs), chromosome structural proteins (CSPs), and chromosome fibrous proteins (CFPs). More than 200 identified proteins have been classified and assigned to the four layers with each layer occupying a distinct region of the chromosome. CCPs are localized at the most outer regions of the chromosomes and they attach to the regions tentatively and occasionally. CCPs include mostly mitochondrial and cytoplasmic proteins, e.g., 70 kDa heat shock protein 9B and Hsp60. CPPs are also localized at the peripheral regions of the chromosomes, but as the essential part of the chromosomes. CPPs include nucleolin, lamin A/C, fibrillarin, etc. CSPs are the primary chromosomal structure proteins, and include topoisomerase IIalpha, condensin subunits, histones, etc. CFPs have a fibrous nature, e.g., beta-actin, vimentin, myosin II, tublin, etc. A data set of these proteins, which we developed, contains essential chromosome proteins with classified information based on this four-layer model and presents useful leads for further studies on chromosomal structure and function.

  19. Strategies for sequencing human chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, G.R.

    1996-06-01

    This project funded for four years (02.92 to 01.96) was a renewal of a project funded for 2.5 years (07.89 to 01.92). This report covers the period 07.89 to 07.94. The original project was entitled {open_quotes}Correlation of physical and genetic maps of Human Chromosome 16{close_quotes}. The aim over this period was to construct a cytogenetic-based physical map of chromosome 16, to enable integration of its physical and genetic maps. This was achieved by collaboration and isolation of new markers until each bin on the physical map contained a polymorphic marker on the linkage map. A further aim was to integrate all mapping data for this chromosome and to achieve contig closure over band q24.

  20. The complete sequence of human chromosome 5

    Energy Technology Data Exchange (ETDEWEB)

    Schmutz, Jeremy; Martin, Joel; Terry, Astrid; Couronne, Olivier; Grimwood, Jane; Lowry, State; Gordon, Laurie A.; Scott, Duncan; Xie, Gary; Huang, Wayne; Hellsten, Uffe; Tran-Gyamfi, Mary; She, Xinwei; Prabhakar, Shyam; Aerts, Andrea; Altherr, Michael; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caoile, Chenier; Challacombe, Jean F.; Chan, Yee Man; Denys, Mirian; Detter, Chris; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Goodstenin, David; Grigoriev, Igor; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimbal, Heather; Kobayashi, Arthur; Lopez, Frederick; Lou, Yunian; Martinez, Diego; Medina, Catherine; Morgan, Jenna; Nandkeshwar, Richard; Noonan, James P.; Pitluck, Sam; Pollard, Martin; Predki, Paul; Priest, James; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanie; Salamov, Asaf; Salazar, Angelica; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wheeler, Jeremy; Wu, Kevin; Yang, Joan; Dickson, Mark; Cheng, Jan-Fang; Eichler, Evan E.; Olsen, Anne; Pennacchio, Len A.; Rokhsar, Daniel S.; Richardson, Paul; Lucas, Susan M.; Myers, Richard M.; Rubin, Edward M.

    2004-04-15

    Chromosome 5 is one of the largest human chromosomes yet has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding and syntenic conservation with non-mammalian vertebrates, suggesting they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-encoding genes including the protocadherin and interleukin gene families and the first complete versions of each of the large chromosome 5 specific internal duplications. These duplications are very recent evolutionary events and play a likely mechanistic role, since deletions of these regions are the cause of debilitating disorders including spinal muscular atrophy (SMA).

  1. Extraction of Chromosomal DNA from Schizosaccharomyces pombe.

    Science.gov (United States)

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-05-02

    Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS-protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation. Finally, DNA is precipitated using isopropanol. At this stage, purity is usually sufficient for PCR. However, for more sensitive procedures, such as restriction enzyme digestion, additional purification steps, including proteinase K digestion and phenol-chloroform extraction, are recommended. All of these steps are described in detail here.

  2. Detecting chromosomal alterations at 1p and 19q by FISH and DNA fragment analysis - a comparative study in human gliomas

    DEFF Research Database (Denmark)

    Broholm, H.; Born, P.W.; Guterbaum, D.;

    2008-01-01

    Histological classification of gliomas is important for treatment and as a prognostic predictor, but classification by histology alone can be a challenge. Molecular genetic investigations, in particular the combined loss of the short arm of chromosome 1 and the long arm of chromosome 19 (LOH1p/19...

  3. Interference in DNA replication can cause mitotic chromosomal breakage unassociated with double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Mari Fujita

    Full Text Available Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs. We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways. Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54(-/-/KU70(-/- DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54(-/-/LIG4(-/- Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.

  4. Human estrogen sulfotransferase gene (STE): Cloning, structure, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Aksoy, I.A.; Weinshilboum, M. [Mayo Foundation, Rochester, MI (United States)] [and others

    1995-09-01

    Sulfation is an important pathway in the metabolism of estrogens. We recently cloned a human liver estrogen sulfotransferase (EST) cDNA. We have now determined the structure and chromosomal localization of the EST gene, STE, as a step toward molecular genetic studies of the regulation of EST in humans. STE spans approximately 20 kb and consists of 8 exons, ranging in length from 95 to 181 bp. The locations of most exon-intron splice junctions within STE are identical to those found in a human phenol ST (PST) gene, STM, and in a rat PST gene. In addition, the locations of five STE introns are also conserved in the human dehydroepiandrosterone (DBEA) ST gene, STD. The 5{prime} flanking region of STE contains one CCAAT and two TATA sequences. The location of one of the TATA box elements is in excellent agreement with the site of transcription initiation as determined by 5{prime}-rapid amplification of cDNA ends. STE was mapped to human chromosome 4q13.1 by fluorescence in situ hybridization. Cloning and structural characterization of STE will now make it possible to study potential molecular genetic mechanisms involved in the regulation of EST in human tissues. 50 refs., 6 figs., 1 tab.

  5. Human Sperm Chromosome Analysis—Study on Human Sperm Chromosome Mutagenesis Induced by Carbon Disulfide

    Institute of Scientific and Technical Information of China (English)

    LEJUN-YI; FUXIAO-MIN

    1996-01-01

    The aim of this study was to investigate the effect CS2 of on human sperm chromosomal aberration.The human sperm/hamster egg fusion techniquse was used to analyze 203 human sperm chromosome complement form 9 healthy volunteers.The incidence of numerical aberration was 1.0%,and that of structural chromosome aberration was 5.9% and total abnormalities was 6.9%.Structural aberrations consisted of breaks,deletions, centric rings,fragments,and chromatid exchange.The results from high concentration group(10μmol·L-1 CS2)showed that the incidence of chromosomal aberration rate was significantly higher than that of the control group.The results indicate that high concentration of CS2 might directly cause mutatenesis f the germ cell.

  6. Directly labeled fluorescent DNA probes for chromosome mapping

    Energy Technology Data Exchange (ETDEWEB)

    Marrone, B.L.; Deaven, L.L.; Chen, D.J.; Park, Min S.; MacInnes, M.A.; Salzman, G.C.; Yoshida, T.M.

    1995-12-31

    A new strategy is briefly described for employing nucleic acid probes that are directly labeled with fluorochromes in fluorescence in situ hybridization techniques. These probes will permit the detection, quantitation, and high-precision spatial analysis of multiple DNA sequences along a single chromosome using video-enhanced fluorescence microscopy and digital image processing and analysis. Potential advantages of direct labeled DNA probes for fluorescence in situ hybridization far surpass currently available, indirect DNA probe labeling techniques in ease of use, versatility, and increased signal- to-noise ratio.

  7. Capillary electrophoresis: Biotechnology for separation of DNA and chromosomes

    Science.gov (United States)

    Williams, George O., Jr.

    1994-01-01

    Electrophoresis has been used for the separation of particles, ions, and molecules for a number of years. The technology for separation and detection of the results has many applications in the life sciences. One of the major goals of the scientific community is to separate DNA molecules and intact chromosomes based upon their different lengths or number of base pairs. This may be achieved by using some of the commercially available and widely used methods, but these processes require a considerable amount of time. The challenge is to achieve separation of intact chromosomes in a short time, preferably in a matter of minutes.

  8. Assessment of aneuploidy in human oocytes and preimplantation embryos by chromosome painting

    Energy Technology Data Exchange (ETDEWEB)

    Rougier, N.; Viegas-Pequignot, E.; Plachot, M. [Hospital Necker, Paris (France)] [and others

    1994-09-01

    The poor quality of chromosome preparations often observed after fixation of oocytes and embryos did not usually allow accurate identification of chromosomes involved in non-disjunctions. We, therefore, used chromosome painting to determine the incidence of abnormalities for chromosomes 1 and 7. A total of 50 oocytes inseminated for IVF and showing no signs of fertilization as well as 37 diploid embryos donated for research were fixed according to the Dyban`s technique. Fluorescence in situ hybridization was carried out using whole chromosome painting DNA probes specific for human chromosome 1 and 7. The incidence of aneuploidy was 28%, 10% and 60% for metaphase II, polar body and sperm chromosomes, respectively. The high incidence of aneuploidy observed in sperm prematurely condensed sperm chromosomes is due to the fact that usually far less than 23 sperm chromatids are observed, maybe as a consequence of incomplete chromosome condensation. Thirty seven embryos were analyzed with the same probes. 48% of early embryos were either monosomic 1 or 7 or mosaics comprising blastomeres with 1, 2 or 3 signals. Thus, 8 among the 11 abnormal embryos had hypodiploid cells (25 to 37 chromosomes) indicating either an artefactual loss of chromosomes or a complex anomaly of nuclear division (maltinucleated blastomeres, abnormal migration of chromosomes at anaphase). We therefore calculated a {open_quotes}corrected{close_quotes} incidence of aneuploidy for chromosomes 1 or 7 in early embryos: 18%. 86% of the blastocysts showed mosaicism 2n/3 or 4n as a consequence of the formation of the syncitiotrophoblast. To conclude, chromosome painting is an efficient method to accurately identify chromosomes involved in aneuploidy. This technique should allow us to evaluate the incidence of non-disjunction for all chromosome pairs. Our results confirm the high incidence of chromosome abnormalities occurring as a consequence of meiotic or mitotic non-disjunctions in human oocytes and embryos.

  9. 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.

  10. Broader utilization of origins of DNA replication in cancer cell lines along a 78 kb region of human chromosome 2q34.

    Science.gov (United States)

    Valenzuela, Manuel S; Hu, Lan; Lueders, John; Walker, Robert; Meltzer, Paul S

    2012-01-01

    Human DNA replication depends on the activation of thousands of origins distributed within the genome. The actual distribution of origins is not known, nor whether this distribution is unique to a cell type, or if it changes with the proliferative state of the cell. In this study, we have employed a real-time PCR-based nascent strand DNA abundance assay, to determine the location of origins along a 78 kb region on Chr2q34. Preliminary studies using nascent DNA strands isolated from either HeLa and normal skin fibroblast cells showed that in both cell lines peaks of high origin activity mapped in similar locations. However, the overall origin profile in HeLa cells corresponded to broad origin activation zones, whereas in fibroblasts a more punctuated profile of origin activation was observed. To investigate the relevance of this differential origin profile, we compared the origin distribution profiles in breast cancer cell lines MDA-MB-231, BT-474, and MCF-7, to their normal counterpart MCF-10A. In addition, the CRL7250 cell line was also used as a normal control. Our results validated our earlier observation and showed that the origin profile in normal cell lines exhibited a punctuated pattern, in contrast to broader zone profiles observed in the cancer cell lines. A quantitative analysis of origin peaks revealed that the number of activated origins in cancer cells is statistically larger than that obtained in normal cells, suggesting that the flexibility of origin usage is significantly increased in cancer cells compared to their normal counterparts.

  11. Human mitochondrial HMG CoA synthase: Liver cDNA and partial genomic cloning, chromosome mapping to 1p12-p13, and possible role in vertebrate evolution

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal (Canada)] [and others

    1994-10-01

    Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholersterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of an HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. the nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistant with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain. 56 refs., 4 figs., 2 tabs.

  12. DNA Cross-Bridging Shapes a Single Nucleus from a Set of Mitotic Chromosomes.

    Science.gov (United States)

    Samwer, Matthias; Schneider, Maximilian W G; Hoefler, Rudolf; Schmalhorst, Philipp S; Jude, Julian G; Zuber, Johannes; Gerlich, Daniel W

    2017-08-24

    Eukaryotic cells store their chromosomes in a single nucleus. This is important to maintain genomic integrity, as chromosomes packaged into separate nuclei (micronuclei) are prone to massive DNA damage. During mitosis, higher eukaryotes disassemble their nucleus and release individualized chromosomes for segregation. How numerous chromosomes subsequently reform a single nucleus has remained unclear. Using image-based screening of human cells, we identified barrier-to-autointegration factor (BAF) as a key factor guiding membranes to form a single nucleus. Unexpectedly, nuclear assembly does not require BAF's association with inner nuclear membrane proteins but instead relies on BAF's ability to bridge distant DNA sites. Live-cell imaging and in vitro reconstitution showed that BAF enriches around the mitotic chromosome ensemble to induce a densely cross-bridged chromatin layer that is mechanically stiff and limits membranes to the surface. Our study reveals that BAF-mediated changes in chromosome mechanics underlie nuclear assembly with broad implications for proper genome function. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

    Directory of Open Access Journals (Sweden)

    Hiroshi Sato

    2017-01-01

    Full Text Available Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV. Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

  14. Cloning chromosome specific genes by reciprocal probing of arrayed cDNA and cosmid libraries

    Energy Technology Data Exchange (ETDEWEB)

    Yazdani, A.; Lee, C.C.; Wehnert, M. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    A human gene map will greatly facilitate the association of genes to single locus diseases and provide candidates for genes involved in complex genetic traits. Given the estimated 100,000 human genes an integrated strategy with a high throughput approach for isolation and mapping of expressed sequences is needed to create such a gene map. We have developed an approach that allows high throughput gene isolation and mapping using arrayed genomic and cDNA lambda libraries. Reciprocal probing of the arrayed genomic and cDNA cosmic libraries can rapidly establish cDNA-cosmid associations. Fluorescence in situ hybridization (FISH) chromosomal mapping and expressed sequence tag/sequence tag site (EST/STS) primers generated from DNA sequence of PCR-based mapping using somatic hybrid cell line mapping panels were utilized to characterize further the hybridization-based cDNA cosmid association. We have applied this approach to chromosome 17 using a placental cDNA library and have identified a total of 30 genes out of which 11 are novel. Furthermore seven cDNAs were mapped to 17q21 in this study, providing novel candidate genes for BRCA-1 gene for early onset breast cancer. The results of our study clearly show that an integration of an expression map into physical and genetic maps can provide candidate genes for human diseases that have been mapped to specific regions. This approach combined with the current physical mapping efforts could efficiently provide a detailed human gene map.

  15. Global DNA Methylation patterns on marsupial and devil facial tumour chromosomes.

    Science.gov (United States)

    Ingles, Emory D; Deakin, Janine E

    2015-01-01

    Despite DNA methylation being one of the most widely studied epigenetic modifications in eukaryotes, only a few studies have examined the global methylation status of marsupial chromosomes. The emergence of devil facial tumour disease (DFTD), a clonally transmissible cancer spreading through the Tasmanian devil population, makes it a particularly pertinent time to determine the methylation status of marsupial and devil facial tumour chromosomes. DNA methylation perturbations are known to play a role in genome instability in human tumours. One of the interesting features of the devil facial tumour is its remarkable karyotypic stability over time as only four strains with minor karyotypic differences having been reported. The cytogenetic monitoring of devil facial tumour (DFT) samples collected over an eight year period and detailed molecular cytogenetic analysis performed on the different DFT strains enables chromosome rearrangements to be correlated with methylation status as the tumour evolves. We used immunofluorescent staining with an antibody to 5-methylcytosine on metaphase chromosomes prepared from fibroblast cells of three distantly related marsupials, including the Tasmanian devil, as well as DFTD chromosomes prepared from samples collected from different years and representing different karyotypic strains. Staining of chromosomes from male and female marsupial cell lines indicate species-specific differences in global methylation patterns but with the most intense staining regions corresponding to telomeric and/or centromeric regions of autosomes. In males, the X chromosome was hypermethylated as was one X in females. Similarly, telomeric regions on DFTD chromosomes and regions corresponding to material from one of the two X chromosomes were hypermethylated. No difference in global methylation in samples of the same strain taken in different years was observed. The methylation patterns on DFTD chromosomes suggests that the hypermethylated active X was

  16. Induction of chromosomal aberrations in human lymphocytes by fission neutrons

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Marcia Augusta da; Coelho, Paulo Rogerio Pinto; Bartolini, Paolo; Okazaki, Kayo [Instituto de Pesquisas Energeticas e Nucleares (IPEN-CNEN/SP), Sao Paulo, SP (Brazil)], e-mail: kokazaki@ipen.br

    2009-07-01

    Chromosome aberrations induced by sparsely ionizing radiation (low-LET) are well known and cytogenetic analyses of irradiated human lymphocytes have been widely applied to biological dosimetry. However, much less is known about chromosome aberrations induced by densely ionizing radiation (high LET), such as that of alpha particles or neutrons. Such particles induce DNA strand breaks, as well as chromosome breakage and rearrangements of high complexity. This damage is more localized and less efficiently repaired than after X- or {gamma}-ray irradiation. This preferential production of complex aberrations by densely ionizing radiation is related to the unique energy deposition patterns, which produces highly localized multiple DNA damage at the chromosomal level. A better knowledge of the interactions between different types of radiation and cellular DNA is of importance, not only from the radiobiological viewpoint but also for dosimetric and therapeutic purposes. The objective of the present study was to analyse the cytogenetic effects of fission neutrons on peripheral blood lymphocytes in order to evaluate structural and numerical aberrations and number of cells in the different mitotic cycles. So, blood samples from five healthy donors, 22-25 years old, of both sexes, were irradiated in the Research Reactor IEA-R1 of our Institute (IPEN/CNEN-SP) with thermal and fast neutrons at doses of 0.2; 0.3; 0.5 and 1.0 Gy. The {gamma} contribution to the total absorbed dose was about 30%. These doses were monitored by thermoluminescent dosemeters: LiF-600 (for neutrons) and LiF-700 (for {gamma}-rays). The data concerning structural aberrations were evaluated with regard to three parameters: percentage of cells with aberrations, number of aberrations/cell and number of dicentric/cell. The cytogenetic results showed an increase in the three parameters after irradiation with neutrons, as a function of radiation dose. Apparently, there was no influence of neutrons on the

  17. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiming [Geneva Univ. Medical School (Switzerland); Morris, M.A.; Rossier, C. [Cantonal Hospital, Geneva (Switzerland)] [and others

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  18. The orphan nuclear receptor ROR{alpha} (RORA) maps to a conserved region of homology on human chromosome 15q21-q22 and mouse chromosome 9

    Energy Technology Data Exchange (ETDEWEB)

    Giguere, V. [McGill Univ., Montreal (Canada); Beatty, B.; Squire, J. [Hospital for Sick Children, Toronto (Canada)] [and others

    1995-08-10

    ROR{alpha} is a novel member of the steroid/thyroid/retinoid receptor superfamily with unique DNA-binding properties. We have mapped the RORA gene by fluorescence in situ hybridization to human chromosome 15q21-q22. To map the mouse Rora gene, a partial mouse cDNA clone was isolated from brain. Using interspecific backcross analysis, we have mapped the Rora gene to mouse chromosome 9. This places the human RORA gene in the proximity of the PML gene, which is involved in a reciprocal chromosomal translocation t(15:17) with the RARA gene in patients with acute promyelocytic leukemia. 13 refs., 2 figs.

  19. New insights into human nondisjunction of chromosome 21 in oocytes.

    Directory of Open Access Journals (Sweden)

    Tiffany Renee Oliver

    2008-03-01

    Full Text Available Nondisjunction of chromosome 21 is the leading cause of Down syndrome. Two risk factors for maternal nondisjunction of chromosome 21 are increased maternal age and altered recombination. In order to provide further insight on mechanisms underlying nondisjunction, we examined the association between these two well established risk factors for chromosome 21 nondisjunction. In our approach, short tandem repeat markers along chromosome 21 were genotyped in DNA collected from individuals with free trisomy 21 and their parents. This information was used to determine the origin of the nondisjunction error and the maternal recombination profile. We analyzed 615 maternal meiosis I and 253 maternal meiosis II cases stratified by maternal age. The examination of meiosis II errors, the first of its type, suggests that the presence of a single exchange within the pericentromeric region of 21q interacts with maternal age-related risk factors. This observation could be explained in two general ways: 1 a pericentromeric exchange initiates or exacerbates the susceptibility to maternal age risk factors or 2 a pericentromeric exchange protects the bivalent against age-related risk factors allowing proper segregation of homologues at meiosis I, but not segregation of sisters at meiosis II. In contrast, analysis of maternal meiosis I errors indicates that a single telomeric exchange imposes the same risk for nondisjunction, irrespective of the age of the oocyte. Our results emphasize the fact that human nondisjunction is a multifactorial trait that must be dissected into its component parts to identify specific associated risk factors.

  20. Exploring function of conserved non-coding DNA in its chromosomal context

    Directory of Open Access Journals (Sweden)

    Delores J. Grant

    2015-11-01

    Full Text Available There is renewed interest in understanding expression of vertebrate genes in their chromosomal context because regulatory sequences that confer tissue-specific expression are often distributed over large distances along the DNA from the gene. One approach inserts a universal sensor/reporter-gene into the mouse or zebrafish genome to identify regulatory sequences in highly conserved non-coding DNA in the vicinity of the integrated reporter-gene. However detailed mechanisms of interaction of these regulatory elements among themselves and/or with the genes they influence remain elusive with the strategy. The inability to associate distant regulatory elements with the genes they regulate makes it difficult to examine the contribution of sequence changes in regulatory DNA to human disease. Such associations have been obtained in favorable circumstances by testing the regulatory potential of highly conserved non-coding DNA individually in small reporter-gene-containing plasmids. Alternative approaches use tiny fragments of chromosomes in Bacterial Artificial Chromosomes, BACs, where the gene of interest is tagged in vitro with a reporter/sensor gene and integrated into the germ-line of animals for expression. Mutational analysis of the BAC DNA identifies regulatory sequences. A recent approach inserts a sensor/reporter-gene into a BAC that is also truncated progressively from an end of genomic insert, and the end-deleted BAC carrying the sensor is then integrated into the genome of a developing animal for expression. The approach allows mechanisms of tissue-specific gene expression to be explored in much greater detail, although the chromosomal context of such mechanisms is limited to the length of the BAC. Here we discuss the relative strengths of the various approaches and explore how the integrated-sensor in the BACs method applied to a contig of BACs spanning a chromosomal region is likely to address mechanistic questions on interactions between

  1. Replication Banding Patterns in Human Chromosomes Detected Using 5-ethynyl-2'-deoxyuridine Incorporation.

    Science.gov (United States)

    Hoshi, Osamu; Ushiki, Tatsuo

    2011-10-26

    A novel technique using the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with incorporated EdU showed a banding pattern similar to G-banding of normal human chromosomes. Imaging by atomic force microscopy (AFM) in liquid conditions showed that the structure of the chromosomes was well preserved even after EdU treatment. Comparison between fluorescence microscopy and AFM images of the same chromosome 1 indicated the presence of ridges and grooves in the chromatid arm, features that have been previously reported in relation to G-banding. These results suggest an intimate relationship between EdU-induced replication bands and G- or R-bands in human chromosomes. This technique is thus useful for analyzing the structure of chromosomes in relation to their banding patterns following DNA replication in the S phase.

  2. Chromosomal aberrations in humans induced by urban air pollution: influence of DNA repair and polymorphisms of glutathione S-transferase M1 and N-acetyltransferase 2

    DEFF Research Database (Denmark)

    Knudsen, Lisbeth E.; Norppa, H; Gamborg, M O

    1999-01-01

    We have studied the influence of individual susceptibility factors on the genotoxic effects of urban air pollution in 106 nonsmoking bus drivers and 101 postal workers in the Copenhagen metropolitan area. We used the frequency of chromosomal aberrations in peripheral blood lymphocytes as a biomar...

  3. Sequence and expression analysis of gaps in human chromosome 20

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Seemann, Stefan; Mang, Yuan;

    2012-01-01

    The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 (chr 20) currently has three unfinished gaps remaining on its q-arm. All three gaps are within gene-dense regions and....../or overlap disease-associated loci, including the DLGAP4 locus. In this study, we sequenced ~99% of all three unfinished gaps on human chr 20, determined their complete genomic sizes and assessed epigenetic profiles using a combination of Sanger sequencing, mate pair paired-end high-throughput sequencing...... and chromatin, methylation and expression analyses. We found histone 3 trimethylated at Lysine 27 to be distributed across all three gaps in immortalized B-lymphocytes. In one gap, five novel CpG islands were predominantly hypermethylated in genomic DNA from peripheral blood lymphocytes and human cerebellum...

  4. Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E.

    1991-09-01

    The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.

  5. Molecular genetic approach to human meningioma: loss of genes on chromosome 22

    Energy Technology Data Exchange (ETDEWEB)

    Seizinger, B.R.; De La Monte, S.; Atkins, L.; Gusella, J.F.; Martuza, R.L.

    1987-08-01

    A molecular genetic approach employing polymorphic DNA markers has been used to investigate the role of chromosomal aberrations in meningioma, one of the most common tumors of the human nervous system. Comparison of the alleles detected by DNA markers in tumor DNA versus DNA from normal tissue revealed chromosomal alterations present in primary surgical specimens. In agreement with cytogenetic studies of cultured meningiomas, the most frequent alteration detected was loss of heterozygosity on chromosome 22. Forty of 51 patients were constitutionally heterozygous for at least one chromosome 22 DNA marker. Seventeen of the 40 constitutionally heterozygotic patients (43%) displayed hemizygosity for the corresponding marker in their meningioma tumor tissues. Loss of heterozygosity was also detected at a significantly lower frequency for markers on several other autosomes. In view of the striking association between acoustic neuroma and meningioma in bilateral acoustic neurofibromatosis and the discovery that acoustic neuromas display specific loss of genes on chromosome 22, the authors propose that a common mechanism involving chromosome 22 is operative in the development of both tumor types. Fine-structure mapping to reveal partial deletions in meningiomas may provide the means to clone and characterize a gene (or genes) of importance for tumorigenesis in this and possibly other clinically associated tumors of the human nervous system.

  6. Construction of human artificial chromosome vectors by recombineering.

    Science.gov (United States)

    Kotzamanis, George; Cheung, Wing; Abdulrazzak, Hassan; Perez-Luz, Sara; Howe, Steven; Cooke, Howard; Huxley, Clare

    2005-05-23

    Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

  7. 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.

  8. Localization of the tight junction protein gene TJP1 to human chromosome 15q13, distal to the Prader-Willi/Angelman region, and to mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Mohandas, T.K. [Darthmouth-Hitchcock Medical Center, Lebanon, NH (United States); Chen, X.N.; Korenberg, J.R. [UCLA School of Medicine, Los Angeles, CA (United States)] [and others

    1995-12-10

    The gene encoding the tight junction (zonula occludens) protein, TJP1, was mapped to human chromosome 15q13 by fluorescence in situ hybridization (FISH) using a cDNA probe. The Jackson Laboratory backcross DNA panel derived from the cross (C57BL/6JEi X SPRET/Ei) F1 females X SPRET/Ei males was used to map the mouse Tjp1 to chromosome 7 near position 30 on the Chromosome Committee Map, a region with conserved homology to human chromosome 15q13. FISH studies on metaphases from patients with the Prader-Willi (PWS) or the Angelman syndrome (AS) showed that TJP1 maps close but distal to the PWS/AS chromosome region. 13 refs., 2 figs.

  9. Differences in DNA curvature-related sequence periodicity between prokaryotic chromosomes and phages, and relationship to chromosomal prophage content

    Directory of Open Access Journals (Sweden)

    Abel Jacob

    2012-05-01

    Full Text Available Abstract Background Periodic spacing of A-tracts (short runs of A or T with the DNA helical period of ~10–11 bp is characteristic of intrinsically bent DNA. In eukaryotes, the DNA bending is related to chromatin structure and nucleosome positioning. However, the physiological role of strong sequence periodicity detected in many prokaryotic genomes is not clear. Results We developed measures of intensity and persistency of DNA curvature-related sequence periodicity and applied them to prokaryotic chromosomes and phages. The results indicate that strong periodic signals present in chromosomes are generally absent in phage genomes. Moreover, chromosomes containing prophages are less likely to possess a persistent periodic signal than chromosomes with no prophages. Conclusions Absence of DNA curvature-related sequence periodicity in phages could arise from constraints associated with DNA packaging in the viral capsid. Lack of prophages in chromosomes with persistent periodic signal suggests that the sequence periodicity and concomitant DNA curvature could play a role in protecting the chromosomes from integration of phage DNA.

  10. Data Mining Empowers the Generation of a Novel Class of Chromosome-specific DNA Probes

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Hui; Weier, Heinz-Ulrich G.; Kwan, Johnson; Wang, Mei; O' Brien, Benjamin

    2011-03-08

    Probes that allow accurate delineation of chromosome-specific DNA sequences in interphase or metaphase cell nuclei have become important clinical tools that deliver life-saving information about the gender or chromosomal make-up of a product of conception or the probability of an embryo to implant, as well as the definition of tumor-specific genetic signatures. Often such highly specific DNA probes are proprietary in nature and have been the result of extensive probe selection and optimization procedures. We describe a novel approach that eliminates costly and time consuming probe selection and testing by applying data mining and common bioinformatics tools. Similar to a rational drug design process in which drug-protein interactions are modeled in the computer, the rational probe design described here uses a set of criteria and publicly available bioinformatics software to select the desired probe molecules from libraries comprised of hundreds of thousands of probe molecules. Examples describe the selection of DNA probes for the human X and Y chromosomes, both with unprecedented performance, but in a similar fashion, this approach can be applied to other chromosomes or species.

  11. Human nicotinamide N-methyltransferase gene: Molecular cloning, structural characterization and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Aksoy, S.; Weinshilboum, R.M. [Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, MN (United States); Brandriff, B.F. [Lawrence Livermore National Lab., CA (United States); Ward, A.; Little, P.F.R. [Imperial College of Science, Technology and Medicine, London (United Kingdom)

    1995-10-10

    Genomic DNA clones for nicotinamide N-methyltransferase (NNMT), an enzyme that catalyzes drug and xenobiotic metabolism, were isolated from a human chromosome 11-specific DNA library. Study of one of those clones, when combined with PCR-based experiments performed with human genomic DNA, made it possible to determine the structure of the human NNMT gene. The gene was approximately 16.5 kb in length and consisted of 3 exons and 2 introns. Transcription initiation for the NNMT gene occurred 105-109 nucleotides 5{prime}-upstream from the cDNA translation initiation codon on the basis of the results of both primer extension and 5{prime}-rapid amplification of cDNA ends. NNMT mapped to chromosome band 11q23.1 by fluorescence in situ hybridization.

  12. Evolution of homologous sequences on the human X and Y chromosomes, outside of the meiotic pairing segment.

    OpenAIRE

    Bickmore, W A; Cooke, H J

    1987-01-01

    A sequence isolated from the long arm of the human Y chromosome detects a highly homologous locus on the X. This homology extends over at least 50 kb of DNA and is postulated to be the result of a transposition event between the X and Y chromosomes during recent human evolution, since homologous sequences are shown to be present on the X chromosome alone in the chimpanzee and gorilla.

  13. Thermolabile phenol sulfotransferase gene (STM): Localization to human chromosome 16p11.2

    Energy Technology Data Exchange (ETDEWEB)

    Aksoy, I.A.; Her, C.; Weinshilboum, M. [Mayo Medical School, Rochester, MN (United States)] [and others

    1994-09-01

    Thermolabile (TL) phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic monoamine neurotransmitters such as dopamine and serotonin. We recently cloned a cDNA for human liver TL PST and expressed it in COS-1 cells. We now report the chromosomal localization of the human TL PST gene (STM) as well as its partial sequence. DNA from NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panels 1 and 2 was screened by use of the PCR, and the STM gene was mapped to chromosome 16. Regional localization to 16p11.2 was performed by PCR analysis of a high-resolution mouse/human somatic cell hybrid panel that contained defined portions of human chromosome 16. 15 refs., 2 figs.

  14. Chromosomal Aberrations and DNA Repair Gene Variants in a Radon-exposed Population

    Energy Technology Data Exchange (ETDEWEB)

    Kiuru, A.; Lindholm, C.; Koivistoinen, A.; Salomaa, S.

    2004-07-01

    Polymorphisms of XRCC1 (X-ray repair cross-complementing group 1), XRCC3 (X-ray repair cross-complementing group 3), and hOGG1 (the human homologue of the yeast OGG1 gene) DNA repair genes have been associated with altered DNA repair capacity and risk of various cancers. In the present study our goal was to clarify the influence of various DNA repair gene variants on the frequency of chromosomal aberrations (CA) in subjects exposed to residential radon. The study group of 84 non-smoking, healthy individuals exposed to domestic radon were analysed using the fluorescence in-situ hybridization (FISH) technique. No association between radon concentration and CA frequencies was observed. However, a significant increase with age was shown as well as a large variability in translocation frequencies between individuals within the same age group. In order to investigate the role of individual susceptibility to this variation genotypes of DNA repair genes XRCC1 (codons 194, 280 and 399), XRCC3 (codon 241) and hOGG1 (codon 326) were determined from leukocyte DNA using methods based on polymerase chain reaction. Multiple regression analysis was applied to evaluate the effect of the polymorphisms and the other confounding factors (age, exposure to randon etc) to the frequency of CA. The preliminary statistical analyses showed that the different gene appeared not to be related to a pronounced increase in chromosome aberration frequencies observed by FISH painting. However, the analysis indicated that the homozygous variant of XRCC3 codon 241 was associated (P<0.05) with two-ways translocations in conjunction with age. Larger studies, both with regard to the cohort and the number of gene variants are needed to elucidate the influence of other DNA repair variants to the yield of chromosomal aberrations. The results indicate that the chromosomal translocations accumulated by age (spontaneous background) may be partly explained by defects in homologous recombination repair. (Author

  15. DNA-damage response during mitosis induces whole-chromosome missegregation.

    Science.gov (United States)

    Bakhoum, Samuel F; Kabeche, Lilian; Murnane, John P; Zaki, Bassem I; Compton, Duane A

    2014-11-01

    Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities. ©2014 American Association for Cancer Research.

  16. Large-scale reconstruction of 3D structures of human chromosomes from chromosomal contact data.

    Science.gov (United States)

    Trieu, Tuan; Cheng, Jianlin

    2014-04-01

    Chromosomes are not positioned randomly within a nucleus, but instead, they adopt preferred spatial conformations to facilitate necessary long-range gene-gene interactions and regulations. Thus, obtaining the 3D shape of chromosomes of a genome is critical for understanding how the genome folds, functions and how its genes interact and are regulated. Here, we describe a method to reconstruct preferred 3D structures of individual chromosomes of the human genome from chromosomal contact data generated by the Hi-C chromosome conformation capturing technique. A novel parameterized objective function was designed for modeling chromosome structures, which was optimized by a gradient descent method to generate chromosomal structural models that could satisfy as many intra-chromosomal contacts as possible. We applied the objective function and the corresponding optimization method to two Hi-C chromosomal data sets of both a healthy and a cancerous human B-cell to construct 3D models of individual chromosomes at resolutions of 1 MB and 200 KB, respectively. The parameters used with the method were calibrated according to an independent fluorescence in situ hybridization experimental data. The structural models generated by our method could satisfy a high percentage of contacts (pairs of loci in interaction) and non-contacts (pairs of loci not in interaction) and were compatible with the known two-compartment organization of human chromatin structures. Furthermore, structural models generated at different resolutions and from randomly permuted data sets were consistent.

  17. Independent intrachromosomal recombination events underlie the pericentric inversions of chimpanzee and gorilla chromosomes homologous to human chromosome 16.

    Science.gov (United States)

    Goidts, Violaine; Szamalek, Justyna M; de Jong, Pieter J; Cooper, David N; Chuzhanova, Nadia; Hameister, Horst; Kehrer-Sawatzki, Hildegard

    2005-09-01

    Analyses of chromosomal rearrangements that have occurred during the evolution of the hominoids can reveal much about the mutational mechanisms underlying primate chromosome evolution. We characterized the breakpoints of the pericentric inversion of chimpanzee chromosome 18 (PTR XVI), which is homologous to human chromosome 16 (HSA 16). A conserved 23-kb inverted repeat composed of satellites, LINE and Alu elements was identified near the breakpoints and could have mediated the inversion by bringing the chromosomal arms into close proximity with each other, thereby facilitating intrachromosomal recombination. The exact positions of the breakpoints may then have been determined by local DNA sequence homologies between the inversion breakpoints, including a 22-base pair direct repeat. The similarly located pericentric inversion of gorilla (GGO) chromosome XVI, was studied by FISH and PCR analysis. The p- and q-arm breakpoints of the inversions in PTR XVI and GGO XVI were found to occur at slightly different locations, consistent with their independent origin. Further, FISH studies of the homologous chromosomal regions in macaque and orangutan revealed that the region represented by HSA BAC RP11-696P19, which spans the inversion breakpoint on HSA 16q11-12, was derived from the ancestral primate chromosome homologous to HSA 1. After the divergence of orangutan from the other great apes approximately 12 million years ago (Mya), a duplication of the corresponding region occurred followed by its interchromosomal transposition to the ancestral chromosome 16q. Thus, the most parsimonious interpretation is that the gorilla and chimpanzee homologs exhibit similar but nonidentical derived pericentric inversions, whereas HSA 16 represents the ancestral form among hominoids.

  18. A worldwide phylogeography for the human X chromosome.

    Directory of Open Access Journals (Sweden)

    Simone S Santos-Lopes

    Full Text Available BACKGROUND: We reasoned that by identifying genetic markers on human X chromosome regions where recombination is rare or absent, we should be able to construct X chromosome genealogies analogous to those based on Y chromosome and mitochondrial DNA polymorphisms, with the advantage of providing information about both male and female components of the population. METHODOLOGY/PRINCIPAL FINDINGS: We identified a 47 Kb interval containing an Alu insertion polymorphism (DXS225 and four microsatellites in complete linkage disequilibrium in a low recombination rate region of the long arm of the human X chromosome. This haplotype block was studied in 667 males from the HGDP-CEPH Human Genome Diversity Panel. The haplotypic diversity was highest in Africa (0.992+/-0.0025 and lowest in the Americas (0.839+/-0.0378, where no insertion alleles of DXS225 were observed. Africa shared few haplotypes with other geographical areas, while those exhibited significant sharing among themselves. Median joining networks revealed that the African haplotypes were numerous, occupied the periphery of the graph and had low frequency, whereas those from the other continents were few, central and had high frequency. Altogether, our data support a single origin of modern man in Africa and migration to occupy the other continents by serial founder effects. Coalescent analysis permitted estimation of the time of the most recent common ancestor as 182,000 years (56,700-479,000 and the estimated time of the DXS225 Alu insertion of 94,400 years (24,300-310,000. These dates are fully compatible with the current widely accepted scenario of the origin of modern mankind in Africa within the last 195,000 years and migration out-of-Africa circa 55,000-65,000 years ago. CONCLUSIONS/SIGNIFICANCE: A haplotypic block combining an Alu insertion polymorphism and four microsatellite markers on the human X chromosome is a useful marker to evaluate genetic diversity of human populations and

  19. Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2----q26.3.

    Science.gov (United States)

    Astrin, K H; Warner, C A; Yoo, H W; Goodfellow, P J; Tsai, S F; Desnick, R J

    1991-05-01

    Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%-53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2----q26.3.

  20. Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes.

    Science.gov (United States)

    Stimpson, Kaitlin M; Song, Ihn Young; Jauch, Anna; Holtgreve-Grez, Heidi; Hayden, Karen E; Bridger, Joanna M; Sullivan, Beth A

    2010-08-12

    Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the alpha-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same alpha-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.

  1. Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes.

    Directory of Open Access Journals (Sweden)

    Kaitlin M Stimpson

    2010-08-01

    Full Text Available Genome rearrangement often produces chromosomes with two centromeres (dicentrics that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the alpha-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same alpha-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.

  2. Nuclear DNA content and chromosome number in Brachiaria spp. genotypes

    Directory of Open Access Journals (Sweden)

    Ana Luiza de Oliveira Timbó

    Full Text Available Breeding programs for Brachiaria spp. use both intraspecies and interspecies crosses between sexual and apomictic plants in order to obtain new cultivars with the desired characteristics. As there are different ploidy levels both within and between species of this genus, it becomes necessary to evaluate the genotypes used in breeding programs, as a guide to breeders when adopting crossing strategies. In this work, DNA content and chromosome number were determined in order to characterise ploidy levels in Brachiaria spp. genotypes. In the analysis of 15 genotypes, DNA content varied with the ploidy levels (2x, 3x and 4x, and between species and/or taxon. The average DNA content was 1.74 pg (2x in B. ruziziensis, 3.74 pg (4x in B. decumbens and 3.52 pg (4x for B. brizantha. For the genotype 86, 2.57 pg of DNA was obtained and 2n = 3x = 27, indicating a triploid accession, probably a natural hybrid. The variation in the total DNA content allowed the differentiation of Brachiaria ruziziensis (2n = 2x = 18 from the tetraploid species Brachiaria Brizantha and Brachiaria decumbens (2n = 4x = 36, as well as the probable hybrid triploid (2n = 3x = 18 of these species.

  3. Chromosome region-specific libraries for human genome analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kao, Fa-Ten.

    1991-01-01

    We have made important progress since the beginning of the current grant year. We have further developed the microdissection and PCR- assisted microcloning techniques using the linker-adaptor method. We have critically evaluated the microdissection libraries constructed by this microtechnology and proved that they are of high quality. We further demonstrated that these microdissection clones are useful in identifying corresponding YAC clones for a thousand-fold expansion of the genomic coverage and for contig construction. We are also improving the technique of cloning the dissected fragments in test tube by the TDT method. We are applying both of these PCR cloning technique to human chromosomes 2 and 5 to construct region-specific libraries for physical mapping purposes of LLNL and LANL. Finally, we are exploring efficient procedures to use unique sequence microclones to isolate cDNA clones from defined chromosomal regions as valuable resources for identifying expressed gene sequences in the human genome. We believe that we are making important progress under the auspices of this DOE human genome program grant and we will continue to make significant contributions in the coming year. 4 refs., 4 figs.

  4. Poetry in motion: Increased chromosomal mobility after DNA damage.

    Science.gov (United States)

    Smith, Michael J; Rothstein, Rodney

    2017-08-01

    Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is homologous recombination (HR), in which information from an undamaged donor site is used as a template for repair. Although many of the biochemical steps of HR are known, the physical movements of chromosomes that must underlie the pairing of homologous sequence during mitotic DSB repair have remained mysterious. Recently, several groups have begun to use a variety of genetic and cell biological tools to study this important question. These studies reveal that both damaged and undamaged loci increase the volume of the nuclear space that they explore after the formation of DSBs. This DSB-induced increase in chromosomal mobility is regulated by many of the same factors that are important during HR, such as ATR-dependent checkpoint activation and the recombinase Rad51, suggesting that this phenomenon may facilitate the search for homology. In this perspective, we review current research into the mobility of chromosomal loci during HR, as well as possible underlying mechanisms, and discuss the critical questions that remain to be answered. Although we focus primarily on recent studies in the budding yeast, Saccharomyces cerevisiae, examples of experiments performed in higher eukaryotes are also included, which reveal that increased mobility of damaged loci is a process conserved throughout evolution. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. 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.

  6. Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene

    Energy Technology Data Exchange (ETDEWEB)

    Tebbs, R.S.; Tucker, J.D.; Hwang, M. [Lawrence Livermore National Lab., CA (United States)] [and others

    1995-07-03

    The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to displatin and {gamma}-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is {approx} 2.5 kb and detects an {approx} 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels. 30 refs., 5 figs., 2 tabs.

  7. Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes.

    Science.gov (United States)

    Holubcová, Zuzana; Blayney, Martyn; Elder, Kay; Schuh, Melina

    2015-06-05

    Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg's progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. We analyzed meiosis in more than 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as a major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs.

  8. Intercalation of psoralen into DNA of plastid chromosomes decreases late during barley chloroplast development.

    OpenAIRE

    Davies, J. P.; Thompson, R.J.; Mosig, G

    1991-01-01

    We have used a DNA crosslinking assay to measure intercalation of the psoralen derivative HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into barley (Hordeum vulgare) plastid chromosomal DNA during chloroplast and etioplast development. Intercalation into DNA in intact plastids in vivo and in plastid lysates in vitro shows that chromosomal DNA in the most mature chloroplasts intercalates HMT less efficiently than DNA in younger chloroplasts. In contrast, there is no change in HMT intercalati...

  9. Scanning conductance microscopy investigations on fixed human chromosomes

    DEFF Research Database (Denmark)

    Clausen, Casper Hyttel; Lange, Jacob Moresco; Jensen, Linda Boye;

    2008-01-01

    Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device...... optimization. The phase shift caused by the electrostatic forces, together with geometrical measurements of the atomic force microscopy (AFM) cantilever and the chromosomes were used to estimate a value,for the dielectric constant of different human chromosomes....

  10. Cloning of a cDNA encoding a putative human very low density lipoprotein/Apolipoprotein E receptor and assignment of the gene to chromosome 9pter-p23[sup 6

    Energy Technology Data Exchange (ETDEWEB)

    Gafvels, M.E.; Strauss, J.F. III (Univ. of Pennyslvania, Philadelphia, PA (United States)); Caird, M.; Patterson, D. (Eleanor Roosevelt Institute, Denver, CO (United States)); Britt, D.; Jackson, C.L. (Brown Univ., Providence, RI (United States))

    1993-11-01

    The authors report the cloning of a 3656-bp cDNA encoding a putative human very low density lipoprotein (VLDL)/apolipoprotein E (ApoE) receptor. The gene encoding this protein was mapped to chromosome 9pter-p23. Northern analysis of human RNA identified cognate mRNAs of 6.0 and 3.8 kb with most abundant expression in heart and skeletal muscle, followed by kidney, placenta, pancreas, and brain. The pattern of expression generally paralleled that of lipoprotein lipase mRNA but differed from that of the low density lipoprotein (LDL) receptor and the low density lipoprotein receptor-related protein/[alpha][sub 2]-macroglobulin receptor (LRP), which are members of the same gene family. VLDL/ApoE receptor message was not detected in liver, whereas mRNAs for both LDL receptor and LRP were found in hepatic tissue. In mouse 3T3-L1 cells, VLDL/ApoE receptor mRNA was induced during the transformation of the cells into adipocytes. Expression was also detected in human choriocarcinoma cells, suggesting that at least part of the expression observed in placenta may be in trophoblasts, cells which would be exposed to maternal blood. Expression in brain may be related to high levels of ApoE expression in that organ, an observation of potential relevance to the recently hypothesized role for ApoE in late onset Alzheimer disease. The results suggest that the putative VLDL/ApoE receptor could play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs including striated and cardiac muscle and adipose tissue and in the transport of maternal lipids across the placenta. The findings presented here, together with recent observations from other laboratories, bring up the possibility that a single gene, the VLDL/ApoE receptor, may play a role in the pathogenesis of certain forms of atherosclerosis, Alzheimer disease, and obesity.

  11. Chromosomal patterns in human malignant astrocytomas.

    Science.gov (United States)

    Rey, J A; Bello, M J; de Campos, J M; Kusak, M E; Ramos, C; Benitez, J

    1987-12-01

    Cytogenetic analysis by direct and/or in vitro preparations was performed on 34 malignant astrocytomas. Thirty tumors showed near-diploid chromosome numbers, whereas, tritetraploid chromosome complements were present in four tumors. The most frequent chromosomal changes implied numerical deviations by a gain of chromosomes #7, #19, and #20, and by losses of #10, #22, and Y. Structural rearrangements were present in stem- or side lines of 24 tumors. Although no common chromosomal rearrangement seems to exist among those tumors, chromosomes #1, #6, #7, and #9 were predominantly involved. Polysomy and structural rearrangements of chromosome #7 could be related to the overexpression of epidermal growth factor gene, previously observed in some malignant gliomas.

  12. Human-chromosome alterations induced by argon laser treatment

    Energy Technology Data Exchange (ETDEWEB)

    Simi, S.; Colella, C. (Consiglio Nazionale delle Ricerche, Pisa (Italy). Lab. di Mutagenesi e Differenziamento); Agati, G.; Fusi, F. (Florence Univ. (Italy). Ist. di Farmacologia); Corsi, M.F.; Pratesi, R. (Consiglio Nazionale delle Ricerche, Florence (Italy). Lab. di Elettronica Quantistica); Tocco, G.A. (Naples Univ. (Italy). Ist. di Istologia ed Embrilogia)

    1984-07-01

    The possible occurrence of genetic damage arising from exposure of human cells to visible laser light has been evaluated in PHA-stimulated human lymphocytes. Aneuploidy and chromosome aberrations have been observed after exposure to an argon laser. These findings appear of special interest in view of the possible role of these chromosome alterations in carcinogenesis.

  13. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

    Science.gov (United States)

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

    De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

  14. Pre-cancerous (DNA and chromosomal) lesions in professional sports.

    Science.gov (United States)

    Sharma, Radhika; Gandhi, Gursatej

    2012-01-01

    Exhaustive exercises may become detrimental, causing disturbance of intracellular oxidant-antioxidant balance and damage to macromolecules, leading to genomic instability when DNA/chromosomes get damaged. As these are precancerous lesions, cancer occurrence is probable. Because professional sports requires high-intensity training and increasing physical demand, there may occur cellular genomic instability. To evaluate genetic damage at DNA and chromosomal levels in hockey and baseball-soft ball players and compare with levels in age- and sex-matched sedentary controls. Players professionally active in their sport from 3 to 11 years on a daily training session of 4h/day were contacted during their camps, and the study was approved by the Institutional Ethics Committee. All participants were healthy individuals, not on any medication and were not taking any supplements. Genetic damage using the single cell gel electrophoresis assay and buccal micronucleus cytome assay of 56 individuals (36 players and 20 controls) was evaluated. Student's t -test, ANOVA, Pearson correlation and linear regression and Chi-square analysis were performed. Players had significantly elevated levels of genetic damage. There were no gender differences and also no significant difference in the genetic damage incurred in both sports types. However, the extent of DNA migration in hockey players was higher. Significantly increased genomic instability in players of both sports was observed. Both repaired and repairable genetic damage cells were observed in different tissues of the same subject. The presence of such genetic damage implies that these players are at an individual risk from cancer- and age-related diseases.

  15. Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach.

    Science.gov (United States)

    Liu, J; Stanton, V P; Fujiwara, T M; Wang, J X; Rezonzew, R; Crumley, M J; Morgan, K; Gros, P; Housman, D; Schurr, E

    1995-03-20

    We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomic DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostly derived from the CEPH Mark I library. Alu-element-mediated PCR was performed for each pool, and amplification products were spotted on hybridization membranes (IRS filters). IRS probes for the screening of the IRS filters were obtained by Alu-element-mediated PCR. Of 708 distinct probes obtained from chromosome 2-specific somatic cell hybrids, 85% were successfully used for library screening. Similarly, 80% of 80 YAC walking probes were successfully used for library screening. Each probe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preliminary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric half of chromosome 2q was considered to be good since 31 of 34 microsatellites and 22 of 23 expressed sequence tags that were chosen from chromosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrary generated by our experiments. We have identified a minimum of 1610 distinct chromosome 2-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome.

  16. Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

    Directory of Open Access Journals (Sweden)

    Paric Enesa

    2003-10-01

    Full Text Available Abstract Background Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci. Results Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal. Conclusion These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres.

  17. Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase.

    Science.gov (United States)

    Schawalder, James; Paric, Enesa; Neff, Norma F

    2003-10-27

    Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci. Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal. These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres.

  18. Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes.

    Science.gov (United States)

    Wu, LiHong; Liu, Yang; Kong, DaoChun

    2014-05-01

    Chromosomal DNA replication is one of the central biological events occurring inside cells. Due to its large size, the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time. Further, eukaryotic DNA replication is sophisticatedly regulated, and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle. The first step of replication initiation is the assembly of pre-replication complex (pre-RC). Since 1973, four proteins, Cdc6/Cdc18, MCM, ORC and Cdt1, have been extensively studied and proved to be pre-RC components. Recently, a novel pre-RC component called Sap1/Girdin was identified. Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells, respectively. At the transition of G1 to S phase, pre-RC is activated by the two kinases, cyclindependent kinase (CDK) and Dbf4-dependent kinase (DDK), and subsequently, RPA, primase-polα, PCNA, topoisomerase, Cdc45, polδ, and polɛ are recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication. As replication forks move along chromatin DNA, they frequently stall due to the presence of a great number of replication barriers on chromatin DNA, such as secondary DNA structures, protein/DNA complexes, DNA lesions, gene transcription. Stalled forks must require checkpoint regulation for their stabilization. Otherwise, stalled forks will collapse, which results in incomplete DNA replication and genomic instability. This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.

  19. 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.

  20. Chromosomal localization of the human apolipoprotein B gene and detection of homologous RNA in monkey intestine

    Energy Technology Data Exchange (ETDEWEB)

    Deeb, S.S.; Disteche, C.; Motulsky, A.G.; Lebo, R.V.; Kan, Y.W.

    1986-01-01

    A cDNA clone of the human apolipoprotein B-100 was used as a hybridization probe to detect homologous sequences in both flow-sorted and in situ metaphase chromosomes. The results indicate that the gene encoding this protein is on the distal end of the short arm of chromosome 2 (2p23-2p24). RNA isolated from monkey small intestine contained sequences (6.5 and 18 kilobases) homologous to the cDNA of apolipoprotein B-100. These results are consistent with the hypothesis that one gene codes for both the intestinal (B-48) and the hepatic (B-100) forms.

  1. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes.

    Science.gov (United States)

    Estandarte, Ana Katrina; Botchway, Stanley; Lynch, Christophe; Yusuf, Mohammed; Robinson, Ian

    2016-08-16

    Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore's fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies.

  2. Chromosomal instability in cell-free DNA is a serum biomarker for prostate cancer.

    Science.gov (United States)

    Schütz, Ekkehard; Akbari, Mohammad R; Beck, Julia; Urnovitz, Howard; Zhang, William W; Bornemann-Kolatzki, Kirsten; Mitchell, William M; Nam, Robert K; Narod, Steven A

    2015-01-01

    Genomic instability resulting in copy number variation is a hallmark of malignant transformation and may be identified through massive parallel sequencing. Tumor-specific cell free DNA (cfDNA) present in serum and plasma provides a real-time, easily accessible surrogate. DNA was extracted from serum of 204 patients with prostate cancer (Gleason score 2-10), 207 male controls, and patients with benign hyperplasia (n = 10) and prostatitis (n = 10). DNA was amplified by use of random primers, tagged with molecular identifiers, sequenced on a SOLID system, and aligned to the human genome. We evaluated the number of sequence reads of cfDNA in sliding 100-kbp intervals for variation from controls. We used chromosomal regions with significant variations in alignment hits for their ability to segregate patients and matched controls. Using ROC curves to assess diagnostic performance, we evaluated the number of regions in a first subset (n = 177), with variations in alignment hits alone, provided an area under the curve (AUC) of 0.81 (95% CI 0.7-0.9, P prostate cancer from controls with an AUC of 0.92 (0.87-0.95), reaching a diagnostic accuracy of 83%. Both benign prostatic hypertrophy and prostatitis could be distinguished from prostate cancer by use of cfDNA, with an accuracy of 90%. Assessment of a limited number of chromosomal structural instabilities by use of massive parallel sequencing of cfDNA was sufficient to distinguish between prostate cancer and controls. This large cohort demonstrates the utility of cfDNA in prostate cancer recently established in other malignant neoplasms. © 2014 American Association for Clinical Chemistry.

  3. Roles of the Y chromosome genes in human cancers

    Directory of Open Access Journals (Sweden)

    Tatsuo Kido

    2015-06-01

    Full Text Available Male and female differ genetically by their respective sex chromosome composition, that is, XY as male and XX as female. Although both X and Y chromosomes evolved from the same ancestor pair of autosomes, the Y chromosome harbors male-specific genes, which play pivotal roles in male sex determination, germ cell differentiation, and masculinization of various tissues. Deletions or translocation of the sex-determining gene, SRY, from the Y chromosome causes disorders of sex development (previously termed as an intersex condition with dysgenic gonads. Failure of gonadal development results not only in infertility, but also in increased risks of germ cell tumor (GCT, such as gonadoblastoma and various types of testicular GCT. Recent studies demonstrate that either loss of Y chromosome or ectopic expression of Y chromosome genes is closely associated with various male-biased diseases, including selected somatic cancers. These observations suggest that the Y-linked genes are involved in male health and diseases in more frequently than expected. Although only a small number of protein-coding genes are present in the male-specific region of Y chromosome, the impacts of Y chromosome genes on human diseases are still largely unknown, due to lack of in vivo models and differences between the Y chromosomes of human and rodents. In this review, we highlight the involvement of selected Y chromosome genes in cancer development in men.

  4. Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation.

    Science.gov (United States)

    Mera, Paola E; Kalogeraki, Virginia S; Shapiro, Lucy

    2014-11-11

    During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation.

  5. Influence of the presence of B chromosomes on DNA damage in Crepis capillaris.

    Directory of Open Access Journals (Sweden)

    Jolanta Kwasniewska

    Full Text Available The sensitivity of different plant species to mutagenic agents is related to the DNA content and organization of the chromatin, which have been described in ABCW and bodyguard hypotheses, respectively. Plant species that have B chromosomes are good models for the study of these hypotheses. This study presents an analysis of the correlation between the occurrence of B chromosomes and the DNA damage that is induced by the chemical mutagen, maleic hydrazide (MH, in Crepis capillaris plants using comet assay. The presence of B chromosomes has a detectable impact on the level of DNA damage. The level of DNA damage after MH treatment was correlated with the number of B chromosomes and it was observed that it increased significantly in plants with 3B chromosomes. We did not find evidence of the protective role from chemical mutagens of the constitutive heterochromatin for euchromatin in relation to DNA damage. The DNA damage involving the 25S rDNA sequences was analyzed using the comet-FISH technique. Fragmentation within or near the 25S rDNA involved the loci on the A and B chromosomes. The presence of B chromosomes in C. capillaris cells had an influence on the level of DNA damage that involves the 25S rDNA region.

  6. Nonrandom involvement of chromosomal segments in human hematologic malignancies

    Energy Technology Data Exchange (ETDEWEB)

    Rowley, J. D.

    1977-01-01

    The consistent occurrence of nonrandom chromosome changes in human malignancies suggests that they are not trivial epiphenomena. Whereas we do not understand their significance at present, one possible role which they may fulfill is to provide the chromosomally aberrant cells with a proliferative advantage as the result of alteration of the number and/or location of genes related to nucleic acid biosynthesis. It would be expected that the proliferative advantage provided by various chromosome aberrations differs in patients with different genetic constitutions.

  7. DNA Commission of the International Society of Forensic Genetics: recommendations on forensic analysis using Y-chromosome short tandem repeats

    DEFF Research Database (Denmark)

    Gill, P.; Brenner, C.; Brinkmann, B.;

    2001-01-01

    During the past few years the DNA commission of the International Society of Forensic Genetics has published a series of documents providing guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. This latest report addresses a relat...... a relatively new area, namely Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). This report addresses nomenclature, use of allelic ladders, population genetics and reporting methods Udgivelsesdato: 2001/12...

  8. Chromosomal locations of four minor rDNA loci and a marker microsatellite sequence in barley

    DEFF Research Database (Denmark)

    Pedersen, C.; Linde-Laursen, I.

    1994-01-01

    is located about 54% out on the short arm of chromosome 4 and it has not previously been reported in barley. We have designated the new locus Nor-I6. rDNA loci on homoeologous group 4 chromosomes have not yet been reported in other Triticeae species. The origin of these 4 minor rDNA loci is discussed...

  9. Human male meiotic sex chromosome inactivation

    NARCIS (Netherlands)

    Vries, M. de; Vosters, S.; Merkx, G.F.M.; Hauwers, K.W.M. d'; Wansink, D.G.; Ramos, L.; Boer, P. de

    2012-01-01

    In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylate

  10. Human male meiotic sex chromosome inactivation

    NARCIS (Netherlands)

    Vries, M. de; Vosters, S.; Merkx, G.F.M.; Hauwers, K.W.M. d'; Wansink, D.G.; Ramos, L.; Boer, P. de

    2012-01-01

    In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly

  11. Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways

    OpenAIRE

    Ichijima, Yosuke; Sin, Ho-Su; Satoshi H Namekawa

    2012-01-01

    Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX). This DDR pathway shares features with the somatic DDR p...

  12. A Model of DNA Repeat-Assembled Mitotic Chromosomal Skeleton

    OpenAIRE

    Shao-Jun Tang

    2011-01-01

    Despite intensive investigation for decades, the principle of higher-order organization of mitotic chromosomes is unclear. Here, I describe a novel model that emphasizes a critical role of interactions of homologous DNA repeats (repetitive elements; repetitive sequences) in mitotic chromosome architecture. According to the model, DNA repeats are assembled, via repeat interactions (pairing), into compact core structures that govern the arrangement of chromatins in mitotic chromosomes. Tandem r...

  13. Conserved synteny between pig chromosome 8 and human chromosome 4 but rearranged and distorted linkage maps

    Energy Technology Data Exchange (ETDEWEB)

    Ellegren, H.; Edfors-Lilja, I.; Anderson, L. (Swedish Univ. of Agricultural Sciences, Uppsala (Sweden)); Wintero, A.K. (Royal Veterinary and Agricultural Univ., Fredriksberg (Denmark))

    1993-09-01

    The porcine genes encoding interleukin 2, alcohol dehydrogenase (class I) gamma polypeptide, and osteopontin were mapped to chromosome 8 by linkage analysis. Together with previous assignments to this chromosome (the albumin, platelet-derived growth factor receptor A, and fibrinogen genes), an extensive syntenic homology with human chromosome 4 was discovered. Loci from about three-quarters of the q arm of human chromosome 4 are on pig chromosome 8. However, the linear order of the markers is not identical in the two species, and there are several examples of interspecific differences in the recombination fractions between adjacent markers. The conserved synteny between man and the pig gives strong support to a previous suggestion that a synteny group present in the ancestor of mammalian species has been retained on human chromosome 4q. Since loci from this synteny group are found on two cattle chromosomes, the bovine rearrangement must have occurred after the split of Suidae and Bovidae within Artiodactyla. 29 refs., 3 figs., 1 tab.

  14. Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

    Directory of Open Access Journals (Sweden)

    Heinz-Ulrich G. Weier

    2012-12-01

    Full Text Available Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols.

  15. Deficit of mitonuclear genes on the human X chromosome predates sex chromosome formation.

    Science.gov (United States)

    Dean, Rebecca; Zimmer, Fabian; Mank, Judith E

    2015-01-29

    Two taxa studied to date, the therian mammals and Caenorhabditis elegans, display underrepresentations of mitonuclear genes (mt-N genes, nuclear genes whose products are imported to and act within the mitochondria) on their X chromosomes. This pattern has been interpreted as the result of sexual conflict driving mt-N genes off of the X chromosome. However, studies in several other species have failed to detect a convergent biased distribution of sex-linked mt-N genes, leading to questions over the generality of the role of sexual conflict in shaping the distribution of mt-N genes. Here we tested whether mt-N genes moved off of the therian X chromosome following sex chromosome formation, consistent with the role of sexual conflict, or whether the paucity of mt-N genes on the therian X is a chance result of an underrepresentation on the ancestral regions that formed the X chromosome. We used a synteny-based approach to identify the ancestral regions in the platypus and chicken genomes that later formed the therian X chromosome. We then quantified the movement of mt-N genes on and off of the X chromosome and the distribution of mt-N genes on the human X and ancestral X regions. We failed to find an excess of mt-N gene movement off of the X. The bias of mt-N genes on ancestral therian X chromosomes was also not significantly different from the biases on the human X. Together our results suggest that, rather than conflict driving mt-N genes off of the mammalian X, random biases on chromosomes that formed the X chromosome could explain the paucity of mt-N genes in the therian lineage.

  16. Chromosomal mosaicism in human preimplantation embryos : a systematic review

    NARCIS (Netherlands)

    van Echten-Arends, Jannie; Mastenbroek, Sebastiaan; Sikkema-Raddatz, Birgit; Korevaar, Johanna C.; Heineman, Maas Jan; van der Veen, Fulco; Repping, Sjoerd

    2011-01-01

    BACKGROUND: Although chromosomal mosaicism in human preimplantation embryos has been described for almost two decades, its exact prevalence is still unknown. The prevalence of mosaicism is important in the context of preimplantation genetic screening in which the chromosomal status of an embryo is d

  17. Chromosomal mosaicism in human preimplantation embryos: a systematic review.

    NARCIS (Netherlands)

    Echten-Arends, J. van; Mastenbroek, S.; Sikkema-Raddatz, B.; Korevaar, J.C.; Heineman, M.J.; Veen, F. van der; Repping, S.

    2011-01-01

    BACKGROUND: Although chromosomal mosaicism in human preimplantation embryos has been described for almost two decades, its exact prevalence is still unknown. The prevalence of mosaicism is important in the context of preimplantation genetic screening in which the chromosomal status of an embryo is d

  18. DNA methylation analysis of chromosome 21 gene promoters at single base pair and single allele resolution.

    Directory of Open Access Journals (Sweden)

    Yingying Zhang

    2009-03-01

    Full Text Available Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii methylation levels of individual cells in one tissue are very similar, and iii methylation patterns follow a relaxed site-specific distribution. Furthermore, iv we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene

  19. MAOA and GYG2 are submitted to X chromosome inactivation in human fibroblasts.

    Science.gov (United States)

    Stabellini, Raquel; Vasques, Luciana R; de Mello, Joana Carvalho Moreira; Hernandes, Lys Molina; Pereira, Lygia V

    2009-08-16

    X chromosome inactivation (XCI) is a comprehensively studied phenomenon that helped to highlight the heritable nature of epigenetic modifications. Although it consists of the transcriptional inactivation of a whole X chromosome in females, some genes escape this process and present bi-allelic expression. Using human fibroblasts with skewed inactivation, we determined allele-specific expression of two X-linked genes previously described to escape XCI in rodent/human somatic cell hybrids, MAOA and GYG2, and the pattern of DNA methylation of their 5' end. Results from these complementary methodologies let us to conclude that both genes are subjected to X inactivation in normal human fibroblasts, indicating that hybrid cells are not an adequate system for studying epigenotypes. We emphasize the need of an analysis of XCI in normal human cell lines, helping us to determine more precisely which X-linked genes contribute to differences among genders and to the phenotypes associated with sex chromosomes aneuploidies.

  20. Absence of Y chromosome in human placental site trophoblastic tumor.

    Science.gov (United States)

    Hui, Pei; Wang, Hanlin L; Chu, Peiguo; Yang, Bin; Huang, Jiaoti; Baergen, Rebecca N; Sklar, Jeffrey; Yang, Ximing J; Soslow, Robert A

    2007-10-01

    Placental site trophoblastic tumor is a neoplasm of extravillous intermediate trophoblast at the implantation site, preceded in the majority of cases by a female gestational event. Our pilot investigation suggested that the development of this tumor might require a paternally derived X chromosome and the absence of a Y chromosome. Twenty cases of placental site trophoblastic tumor were included in this study. Genotyping at 15 polymorphic loci and one sex determination locus was performed by multiplex PCR followed by capillary electrophoresis. X chromosome polymorphisms were determined by PCR amplification of exon 1 of the human androgen receptor gene using primers flanking the polymorphic CAG repeats within this region. Genotyping at 15 polymorphic loci was informative and paternal alleles were present in all tumors, confirming the trophoblastic origin of the tumors. The presence of an X chromosome and the absence of a Y chromosome were observed in all tumors. Among 13 cases in which analysis of the X chromosome polymorphism was informative, all but one demonstrated at least two X alleles and seven cases showed one identifiable paternal X allele. These results confirm a unique pathogenetic mechanism in placental site trophoblastic tumor, involving an exclusion of the Y chromosome from the genome and, therefore, a tumor arising from the trophectoderm of a female conceptus. As epigenetic regulations of imprinting during X chromosome inactivation are of significant biological implications, placental site trophoblastic tumor may provide an important model for studying the sex chromosome biology and the proliferative advantage conferred by the paternal X chromosome.

  1. Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes.

    Directory of Open Access Journals (Sweden)

    Duílio M Z de A Silva

    Full Text Available Supernumerary (B chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.

  2. Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes).

    Science.gov (United States)

    Silva, Duílio M Z de A; Pansonato-Alves, José Carlos; Utsunomia, Ricardo; Araya-Jaime, Cristian; Ruiz-Ruano, Francisco J; Daniel, Sandro Natal; Hashimoto, Diogo Teruo; Oliveira, Cláudio; Camacho, Juan Pedro M; Porto-Foresti, Fábio; Foresti, Fausto

    2014-01-01

    Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.

  3. Palindrome-mediated Translocations in Humans: A New Mechanistic Model for Gross Chromosomal Rearrangements

    Directory of Open Access Journals (Sweden)

    Hidehito Inagaki

    2016-07-01

    Full Text Available Palindromic DNA sequences, which can form secondary structures, are widely distributed in the human genome. Although the nature of the secondary structure—single-stranded hairpin or double-stranded cruciform—has been extensively investigated in vitro, the existence of such unusual non-B DNA in vivo remains controversial. Here, we review palindrome-mediated gross chromosomal rearrangements possibly induced by non-B DNA in humans. Recent advances in next-generation sequencing have not yet overcome the difficulty of palindromic sequence analysis. However, a dozen palindromic AT-rich repeat (PATRR sequences have been identified at the breakpoints of recurrent or non-recurrent chromosomal translocations in humans. The breakages always occur at the center of the palindrome. Analyses of polymorphisms within the palindromes indicate that the symmetry and length of the palindrome affect the frequency of the de novo occurrence of these palindrome-mediated translocations, suggesting the involvement of non-B DNA. Indeed, experiments using a plasmid-based model system showed that the formation of non-B DNA is likely the key to palindrome-mediated genomic rearrangements. Some evidence implies a new mechanism that cruciform DNAs may come close together first in nucleus and illegitimately joined. Analysis of PATRR-mediated translocations in humans will provide further understanding of gross chromosomal rearrangements in many organisms.

  4. Genomic structure and paralogous regions of the inversion breakpoint occurring between human chromosome 3p12.3 and orangutan chromosome 2.

    Science.gov (United States)

    Yue, Y; Grossmann, B; Tsend-Ayush, E; Grützner, F; Ferguson-Smith, M A; Yang, F; Haaf, T

    2005-01-01

    Intrachromosomal duplications play a significant role in human genome pathology and evolution. To better understand the molecular basis of evolutionary chromosome rearrangements, we performed molecular cytogenetic and sequence analyses of the breakpoint region that distinguishes human chromosome 3p12.3 and orangutan chromosome 2. FISH with region-specific BAC clones demonstrated that the breakpoint-flanking sequences are duplicated intrachromosomally on orangutan 2 and human 3q21 as well as at many pericentromeric and subtelomeric sites throughout the genomes. Breakage and rearrangement of the human 3p12.3-homologous region in the orangutan lineage were associated with a partial loss of duplicated sequences in the breakpoint region. Consistent with our FISH mapping results, computational analysis of the human chromosome 3 genomic sequence revealed three 3p12.3-paralogous sequence blocks on human chromosome 3q21 and smaller blocks on the short arm end 3p26-->p25. This is consistent with the view that sequences from an ancestral site at 3q21 were duplicated at 3p12.3 in a common ancestor of orangutan and humans. Our results show that evolutionary chromosome rearrangements are associated with microduplications and microdeletions, contributing to the DNA differences between closely related species. Copyright (c) 2005 S. Karger AG, Basel.

  5. Repair-mediated duplication by capture of proximal chromosomal DNA has shaped vertebrate genome evolution.

    Directory of Open Access Journals (Sweden)

    John K Pace

    2009-05-01

    Full Text Available DNA double-strand breaks (DSBs are a common form of cellular damage that can lead to cell death if not repaired promptly. Experimental systems have shown that DSB repair in eukaryotic cells is often imperfect and may result in the insertion of extra chromosomal DNA or the duplication of existing DNA at the breakpoint. These events are thought to be a source of genomic instability and human diseases, but it is unclear whether they have contributed significantly to genome evolution. Here we developed an innovative computational pipeline that takes advantage of the repetitive structure of genomes to detect repair-mediated duplication events (RDs that occurred in the germline and created insertions of at least 50 bp of genomic DNA. Using this pipeline we identified over 1,000 probable RDs in the human genome. Of these, 824 were intra-chromosomal, closely linked duplications of up to 619 bp bearing the hallmarks of the synthesis-dependent strand-annealing repair pathway. This mechanism has duplicated hundreds of sequences predicted to be functional in the human genome, including exons, UTRs, intron splice sites and transcription factor binding sites. Dating of the duplication events using comparative genomics and experimental validation revealed that the mechanism has operated continuously but with decreasing intensity throughout primate evolution. The mechanism has produced species-specific duplications in all primate species surveyed and is contributing to genomic variation among humans. Finally, we show that RDs have also occurred, albeit at a lower frequency, in non-primate mammals and other vertebrates, indicating that this mechanism has been an important force shaping vertebrate genome evolution.

  6. Replication Banding Patterns in Human Chromosomes Detected Using 5-ethynyl-2'-deoxyuridine Incorporation

    OpenAIRE

    Hoshi, Osamu; Ushiki, Tatsuo

    2011-01-01

    A novel technique using the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acety...

  7. Fragile sites, dysfunctional telomere and chromosome fusions: What is 5S rDNA role?

    Science.gov (United States)

    Barros, Alain Victor; Wolski, Michele Andressa Vier; Nogaroto, Viviane; Almeida, Mara Cristina; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo

    2017-04-15

    Repetitive DNA regions are known as fragile chromosomal sites which present a high flexibility and low stability. Our focus was characterize fragile sites in 5S rDNA regions. The Ancistrus sp. species shows a diploid number of 50 and an indicative Robertsonian fusion at chromosomal pair 1. Two sequences of 5S rDNA were identified: 5S.1 rDNA and 5S.2 rDNA. The first sequence gathers the necessary structures to gene expression and shows a functional secondary structure prediction. Otherwise, the 5S.2 rDNA sequence does not contain the upstream sequences that are required to expression, furthermore its structure prediction reveals a nonfunctional ribosomal RNA. The chromosomal mapping revealed several 5S.1 and 5S.2 rDNA clusters. In addition, the 5S.2 rDNA clusters were found in acrocentric and metacentric chromosomes proximal regions. The pair 1 5S.2 rDNA cluster is co-located with interstitial telomeric sites (ITS). Our results indicate that its clusters are hotspots to chromosomal breaks. During the meiotic prophase bouquet arrangement, double strand breaks (DSBs) at proximal 5S.2 rDNA of acrocentric chromosomes could lead to homologous and non-homologous repair mechanisms as Robertsonian fusions. Still, ITS sites provides chromosomal instability, resulting in telomeric recombination via TRF2 shelterin protein and a series of breakage-fusion-bridge cycles. Our proposal is that 5S rDNA derived sequences, act as chromosomal fragile sites in association with some chromosomal rearrangements of Loricariidae. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Maternal uniparental disomy for human chromosome 14, due to loss of a chromosome 14 from somatic cells with t(13;14) trisomy 14.

    Science.gov (United States)

    Antonarakis, S E; Blouin, J L; Maher, J; Avramopoulos, D; Thomas, G; Talbot, C C

    1993-06-01

    Uniparental disomy (UPD) for particular chromosomes is increasingly recognized as a cause of abnormal phenotypes in humans. We recently studied a 9-year-old female with a de novo Robertsonian translocation t(13;14), short stature, mild developmental delay, scoliosis, hyperextensible joints, hydrocephalus that resolved spontaneously during the first year of life, and hypercholesterolemia. To determine the parental origin of chromosomes 13 and 14 in the proband, we have studied the genotypes of DNA polymorphic markers due to (GT)n repeats in the patient and her parents' blood DNA. The genotypes of markers D14S43, D14S45, D14S49, and D14S54 indicated maternal UPD for chromosome 14. There was isodisomy for proximal markers and heterodisomy for distal markers, suggesting a recombination event on maternal chromosomes 14. In addition, DNA analysis first revealed--and subsequent cytogenetic analysis confirmed--that there was mosaic trisomy 14 in 5% of blood lymphocytes. There was normal (biparental) inheritance for chromosome 13, and there was no evidence of false paternity in genotypes of 11 highly polymorphic markers on human chromosome 21. Two cases of maternal UPD for chromosome 14 have previously been reported, one with a familial rob t(13;14) and the other with a t(14;14). There are several similarities among these patients, and a "maternal UPD chromosome 14 syndrome" is emerging; however, the contribution of the mosaic trisomy 14 to the phenotype cannot be evaluated. The study of de novo Robertsonian translocations of the type reported here should reveal both the extent of UPD in these events and the contribution of particular chromosomes involved in certain phenotypes.

  9. Maternal uniparental disomy for human chromosome 14, due to loss of a chromosome 14 from somatic cells with t(13; 14) trisomy 14

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E.; Blouin, J.L.; Maher, J.; Avramopoulos, D.; Thomas, G.; Talbot, C.C. Jr. (Johns Hopkins Univ., Baltimore (United States))

    1993-06-01

    Uniparental disomy (UPD) for particular chromosomes is increasingly recognized as a cause of abnormal phenotypes in humans. The authors recently studied a 9-year-old female with a de novo Robertsonian translocation t(13;14), short stature, mild developmental delay, scoliosis, hyperextensible joints, hydrocephalus that resolved spontaneously during the first year of life, and hyperchloesterolemia. To determine the parental origin of chromosomes 13 and 14 in the proband, they have studied the genotypes of DNA polymorphic markers due to (GT)n repeats in the patient and her parents' blood DNA. The genotypes of markers D14S43, D14S45, D14S49, and D14S54 indicated maternal UPD for chromosome 14. There was isodisomy for proximal markers and heterodisomy for distal markers, suggesting a recombination event on maternal chromosomes 14. In addition, DNA analysis first revealed -- and subsequent cytogenetic analysis confirmed -- that there was mosaic trisomy 14 in 5% of blood lymphocytes. There was normal (biparental) inheritance for chromosome 13, and there was no evidence of false paternity in genotypes of 11 highly polymorphic markers on human chromosome 21. Two cases of maternal UPD for chromosome 14 have previously been reported, one with a familial rob t(13;14) and the other with a t(14;14). There are several similarities among these patients, and a [open quotes]maternal UPD chromosome 14 syndrome[close quotes] is emerging; however, the contribution of the mosaic trisomy 14 to the phenotype cannot be evaluated. The study of de novo Robertsonian translocations of the type reported here should reveal both the extent of UPD in these events and the contribution of particular chromosomes involved in certain phenotypes. 33 refs., 3 figs., 1 tab.

  10. Dielectrophoretic manipulation of human chromosomes in microfluidic channels: extracting chromosome dielectric properties

    DEFF Research Database (Denmark)

    Clausen, Casper Hyttel; Dimaki, Maria; Buckley, Sonia;

    2011-01-01

    An investigation of the dielectric properties of polyamine buffer prepared human chromosomes is presented in this paper. Chromosomes prepared in this buffer are only a few micrometers in size and shaped roughly like spherical discs. Dielectrophoresis was therefore chosen as the method...... of manipulation combined with a custom designed microfluidic system containing the required electrodes for dielectrophoresis experiments. Our results show that although this system is presently not able to distinguish between the different chromosomes, it can provide average data for the dielectric properties...... of human chromosomes in polyamine buffer. These can then be used to optimize system designs for further characterization and even sorting. The experimental data from the dielectrophoretic manipulation were combined with theoretical calculations to extract a range of values for the permittivity...

  11. A new light on DNA replication from the inactive X chromosome.

    Science.gov (United States)

    Aladjem, Mirit I; Fu, Haiqing

    2014-06-01

    While large portions of the mammalian genome are known to replicate sequentially in a distinct, tissue-specific order, recent studies suggest that the inactive X chromosome is duplicated rapidly via random, synchronous DNA synthesis at numerous adjacent regions. The rapid duplication of the inactive X chromosome was observed in high-resolution studies visualizing DNA replication patterns in the nucleus, and by allele-specific DNA sequencing studies measuring the extent of DNA synthesis. These studies conclude that inactive X chromosomes complete replication earlier than previously thought and suggest that the strict order of DNA replication detected in the majority of genomic regions is not preserved in non-transcribed, "silent" chromatin. These observations alter current concepts about the regulation of DNA replication in non-transcribed portions of the genome in general and in the inactive X-chromosome in particular.

  12. Three-dimensional genome architecture influences partner selection for chromosomal translocations in human disease.

    Directory of Open Access Journals (Sweden)

    Jesse M Engreitz

    Full Text Available Chromosomal translocations are frequent features of cancer genomes that contribute to disease progression. These rearrangements result from formation and illegitimate repair of DNA double-strand breaks (DSBs, a process that requires spatial colocalization of chromosomal breakpoints. The "contact first" hypothesis suggests that translocation partners colocalize in the nuclei of normal cells, prior to rearrangement. It is unclear, however, the extent to which spatial interactions based on three-dimensional genome architecture contribute to chromosomal rearrangements in human disease. Here we intersect Hi-C maps of three-dimensional chromosome conformation with collections of 1,533 chromosomal translocations from cancer and germline genomes. We show that many translocation-prone pairs of regions genome-wide, including the cancer translocation partners BCR-ABL and MYC-IGH, display elevated Hi-C contact frequencies in normal human cells. Considering tissue specificity, we find that translocation breakpoints reported in human hematologic malignancies have higher Hi-C contact frequencies in lymphoid cells than those reported in sarcomas and epithelial tumors. However, translocations from multiple tissue types show significant correlation with Hi-C contact frequencies, suggesting that both tissue-specific and universal features of chromatin structure contribute to chromosomal alterations. Our results demonstrate that three-dimensional genome architecture shapes the landscape of rearrangements directly observed in human disease and establish Hi-C as a key method for dissecting these effects.

  13. Extensive polymorphism and chromosomal characteristics of ribosomal DNA in the characid fish Triportheus venezuelensis (Characiformes, Characidae

    Directory of Open Access Journals (Sweden)

    Mauro Nirchio

    2007-01-01

    Full Text Available The karyotype and chromosomal characteristics of the characid fish Triportheus venezuelensis were investigated using differential staining techniques (C-banding, Ag-NOR staining and fluorescent in situ hybridization (FISH with an 18S rDNA probe. The diploid chromosome number (2n = 52, karyotype composition and sex chromosome determination system of the ZZ/ZW type were the same as previously described in other species of the genus Triportheus. However, extensive variation regarding nucleolus organizer regions (NOR different from other species was observed. 18S rDNA sequences were distributed on nine chromosome pairs, but the number of chromosomes with Ag-NORs was usually lower, reaching a maximum of four chromosomes. When sequential staining experiments were performed, it was demonstrated that: 1. active NORs usually corresponded to segments with 18S rDNA genes identified in FISH experiments; 2. several 18S rDNA sequences were not silver-stained, suggesting that they do not correspond to active NORs; and 3. some chromosomes with silver-stained regions did not display any 18S rDNA signals. These findings characterize an extensive polymorphism associated with the NOR-bearing chromosomes of T. venezuelensis and emphasize the importance of combining traditional and molecular techniques in chromosome studies.

  14. Localization of the kappa opioid receptor gene to human chromosome band 8q11. 2

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Kazuki; Takeda, Jun; Bell, G.I.; Espinosa, R.; Le Beau, M.M. (Univ. of Chicago, IL (United States))

    1994-02-01

    Using the cloned mouse kappa opioid receptor cDNA clone as a probe, screened a human genomic library and isolated a clone containing part of the human kappa opioid receptor gene (OPRK1), designated [lambda]hSR4-1. To determine the chromosomal localization of OPRK1, [lambda]hSR4-1 DNA was labeled with biotin by nick-translation in the presence of bio-11-dUTP and hybridized to human metaphase cells prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes as described previously. Hybridization of the OPRK1-specific probe [lambda]hSR4-1 DNA to normal human metaphase chromosomes resulted in specific labeling only of chromosome 8. Specific labeling of 8q11 was observed on all 4 (6 cells), 3 (9 cells), 2 (9 cells), or 1 (1 cell) chromatid of the chromosome 8 homologs in 25 cells examined. Of 72 signals observed, 70 were located at 8q11. 1 signal was located at 7q11 and at 12p11. In most cells, the signal on 8q was located at 8q11.2. 7 refs., 1 fig.

  15. Nanodissection of human chromosomes with near-infrared femtosecond laser pulses.

    Science.gov (United States)

    König, K; Riemann, I; Fritzsche, W

    2001-06-01

    Near-infrared laser pulses of a compact 80-MHz femtosecond laser source at 800 nm, a mean power of 15-100 mW, 170-fs pulse width, and millisecond beam dwell times at the target have been used for multiphoton-mediated nanoprocessing of human chromosomes. By focusing of the laser beam with high-numerical-aperture objectives of a scanning microscope to diffraction-limited spots and with light intensities of terawatts per cubic centimeter, precise submicrometer holes and cuts in human chromosomes have been processed by single-point exposure and line scans. A minimum FWHM cut size of ~100 nm during a partial dissection of chromosome 1, which is below the diffraction-limited spot size, and a minimum material removal of ~0.003mum (3) were determined by a scanning-force microscope. The plasma-induced ablated material corresponds to ~1/400 of the chromosome 1 volume and to ~65x10(3) base pairs of chromosomal DNA. A complete dissection could be performed with FWHM cut sizes below 200 nm. High-repetition-frequency femtosecond lasers at low mean power in combination with high-numerical-aperture focusing optics appear therefore as appropriate noncontact tools for nanoprocessing of bulk and (or) surfaces of transparent materials such as chromosomes. In particular, the noninvasive inactivation of certain genomic regions on single chromosomes within living cells becomes possible.

  16. Intercalation of psoralen into DNA of plastid chromosomes decreases late during barley chloroplast development.

    Science.gov (United States)

    Davies, J P; Thompson, R J; Mosig, G

    1991-01-01

    We have used a DNA crosslinking assay to measure intercalation of the psoralen derivative HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into barley (Hordeum vulgare) plastid chromosomal DNA during chloroplast and etioplast development. Intercalation into DNA in intact plastids in vivo and in plastid lysates in vitro shows that chromosomal DNA in the most mature chloroplasts intercalates HMT less efficiently than DNA in younger chloroplasts. In contrast, there is no change in HMT intercalation during etioplast differentiation in the dark. Our results also show that DNA in higher plant plastid chromosomes is under superhelical tension in vivo. The lower susceptibility to HMT intercalation of DNA in the most mature chloroplasts indicates that late during chloroplast development the superhelical tension or the binding of proteins to the DNA or both change. Images PMID:1923805

  17. Mapping of the phenol sulfotransferase gene (STP) to human chromosome 16p12. 1-p11. 2 and to mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Dooley, T.P.; Obermoeller, R.D. (Southwest Foundation for Biomedical Research, San Antonio, TX (United States)); Leiter, E.H.; Chapman, H.D. (Jackson Lab., Bar Harbor, ME (United States)); Falany, C.N. (Univ. of Alabama, Birmingham, AL (United States)); Deng, Z.; Siciliano, M.J. (Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States))

    1993-11-01

    The authors have recently cloned a cDNA encoding the human phenol-preferring phenol sulfotransferase (P-PST) enzyme. An oligonucleotide primer pair based on the human STP (representing sulfotransferase, phenol-preferring) cDNA sequence was synthesized and was employed in polymerase chain reaction (PCR) amplification of human genomic DNA to identify a 525-bp DNA fragment. The DNA sequence of this portion of the STP gene, near the 5[prime] end of the coding region, was determined. The amplified genomic fragment contained two small introns of 104 and 89 bp. When DNA samples from a human-hamster somatic cell hybrid panel were screened by PCR using these primers, only those hybrids that contained human chromosome 16 were positive for the 525-bp genomic fragment. To identify the specific region on chromosome 16 that contained the STP gene, PCR amplification reactions were performed on a human-mouse somatic cell hybrid panel containing defined portions of human chromosome 16. The results indicated that STP is localized proximal to the gene for protein kinase C, [beta]1 polypeptide (PRKCB1), in the region from the distal portion of 16p11.2 to p12.1. The human STP gene maps near the locus for Batten disease (CLN3). Furthermore, the authors have determined by genotyping of murine interspecific backcross progeny that the homologous gene in mouse (Stp) localizes to the syntenic region of mouse chromosome 7 near the D7Mit8 (at 54 cM) and D7Bir1 markers. 18 refs., 2 figs., 1 tab.

  18. Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos.

    Science.gov (United States)

    Sonneville, Remi; Craig, Gillian; Labib, Karim; Gartner, Anton; Blow, J Julian

    2015-07-21

    During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG) DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2-7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication.

  19. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  20. Frequency of chromosomally-integrated human herpesvirus 6 in children with acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Annie Gravel

    Full Text Available INTRODUCTION: Human herpesvirus 6 (HHV-6 is a ubiquitous pathogen infecting nearly 100% of the human population. Of these individuals, between 0.2% and 1% of them carry chromosomally-integrated HHV-6 (ciHHV-6. The biological consequences of chromosomal integration by HHV-6 remain unknown. OBJECTIVE: To determine and compare the frequency of ciHHV-6 in children with acute lymphoblastic leukemia to healthy blood donors. METHODOLOGY: A total of 293 DNA samples from children with pre-B (n=255, pre-pre-B (n=4, pre-T (n=26 and undetermined (n=8 leukemia were analyzed for ciHHV-6 by quantitative TaqMan PCR (QPCR using HHV-6 specific primers and probe. As control, DNA samples from 288 healthy individuals were used. Primers and probe specific to the cellular GAPDH gene were used to estimate integrity and DNA content. RESULTS: Out of 293 DNA samples from the leukemic cohort, 287 contained amplifiable DNA. Of these, only 1 (0.35% contained ciHHV-6. Variant typing indicates that the ci-HHV-6 corresponds to variant A. None of the 288 DNA samples from healthy individuals contained ciHHV-6. CONCLUSION: The frequency of ciHHV-6 in children with acute lymphoblastic leukemia is similar (p=0.5 to that of healthy individuals. These results suggest that acute lymphoblastic leukemia does not originate as a consequence to integration of HHV-6 within the chromosomes.

  1. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells

    Science.gov (United States)

    Mandegar, Mohammad A.; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y.L.; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P.; Volpi, Emanuela V.; Thrasher, Adrian J.; James, William; Monaco, Zoia L.

    2011-01-01

    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  2. The third international workshop of human chromosome 5. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-12-31

    The Third International Workshop on Human Chromosome 5 was held in Laguna Beach, California, March 5-8, 1994. The pace at which new mapping information has been published in the last year make almost any report outdated before publication. Much of the information in this report and the most recent data from the Human chromosome 5 Genome Center at U.C. Irvine on the physical map of chromosome 5 are accessible via a WWW server. For most loci referred to in this report that can be detected by Polymerase Chain Reaction, the sequences of the oligonucleotide primers are available and some primer sequences are provided in this report.

  3. DNA double strand break repair, chromosome synapsis and transcriptional silencing in meiosis.

    Science.gov (United States)

    Inagaki, Akiko; Schoenmakers, Sam; Baarends, Willy M

    2010-05-16

    Chromosome pairing and synapsis during meiotic prophase requires the formation and repair of DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme SPO11. Chromosomes, or chromosomal regions, that lack a pairing partner, such as the largely heterologous X and Y chromosomes, show delayed meiotic DSB repair and are transcriptionally silenced. Herein, we review meiosis-specific aspects of DSB repair in relation to homology recognition and meiotic silencing of heterologous regions. We propose a dynamic interplay between progression of synapsis and persistent meiotic DSBs. Signaling from these persistent breaks could inhibit heterologous synapsis and stimulate meiotic silencing of the X and Y chromosomes.

  4. 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...

  5. Human oocyte chromosome analysis: complicated cases and major pitfalls

    Indian Academy of Sciences (India)

    Bernd Rosenbusch; Michael Schneider; Hans Wilhelm Michelmann

    2008-08-01

    Human oocytes that remained unfertilized in programmes of assisted reproduction have been analysed cytogenetically for more than 20 years to assess the incidence of aneuploidy in female gametes. However, the results obtained so far are not indisputable as a consequence of difficulties in evaluating oocyte chromosome preparations. Because of the lack of guidelines, we decided to summarize for the first time, the possible pitfalls in human oocyte chromosome analysis. Therefore, we screened the material from our previous studies and compiled representative, complicated cases with recommendations for their cytogenetic classification. We point out that maturity and size of the oocyte are important parameters and that fixation artefacts, as well as the particular structure of oocyte chromosomes, may predispose one to misinterpretations. Moreover, phenomena related to oocyte activation and fertilization are illustrated and explained. This compilation may help to avoid major problems in future studies and contribute to a more precise, and uniform assessment of human oocyte chromosomes.

  6. Fourth international workshop on human chromosome 5. Final progress report

    Energy Technology Data Exchange (ETDEWEB)

    McPherson, J.D.

    1996-12-31

    The Fourth International Workshop on Human Chromosome 5 was held in Manchester, UK on November 9--10, 1996 and was hosted by the University of Manchester. The major goals of the workshop were: (1) to collate the various genetic, cytogenetic and physical maps of human chromosome 5; (2) to integrate these maps and identify/correct discrepancies between them wherever possible; (3) to catalogue the sequence-ready contigs of the chromosome; (4) to co-ordinate the various sequencing efforts to avoid future duplication; (5) to establish the first (to the author`s knowledge) web site for the human chromosome 5 community which contains the above information in a readily accessible form.

  7. B chromosome in the beetle Coprophanaeus cyanescens (Scarabaeidae: emphasis in the organization of repetitive DNA sequences

    Directory of Open Access Journals (Sweden)

    Gomes de Oliveira Sarah

    2012-11-01

    Full Text Available Abstract Background To contribute to the knowledge of coleopteran cytogenetics, especially with respect to the genomic content of B chromosomes, we analyzed the composition and organization of repetitive DNA sequences in the Coprophanaeus cyanescens karyotype. We used conventional staining and the application of fluorescence in situ hybridization (FISH mapping using as probes C0t-1 DNA fraction, the 18S and 5S rRNA genes, and the LOA-like non-LTR transposable element (TE. Results The conventional analysis detected 3 individuals (among 50 analyzed carrying one small metacentric and mitotically unstable B chromosome. The FISH analysis revealed a pericentromeric block of C0t-1 DNA in the B chromosome but no 18S or 5S rDNA clusters in this extra element. Using the LOA-like TE probe, the FISH analysis revealed large pericentromeric blocks in eight autosomal bivalents and in the B chromosome, and a pericentromeric block extending to the short arm in one autosomal pair. No positive hybridization signal was observed for the LOA-like element in the sex chromosomes. Conclusions The results indicate that the origin of the B chromosome is associated with the autosomal elements, as demonstrated by the hybridization with C0t-1 DNA and the LOA-like TE. The present study is the first report on the cytogenetic mapping of a TE in coleopteran chromosomes. These TEs could have been involved in the origin and evolution of the B chromosome in C. cyanescens.

  8. Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley

    Directory of Open Access Journals (Sweden)

    Bartoš Jan

    2008-06-01

    Full Text Available Abstract Background Flow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis. However, the recovery of microgram amounts of DNA needed for some assays requires sorting of millions of chromosomes which is laborious and time consuming. Yet, many genomic applications such as development of genetic maps or physical mapping do not require large DNA fragments. In such cases time-consuming de novo sorting can be minimized by utilizing whole-genome amplification. Results Here we report a protocol optimized in barley including amplification of DNA from only ten thousand chromosomes, which can be isolated in less than one hour. Flow-sorted chromosomes were treated with proteinase K and amplified using Phi29 multiple displacement amplification (MDA. Overnight amplification in a 20-microlitre reaction produced 3.7 – 5.7 micrograms DNA with a majority of products between 5 and 30 kb. To determine the purity of sorted fractions and potential amplification bias we used quantitative PCR for specific genes on each chromosome. To extend the analysis to a whole genome level we performed an oligonucleotide pool assay (OPA for interrogation of 1524 loci, of which 1153 loci had known genetic map positions. Analysis of unamplified genomic DNA of barley cv. Akcent using this OPA resulted in 1426 markers with present calls. Comparison with three replicates of amplified genomic DNA revealed >99% concordance. DNA samples from amplified chromosome 1H and a fraction containing chromosomes 2H – 7H were examined. In addition to loci with known map positions, 349 loci with unknown map positions were included. Based on this analysis 40 new loci were mapped to 1H. Conclusion The results indicate a significant potential of using this approach for physical mapping. Moreover, the study showed that multiple displacement amplification of flow-sorted chromosomes is highly efficient and representative which

  9. Circular chromosomal DNA in the sulfur-dependent archaebacterium Sulfolobus acidocaldarius.

    OpenAIRE

    Yamagishi, A; Oshima, T

    1990-01-01

    The shape of the chromosomal DNA of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius was analyzed by the pulsed-field gel electrophoresis(PFGE). S.acidocaldarius DNA digested with Notl showed two DNA bands at around 1.0 Mbp and 2.1 Mbp. Notl-linking clones were isolated from the library of S.acidocaldarius chromosomal DNA. It contained two Notl sites. Both 1.0 and 2.1 Mbp DNA band separated by PFGE were hybridized with the two independent Notl-linking fragment. Each right and le...

  10. Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.

    Science.gov (United States)

    Schalbetter, Stephanie A; Mansoubi, Sahar; Chambers, Anna L; Downs, Jessica A; Baxter, Jonathan

    2015-08-18

    Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

  11. A Model of DNA Repeat-Assembled Mitotic Chromosomal Skeleton

    Directory of Open Access Journals (Sweden)

    Shao-Jun Tang

    2011-09-01

    Full Text Available Despite intensive investigation for decades, the principle of higher-order organization of mitotic chromosomes is unclear. Here, I describe a novel model that emphasizes a critical role of interactions of homologous DNA repeats (repetitive elements; repetitive sequences in mitotic chromosome architecture. According to the model, DNA repeats are assembled, via repeat interactions (pairing, into compact core structures that govern the arrangement of chromatins in mitotic chromosomes. Tandem repeat assemblies form a chromosomal axis to coordinate chromatins in the longitudinal dimension, while dispersed repeat assemblies form chromosomal nodes around the axis to organize chromatins in the halo. The chromosomal axis and nodes constitute a firm skeleton on which non-skeletal chromatins can be anchored, folded, and supercoiled.

  12. A model of DNA repeat-assembled mitotic chromosomal skeleton.

    Science.gov (United States)

    Tang, Shao-Jun

    2011-01-01

    Despite intensive investigation for decades, the principle of higher-order organization of mitotic chromosomes is unclear. Here, I describe a novel model that emphasizes a critical role of interactions of homologous DNA repeats (repetitive elements; repetitive sequences) in mitotic chromosome architecture. According to the model, DNA repeats are assembled, via repeat interactions (pairing), into compact core structures that govern the arrangement of chromatins in mitotic chromosomes. Tandem repeat assemblies form a chromosomal axis to coordinate chromatins in the longitudinal dimension, while dispersed repeat assemblies form chromosomal nodes around the axis to organize chromatins in the halo. The chromosomal axis and nodes constitute a firm skeleton on which non-skeletal chromatins can be anchored, folded, and supercoiled.

  13. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  14. [Molecular cytogenetic methods for studying interphase chromosomes in human brain cells].

    Science.gov (United States)

    Iurov, I Iu; Vorsanova, S G; Solov'ev, I V; Iurov, Iu B

    2010-09-01

    One of the main genetic factors determining the functional activity of the genome in somatic cells, including brain nerve cells, is the spatial organization of chromosomes in the interphase nucleus. For a long time, no studies of human brain cells were carried out until high-resolution methods of molecular cytogenetics were developed to analyze interphase chromosomes in nondividing somatic cells. The purpose of the present work was to assess the potential of high-resolution methods of interphase molecular cytogenetics for studying chromosomes and the nuclear organization in postmitotic brain cells. A high efficiency was shown by such methods as multiprobe and quantitative fluorescence in situ hybridization (Multiprobe FISH and QFISH), ImmunoMFISH (analysis of the chromosome organization in different types of brain cells), and interphase chromosome-specific multicolor banding (ICS-MCB). These approaches allowed studying the nuclear organization depending on the gene composition and types of repetitive DNA of specific chromosome regions in certain types of brain cells (in neurons and glial cells, in particular). The present work demonstrates a high potential of interphase molecular cytogenetics for studying the structural and functional organizations of the cell nucleus in highly differentiated nerve cells. Analysis of interphase chromosomes of brain cells in the normal and pathological states can be considered as a promising line of research in modern molecular cytogenetics and cell neurobiology, i. e., molecular neurocytogenetics.

  15. 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.

  16. Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy.

    Directory of Open Access Journals (Sweden)

    Ji Hyae Lim

    Full Text Available BACKGROUND: Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy. METHODOLOGY/PRINCIPAL FINDINGS: A nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both. The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001 than in SA women with normal karyotype (r = 0.465, P = 0.002 and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037. The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852-0.945 and 0.939 (95% CI, 0.903-0.975, respectively. CONCLUSIONS: Significantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA

  17. Repetitive DNA Sequences and Evolution of ZZ/ZW Sex Chromosomes in Characidium (Teleostei: Characiformes).

    Science.gov (United States)

    Scacchetti, Priscilla Cardim; Utsunomia, Ricardo; Pansonato-Alves, José Carlos; da Costa Silva, Guilherme José; Vicari, Marcelo Ricardo; Artoni, Roberto Ferreira; Oliveira, Claudio; Foresti, Fausto

    2015-01-01

    Characidium constitutes an interesting model for cytogenetic studies, since a large degree of karyotype variation has been detected in this group, like the presence/absence of sex and supernumerary chromosomes and variable distribution of repetitive sequences in different species/populations. In this study, we performed a comparative cytogenetic analysis in 13 Characidium species collected at different South American river basins in order to investigate the karyotype diversification in this group. Chromosome analyses involved the karyotype characterization, cytogenetic mapping of repetitive DNA sequences and cross-species chromosome painting using a W-specific probe obtained in a previous study from Characidium gomesi. Our results evidenced a conserved diploid chromosome number of 2n = 50, and almost all the species exhibited homeologous ZZ/ZW sex chromosomes in different stages of differentiation, except C. cf. zebra, C. tenue, C. xavante and C. stigmosum. Notably, some ZZ/ZW sex chromosomes showed 5S and/or 18S rDNA clusters, while no U2 snDNA sites could be detected in the sex chromosomes, being restricted to a single chromosome pair in almost all the analyzed species. In addition, the species Characidium sp. aff. C. vidali showed B chromosomes with an inter-individual variation of 1 to 4 supernumerary chromosomes per cell. Notably, these B chromosomes share sequences with the W-specific probe, providing insights about their origin. Results presented here further confirm the extensive karyotype diversity within Characidium in contrast with a conserved diploid chromosome number. Such chromosome differences seem to constitute a significant reproductive barrier, since several sympatric Characidium species had been described during the last few years and no interespecific hybrids were found.

  18. Retrieval of human DNA from rodent-human genomic libraries by a recombination process.

    Science.gov (United States)

    Neve, R L; Bruns, G A; Dryja, T P; Kurnit, D M

    1983-09-01

    Human Alu repeat ("BLUR") sequences have been cloned into the mini-plasmid vector piVX. The resulting piBLUR clones have been used to rescue selectively, by recombination, bacteriophage carrying human DNA sequences from genomic libraries constructed using DNA from rodent-human somatic cell hybrids. piBLUR clones are able to retrieve human clones from such libraries because at least one Alu family repeat is present on most 15 to 20 kb fragments of human DNA and because of the relative species-specificity of the sequences comprising the Alu family. The rapid, selective plaque purification achieved results in the construction of a collection of recombinant phage carrying diverse human DNA inserts from a specific subset of the human karyotype. Subfragments of two recombinants rescued from a mouse-human somatic cell hybrid containing human chromosomes X, 10, 13, and 22 were mapped to human chromosomes X and 13, respectively, demonstrating the utility of this protocol for the isolation of human chromosome-specific DNA sequences from appropriate somatic cell hybrids.

  19. Regulation of DNA Replication Initiation by Chromosome Structure

    OpenAIRE

    Magnan, David; Bates, David

    2015-01-01

    Recent advancements in fluorescence imaging have shown that the bacterial nucleoid is surprisingly dynamic in terms of both behavior (movement and organization) and structure (density and supercoiling). Links between chromosome structure and replication initiation have been made in a number of species, and it is universally accepted that favorable chromosome structure is required for initiation in all cells. However, almost nothing is known about whether cells use changes in chromosome struct...

  20. Assignment of ten DNA repair genes from Schizosaccharomyces pombe to chromosomal NotI restriction fragments

    NARCIS (Netherlands)

    B.C. Broughton; N.C. Barbet; J. Murray (Johanne); F.Z. Watts (Felicity); M.H.M. Koken (Marcel); A.R. Lehmann (Alan); A.M. Carr (Anthony)

    1991-01-01

    textabstractTen DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.

  1. Molecular cloaking of H2A.Z on mortal DNA chromosomes during nonrandom segregation.

    Science.gov (United States)

    Huh, Yang Hoon; Sherley, James L

    2011-10-01

    Although nonrandom sister chromatid segregation is a singular property of distributed stem cells (DSCs) that are responsible for renewing and repairing mature vertebrate tissues, both its cellular function and its molecular mechanism remain unknown. This situation persists in part because of the lack of facile methods for detecting and quantifying nonrandom segregating cells and for identifying chromosomes with immortal DNA strands, the cellular molecules that signify nonrandom segregation. During nonrandom segregation, at each mitosis, asymmetrically self-renewing DSCs continuously cosegregate to themselves the set of chromosomes that contain immortal DNA strands, which are the oldest DNA strands. Here, we report the discovery of a molecular asymmetry between segregating sets of immortal chromosomes and opposed mortal chromosomes (i.e., containing the younger set of DNA template strands) that constitutes a new convenient biomarker for detection of cells undergoing nonrandom segregation and direct delineation of chromosomes that bear immortal DNA strands. In both cells engineered with DSC-specific properties and ex vivo-expanded mouse hair follicle stem cells, the histone H2A variant H2A.Z shows specific immunodetection on immortal DNA chromosomes. Cell fixation analyses indicate that H2A.Z is present on mortal chromosomes as well but is cloaked from immunodetection, and the cloaking entity is acid labile. The H2A.Z chromosomal asymmetry produced by molecular cloaking provides a first direct assay for nonrandom segregation and for chromosomes with immortal DNA strands. It also seems likely to manifest an important aspect of the underlying mechanism(s) responsible for nonrandom sister chromatid segregation in DSCs.

  2. Origin and molecular organization of supernumerary chromosomes of Prochilodus lineatus (characiformes, prochilodontidae) obtained by DNA probes.

    Science.gov (United States)

    Voltolin, Tatiana Aparecida; Laudicina, Alejandro; Senhorini, José Augusto; Bortolozzi, Jehud; Oliveira, Cláudio; Foresti, Fausto; Porto-Foresti, Fábio

    2010-12-01

    In Prochilodus lineatus B-chromosomes are visualized as reduced size extra elements identified as microchromosomes and are variable in morphology and number. We describe the specific total probe (B-chromosome probe) in P. lineatus obtained by chromosome microdissection and a whole genomic probe (genomic probe) from an individual without B-chromosome. The specific B-chromosome was scraped and processed to obtain DNA with amplification by DOP-PCR, and so did the genomic probe DNA. Fluorescence in situ hybridization using the B-chromosome probe labeled with dUTP-Tetramethyl-rhodamine and the genomic probe labeled with digoxigenin-FITC permitted to establish that in this species supernumerary chromosomes with varying number and morphology had different structure of chromatin when compared to that of the regular chromosomes or A complement, since only these extra elements were labeled in the metaphases. The present findings suggest that modifications in the chromatin structure of B-chromosomes to differentiate them from the A chromosomes could occur along their dispersion in the individuals of the population.

  3. 人类染色体的原子力显微术研究%Atomic force microscopy of human chromosomes

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ The atomic force microscope(AFM),invented by Binnig et al.in 1986,can obtain topographic images at high spatial resolution in various(vacuum,air and liquid)environments,and has been applied to studies for imaging various biological samples from DNA to living cells [1,2].We have been especially interested in AFM imaging of human chromosomes for analyzing their three-dimensional structure.In this paper,we briefly reviewed our AFM studies of human metaphase chromosomes,and introduced our results on the high-order structure of the chromosomes.

  4. Correlation of physical and genetic maps of human chromosome 16. Annual progress report, October 1, 1990--July 31, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, G.R.

    1991-12-31

    This project aimed to divide chromosome 16 into approximately 50 intervals of {approximately}2Mb in size by constructing a series of mouse/human somatic cell hybrids each containing a rearranged chromosome 16. Using these hybrids, DNA probes would be regionally mapped by Southern blot or PCR analysis. Preference would be given to mapping probes which demonstrated polymorphisms for which the CEPH panel of families had been typed. This would allow a correlation of the physical and linkage maps of this chromosome. The aims have been substantially achieved. 49 somatic cell hybrids have been constructed which have allowed definition of 46, and potentially 57, different physical intervals on the chromosome. 164 loci have been fully mapped into these intervals. A correlation of the physical and genetic maps of the chromosome is in an advanced stage of preparation. The somatic cell hybrids constructed have been widely distributed to groups working on chromosome 16 and other genome projects.

  5. The sequence and analysis of duplication rich human chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Martin, J; Han, C; Gordon, L A; Terry, A; Prabhakar, S; She, X; Xie, G; Hellsten, U; Chan, Y M; Altherr, M; Couronne, O; Aerts, A; Bajorek, E; Black, S; Blumer, H; Branscomb, E; Brown, N; Bruno, W J; Buckingham, J; Callen, D F; Campbell, C S; Campbell, M L; Campbell, E W; Caoile, C; Challacombe, J F; Chasteen, L A; Chertkov, O; Chi, H C; Christensen, M; Clark, L M; Cohn, J D; Denys, M; Detter, J C; Dickson, M; Dimitrijevic-Bussod, M; Escobar, J; Fawcett, J J; Flowers, D; Fotopulos, D; Glavina, T; Gomez, M; Gonzales, E; Goodstein, D; Goodwin, L A; Grady, D L; Grigoriev, I; Groza, M; Hammon, N; Hawkins, T; Haydu, L; Hildebrand, C E; Huang, W; Israni, S; Jett, J; Jewett, P B; Kadner, K; Kimball, H; Kobayashi, A; Krawczyk, M; Leyba, T; Longmire, J L; Lopez, F; Lou, Y; Lowry, S; Ludeman, T; Manohar, C F; Mark, G A; McMurray, K L; Meincke, L J; Morgan, J; Moyzis, R K; Mundt, M O; Munk, A C; Nandkeshwar, R D; Pitluck, S; Pollard, M; Predki, P; Parson-Quintana, B; Ramirez, L; Rash, S; Retterer, J; Ricke, D O; Robinson, D; Rodriguez, A; Salamov, A; Saunders, E H; Scott, D; Shough, T; Stallings, R L; Stalvey, M; Sutherland, R D; Tapia, R; Tesmer, J G; Thayer, N; Thompson, L S; Tice, H; Torney, D C; Tran-Gyamfi, M; Tsai, M; Ulanovsky, L E; Ustaszewska, A; Vo, N; White, P S; Williams, A L; Wills, P L; Wu, J; Wu, K; Yang, J; DeJong, P; Bruce, D; Doggett, N A; Deaven, L; Schmutz, J; Grimwood, J; Richardson, P; Rokhsar, D S; Eichler, E E; Gilna, P; Lucas, S M; Myers, R M; Rubin, E M; Pennacchio, L A

    2005-04-06

    Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes, and 3 RNA pseudogenes. These genes include metallothionein, cadherin, and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. While the segmental duplications of chromosome 16 are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events likely to have had an impact on the evolution of primates and human disease susceptibility.

  6. A 2-megabase physical contig incorporating 43 DNA markers on the human X chromosome at p11.23-p11.22 from ZNF21 to DXS255

    Energy Technology Data Exchange (ETDEWEB)

    Boycott, K.M.; Bech-Hansen, N.T. [Univ. of Calgary, Alberta (Canada); Halley, G.R.; Schlessinger, D. [Washington Univ. School of Medicine, St. Louis, MO (United States)

    1996-05-01

    A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23-p11.22. As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids. The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internal Alu-PCR product. The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb. The relative order of previously known gene and expressed sequence tags in this region is predicted to be Xpter-ZNF21-DXS7465E (MG66)-DXS7927E (MG81)-WASP, DXS1011E, DXS7467E (MG21)-DXS-7466E (MG44)-GATA1-DXS7469E (Xp664)-TFE3-SYP (DXS1007E)-Xcen. This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates. 47 refs., 1 fig., 4 tabs.

  7. Independent control of replication initiation of the two Vibrio cholerae chromosomes by DnaA and RctB

    DEFF Research Database (Denmark)

    Duigou, Stephane; Knudsen, Kristine Groth; Skovgaard, Ole

    2006-01-01

    Although the two Vibrio cholerae chromosomes initiate replication in a coordinated fashion, we show here that each chromosome appears to have a specific replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of Rct...

  8. The 5S rDNA in two Abracris grasshoppers (Ommatolampidinae: Acrididae): molecular and chromosomal organization.

    Science.gov (United States)

    Bueno, Danilo; Palacios-Gimenez, Octavio Manuel; Martí, Dardo Andrea; Mariguela, Tatiane Casagrande; Cabral-de-Mello, Diogo Cavalcanti

    2016-08-01

    The 5S ribosomal DNA (rDNA) sequences are subject of dynamic evolution at chromosomal and molecular levels, evolving through concerted and/or birth-and-death fashion. Among grasshoppers, the chromosomal location for this sequence was established for some species, but little molecular information was obtained to infer evolutionary patterns. Here, we integrated data from chromosomal and nucleotide sequence analysis for 5S rDNA in two Abracris species aiming to identify evolutionary dynamics. For both species, two arrays were identified, a larger sequence (named type-I) that consisted of the entire 5S rDNA gene plus NTS (non-transcribed spacer) and a smaller (named type-II) with truncated 5S rDNA gene plus short NTS that was considered a pseudogene. For type-I sequences, the gene corresponding region contained the internal control region and poly-T motif and the NTS presented partial transposable elements. Between the species, nucleotide differences for type-I were noticed, while type-II was identical, suggesting pseudogenization in a common ancestor. At chromosomal point to view, the type-II was placed in one bivalent, while type-I occurred in multiple copies in distinct chromosomes. In Abracris, the evolution of 5S rDNA was apparently influenced by the chromosomal distribution of clusters (single or multiple location), resulting in a mixed mechanism integrating concerted and birth-and-death evolution depending on the unit.

  9. DNA tandem repeat instability in the Escherichia coli chromosome is stimulated by mismatch repair at an adjacent CAG·CTG trinucleotide repeat

    Science.gov (United States)

    Blackwood, John K.; Okely, Ewa A.; Zahra, Rabaab; Eykelenboom, John K.; Leach, David R. F.

    2010-01-01

    Approximately half the human genome is composed of repetitive DNA sequences classified into microsatellites, minisatellites, tandem repeats, and dispersed repeats. These repetitive sequences have coevolved within the genome but little is known about their potential interactions. Trinucleotide repeats (TNRs) are a subclass of microsatellites that are implicated in human disease. Expansion of CAG·CTG TNRs is responsible for Huntington disease, myotonic dystrophy, and a number of spinocerebellar ataxias. In yeast DNA double-strand break (DSB) formation has been proposed to be associated with instability and chromosome fragility at these sites and replication fork reversal (RFR) to be involved either in promoting or in preventing instability. However, the molecular basis for chromosome fragility of repetitive DNA remains poorly understood. Here we show that a CAG·CTG TNR array stimulates instability at a 275-bp tandem repeat located 6.3 kb away on the Escherichia coli chromosome. Remarkably, this stimulation is independent of both DNA double-strand break repair (DSBR) and RFR but is dependent on a functional mismatch repair (MMR) system. Our results provide a demonstration, in a simple model system, that MMR at one type of repetitive DNA has the potential to influence the stability of another. Furthermore, the mechanism of this stimulation places a limit on the universality of DSBR or RFR models of instability and chromosome fragility at CAG·CTG TNR sequences. Instead, our data suggest that explanations of chromosome fragility should encompass the possibility of chromosome gaps formed during MMR. PMID:21149728

  10. DNA amount of X and B chromosomes in the grasshoppers Eyprepocnemis plorans and Locusta migratoria.

    Science.gov (United States)

    Ruiz-Ruano, F J; Ruiz-Estévez, M; Rodríguez-Pérez, J; López-Pino, J L; Cabrero, J; Camacho, J P M

    2011-01-01

    We analyzed the DNA amount in X and B chromosomes of 2 XX/X0 grasshopper species (Eyprepocnemis plorans and Locusta migratoria), by means of Feulgen image analysis densitometry (FIAD), using previous estimates in L. migratoria as standard (5.89 pg). We first analyzed spermatids of 0B males and found a bimodal distribution of integrated optical densities (IODs), suggesting that one peak corresponded to +X and the other to -X spermatids. The difference between the 2 peaks corresponded to the X chromosome DNA amount, which was 1.28 pg in E. plorans and 0.80 pg in L. migratoria. In addition, the +X peak in E. plorans gave an estimate of the C-value in this species (10.39 pg). We next analyzed diplotene cells from 1B males in E. plorans and +B males in L. migratoria (a species where Bs are mitotically unstable and no integer B number can be defined for an individual) and measured B chromosome IOD relative to X chromosome IOD, within the same cell, taking advantage of the similar degree of condensation for both positively heteropycnotic chromosomes at this meiotic stage. From this proportion, we estimated the DNA amount for 3 different B chromosome variants found in individuals from 3 E. plorans Spanish populations (0.54 pg for B1 from Saladares, 0.51 pg for B2 from Salobreña and 0.64 for B24 from Torrox). Likewise, we estimated the DNA amount of the B chromosome in L. migratoria to be 0.15 pg. To automate measurements, we wrote a GPL3 licensed Python program (pyFIA). We discuss the utility of the present approach for estimating X and B chromosome DNA amount in a variety of situations, and the meaning of the DNA amount estimates for X and B chromosomes in these 2 species.

  11. Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach

    Energy Technology Data Exchange (ETDEWEB)

    Liu, J.; Rezonzew, R. [McGill Centre for the Study of Host Resistance, Montreal, Quebec (Canada)]|[McGill Univ., Montreal, Quebec (Canada); Stanton, V.P. Jr. [Massachusetts Institute of Technology, Cambridge, MA (United States)] [and others

    1995-03-20

    We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomic DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostly derived from the CEPH Mark I library. Alu-element-mediated PCR was performed for each pool, and amplification products were spotted on hybridization membranes (IRS filters). IRS probes for the screening of the IRS filters were obtained by Alu-element-mediated PCR. Of 708 distinct probes obtained from chromosome 2-specific somatic cell hybrids, 85% were successfully used for library screening. Similarly, 80% of 80 YAC walking probes were successfully used for library screening. Each probe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preliminary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric half of chromosome 2q was considered to be good since 31 of 34 microsatellites and 22 of 23 expressed sequence tags that were chosen from chromosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrary generated by our experiments. We have identified a minimum of 1610 distinct chromosome 2-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome. 81 refs., 8 figs., 3 tabs.

  12. A novel tandem repeat sequence located on human chromosome 4p: isolation and characterization.

    Science.gov (United States)

    Kogi, M; Fukushige, S; Lefevre, C; Hadano, S; Ikeda, J E

    1997-06-01

    In an effort to analyze the genomic region of the distal half of human chromosome 4p, to where Huntington disease and other diseases have been mapped, we have isolated the cosmid clone (CRS447) that was likely to contain a region with specific repeat sequences. Clone CRS447 was subjected to detailed analysis, including chromosome mapping, restriction mapping, and DNA sequencing. Chromosome mapping by both a human-CHO hybrid cell panel and FISH revealed that CRS447 was predominantly located in the 4p15.1-15.3 region. CRS447 was shown to consist of tandem repeats of 4.7-kb units present on chromosome 4p. A single EcoRI unit was subcloned (pRS447), and the complete sequence was determined as 4752 nucleotides. When pRS447 was used as a probe, the number of copies of this repeat per haploid genome was estimated to be 50-70. Sequence analysis revealed that it contained two internal CA repeats and one putative ORF. Database search established that this sequence was unreported. However, two homologous STS markers were found in the database. We concluded that CRS447/pRS447 is a novel tandem repeat sequence that is mainly specific to human chromosome 4p.

  13. Dynamic in situ chromosome immobilisation and DNA extraction using localized poly(N-isopropylacrylamide) phase transition

    DEFF Research Database (Denmark)

    Eriksen, Johan; Thilsted, Anil Haraksingh; Marie, Rodolphe

    2011-01-01

    A method of in situ chromosome immobilisation and DNA extraction in a microfluidic polymer chip was presented. Light-induced local heating was used to induce poly(N-isopropylacrylamide) phase transition in order to create a hydrogel and embed a single chromosome such that it was immobilised....... This was achieved with the use of a near-infrared laser focused on an absorption layer integrated in the polymer chip in close proximity to the microchannel. It was possible to proceed to DNA extraction while holding on the chromosome at an arbitrary location by introducing protease K into the microchannel. © 2011...

  14. Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells

    OpenAIRE

    Prashant K. Mishra; Wei-Chun Au; John S. Choy; P Henning Kuich; Baker, Richard E.; Foltz, Daniel R.; Basrai, Munira A.

    2011-01-01

    The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevanc...

  15. Evaluating the relationship between spermatogenic silencing of the X chromosome and evolution of the Y chromosome in chimpanzee and human.

    Directory of Open Access Journals (Sweden)

    Eskeatnaf Mulugeta Achame

    Full Text Available Chimpanzees and humans are genetically very similar, with the striking exception of their Y chromosomes, which have diverged tremendously. The male-specific region (MSY, representing the greater part of the Y chromosome, is inherited from father to son in a clonal fashion, with natural selection acting on the MSY as a unit. Positive selection might involve the performance of the MSY in spermatogenesis. Chimpanzees have a highly polygamous mating behavior, so that sperm competition is thought to provide a strong selective force acting on the Y chromosome in the chimpanzee lineage. In consequence of evolution of the heterologous sex chromosomes in mammals, meiotic sex chromosome inactivation (MSCI results in a transcriptionally silenced XY body in male meiotic prophase, and subsequently also in postmeiotic repression of the sex chromosomes in haploid spermatids. This has evolved to a situation where MSCI has become a prerequisite for spermatogenesis. Here, by analysis of microarray testicular expression data representing a small number of male chimpanzees and men, we obtained information indicating that meiotic and postmeiotic X chromosome silencing might be more effective in chimpanzee than in human spermatogenesis. From this, we suggest that the remarkable reorganization of the chimpanzee Y chromosome, compared to the human Y chromosome, might have an impact on its meiotic interactions with the X chromosome and thereby on X chromosome silencing in spermatogenesis. Further studies will be required to address comparative functional aspects of MSCI in chimpanzee, human, and other placental mammals.

  16. Evaluating the relationship between spermatogenic silencing of the X chromosome and evolution of the Y chromosome in chimpanzee and human.

    Science.gov (United States)

    Mulugeta Achame, Eskeatnaf; Baarends, Willy M; Gribnau, Joost; Grootegoed, J Anton

    2010-12-14

    Chimpanzees and humans are genetically very similar, with the striking exception of their Y chromosomes, which have diverged tremendously. The male-specific region (MSY), representing the greater part of the Y chromosome, is inherited from father to son in a clonal fashion, with natural selection acting on the MSY as a unit. Positive selection might involve the performance of the MSY in spermatogenesis. Chimpanzees have a highly polygamous mating behavior, so that sperm competition is thought to provide a strong selective force acting on the Y chromosome in the chimpanzee lineage. In consequence of evolution of the heterologous sex chromosomes in mammals, meiotic sex chromosome inactivation (MSCI) results in a transcriptionally silenced XY body in male meiotic prophase, and subsequently also in postmeiotic repression of the sex chromosomes in haploid spermatids. This has evolved to a situation where MSCI has become a prerequisite for spermatogenesis. Here, by analysis of microarray testicular expression data representing a small number of male chimpanzees and men, we obtained information indicating that meiotic and postmeiotic X chromosome silencing might be more effective in chimpanzee than in human spermatogenesis. From this, we suggest that the remarkable reorganization of the chimpanzee Y chromosome, compared to the human Y chromosome, might have an impact on its meiotic interactions with the X chromosome and thereby on X chromosome silencing in spermatogenesis. Further studies will be required to address comparative functional aspects of MSCI in chimpanzee, human, and other placental mammals.

  17. Human embryonic stem cells as models for aneuploid chromosomal syndromes.

    Science.gov (United States)

    Biancotti, Juan-Carlos; Narwani, Kavita; Buehler, Nicole; Mandefro, Berhan; Golan-Lev, Tamar; Yanuka, Ofra; Clark, Amander; Hill, David; Benvenisty, Nissim; Lavon, Neta

    2010-09-01

    Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.

  18. Targeted sequencing of the human X chromosome exome.

    Science.gov (United States)

    Mondal, Kajari; Shetty, Amol Carl; Patel, Viren; Cutler, David J; Zwick, Michael E

    2011-10-01

    We used a RainDance Technologies (RDT) expanded content library to enrich the human X chromosome exome (2.5 Mb) from 26 male samples followed by Illumina sequencing. Our multiplex primer library covered 98.05% of the human X chromosome exome in a single tube with 11,845 different PCR amplicons. Illumina sequencing of 24 male samples showed coverage for 97% of the targeted sequences. Sequence from 2 HapMap samples confirmed missing data rates of 2-3% at sites successfully typed by the HapMap project, with an accuracy of at least ~99.5% as compared to reported HapMap genotypes. Our demonstration that a RDT expanded content library can efficiently enrich and enable the routine sequencing of the human X chromosome exome suggests a wide variety of potential research and clinical applications for this platform.

  19. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.

    Science.gov (United States)

    Ghezraoui, Hind; Piganeau, Marion; Renouf, Benjamin; Renaud, Jean-Baptiste; Sallmyr, Annahita; Ruis, Brian; Oh, Sehyun; Tomkinson, Alan E; Hendrickson, Eric A; Giovannangeli, Carine; Jasin, Maria; Brunet, Erika

    2014-09-18

    Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

  20. Chromosomal mapping of the human and murine orphan receptors ERRalpha (ESRRA) and ERRbeta (ESRRB) and identification of a novel human ERRalpha-related pseudogene.

    Science.gov (United States)

    Sladek, R; Beatty, B; Squire, J; Copeland, N G; Gilbert, D J; Jenkins, N A; Giguère, V

    1997-10-15

    The estrogen-related receptors ERRalpha and ERRbeta (formerly ERR1 and ERR2) form a subgroup of the steroid/thyroid/retinoid receptor family. ERRalpha and ERRbeta are homologous to the estrogen receptor and bind similar DNA targets; however, they are unable to activate gene transcription in response to estrogens. We have used interspecific backcross analysis to map the murine Estrra locus to chromosome 19 and Estrrb to mouse chromosome 12. Using fluorescence in situ hybridization, we have mapped the human ESRRA gene to chromosome 11q12-q13 and the human ESRRB gene to chromosome 14q24.3. In addition, we report the isolation of a processed human ERRalpha pseudogene mapping to chromosome 13q12.1. To our knowledge, this represents the first report of a pseudogene associated with a member of the nuclear receptor superfamily.

  1. Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways.

    Science.gov (United States)

    Ichijima, Yosuke; Sin, Ho-Su; Namekawa, Satoshi H

    2012-08-01

    Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX). This DDR pathway shares features with the somatic DDR pathway recognizing DNA replication stress in the S phase. Additionally, it is likely to be distinct from the DDR pathway that recognizes meiosis-specific double-strand breaks. This review article extensively discusses the underlying mechanism of sex chromosome inactivation.

  2. Chromosomal DNA probes for the identification of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity of cats.

    Science.gov (United States)

    Love, D N; Bailey, G D; Bastin, D

    1992-06-01

    A dot-blot hybridisation assay using isolated high molecular weight DNA as whole chromosomal probes of the cat pigmented asaccharolytic Bacteroides/Porphyromonas species was used against both purified high molecular weight DNA and DNA released on membranes from whole cells for the identification of B. salivosus and for its differentiation from the other anaerobic species isolated from normal and diseased mouths of cats and horses. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes (Boehringer-Mannheim). The whole chromosomal probes were specific--differentiating B. salivosus from a variety of species (including members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. Likewise, asaccharolytic black pigmented Group 2 strains were distinguishable from all strains tested. However, cat strains of P. gingivalis which show 68-76% DNA-DNA homology with human strain P. gingivalis ATCC 33277T, were not distinguishable from each other using either 32P-labelled or DIG-labelled probes. The minimum amount of pure Bacteroides DNA which could be detected by the 32P-labelled probe was 100-300 pg, while the amount of pure DNA detected by the DIG system was 1-3 mg after room temperature colour development for 1 h and 100-300 pg after 6 h colour development.

  3. Modified C-band technique for the analysis of chromosome abnormalities in irradiated human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Nakata, Akifumi; Akiyama, Miho; Yamada, Yuji [Biodosimetry Section, Department of Radiation Dosimetry, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yoshida, Mitsuaki A., E-mail: myoshida@cc.hirosaki-u.ac.jp [Biodosimetry Section, Department of Radiation Dosimetry, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2011-10-15

    A modified C-band technique was developed in order to analyze more accurately dicentric, tricentric, and ring chromosomes in irradiated human peripheral lymphocytes. Instead of the original method relying on treatment with barium hydroxide Ba(OH){sub 2}, C-bands were obtained using a modified form of heat treatment in formamide followed with DAPI staining. This method was tentatively applied to the analysis of dicentric chromosomes in irradiated human lymphocytes to examine its availability. The frequency of dicentric chromosome was almost the same with conventional Giemsa staining and the modified C-band technique. In the analysis using Giemsa staining, it is relatively difficult to identify the centromere on the elongated chromosomes, over-condensed chromosomes, fragment, and acentric ring. However, the modified C-band method used in this study makes it easier to identify the centromere on such chromosomes than with the use of Giemsa staining alone. Thus, the modified C-band method may give more information about the location of the centromere. Therefore, this method may be available and more useful for biological dose estimation due to the analysis of the dicentric chromosome in human lymphocytes exposed to the radiation. Furthermore, this method is simpler and faster than the original C-band protocol and fluorescence in situ hybridization (FISH) method with the centromeric DNA probe. - Highlights: > The dicentric (dic) assay is the most effective for the radiation biodosimetry. > It is important to recognize the centromere of the dic. > We improved a C-band technique based on heat denaturation. > This technique enables the accurate detection of a centromere. > This method may be available and more useful for biological dose estimation.

  4. The escherichia coli chromosome replication initiator protein, DnaA

    DEFF Research Database (Denmark)

    Nyborg, Malene

    The experimental work presented in this thesis involve mutational analysis of the DNA binding domain of the DnaA protein and analysis of the A184V substitution in the ATP area of domain III and other amino acid substitutions found in the DnaA5 and DnaA4G proteins....

  5. 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.

  6. Assignment of the protein kinase C [delta] polypeptide gene (PRKCD) to human chromosome 3 and mouse chromosome 14

    Energy Technology Data Exchange (ETDEWEB)

    Huppi, K.; Siwarski, D.; Goodnight, J.; Mischak, H. (Molecular Genetics Section Lab. of Genetics, Bethesda, MD (United States))

    1994-01-01

    The protein kinase C (pkc) enzymes are a family of serine-threonine protein kinases, each encoded by a distinct and separate gene. The chromosomal locations of human PRKCA, PRKCB, and PRKCG have previously been established. The authors now report that PRKCD, a novel member of the pkc gene family, maps to human chromosome 3. The chromosomal location of Pkcd has also been determined in the mouse by analysis of recombination frequency in an interspecific panel of back-cross mice. They find that the locus encoding pkcd resides proximal to nucleoside phosphorylase (Np-2) and Tcra on mouse chromosome 14 in a region syntenic with human 3p. 9 refs., 2 tabs.

  7. Assignment of the protein kinase C delta polypeptide gene (PRKCD) to human chromosome 3 and mouse chromosome 14.

    Science.gov (United States)

    Huppi, K; Siwarski, D; Goodnight, J; Mischak, H

    1994-01-01

    The protein kinase C (pkc) enzymes are a family of serine-threonine protein kinases, each encoded by a distinct and separate gene. The chromosomal locations of human PRKCA, PRKCB, and PRKCG have previously been established. We now report that PRKCD, a novel member of the pkc gene family, maps to human chromosome 3. The chromosomal location of Pkcd has also been determined in the mouse by analysis of recombination frequency in an interspecific panel of backcross mice. We find that the locus encoding pkcd resides proximal to nucleoside phosphorylase (Np-2) and Tcra on mouse chromosome 14 in a region syntenic with human 3p.

  8. Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S. [Institute of Genetics, Taipei (Taiwan, Province of China)

    1994-09-01

    Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.

  9. Nerve growth factor receptor gene is at human chromosome region 17q12-17q22, distal to the chromosome 17 breakpoint in acute leukemias

    Energy Technology Data Exchange (ETDEWEB)

    Huebner, K.; Isobe, M.; Chao, M.; Bothwell, M.; Ross, A.H.; Finan, J.; Hoxie, J.A.; Sehgal, A.; Buck, C.R.; Lanahan, A.

    1986-03-01

    Genomic and cDNA clones for the human nerve growth factor receptor have been used in conjunction with somatic cell hybrid analysis and in situ hybridization to localize the nerve growth factor receptor locus to human chromosome region 17q12-q22. Additionally, part, if not all, of the nerve growth factor receptor locus is present on the translocated portion of 17q (17q21-qter) from a poorly differential acute leukemia in which the chromosome 17 breakpoint was indistinguishable cytogenetically from the 17 breakpoint observed in the t(15;17)(q22;q21) translocation associated with acute promyelocytic leukemia. Thus the nerve growth factor receptor locus may be closely distal to the acute promyelocytic leukemia-associated chromosome 17 breakpoint at 17q21.

  10. [Loop organization of eukaryotic chromosomes and triple-stranded DNA structures].

    Science.gov (United States)

    Glazkov, M V

    2011-01-01

    To study possible involvement of polypurine and polypyrimidine DNA tracks potentially capable to form triple-stranded structures (H-form of DNA) in compactization of eukaryotic chromosomes a search in silico for "complementary" polypurine and polypyrimidine tracks within 12 eukaryotic gene nucleotide sequences was carried out. Polypurine and polypyrimidine tracks (10-11 b.p.) potentially capable of interacting with each other with the formation of triplex structures ("structurizing" regions) has been shown to be located in chromosomal locus of genes, predominantly in introns and flanking regions. In the case of in vivo realization of such DNA-DNA interactions the chromosomal gene domains can be folded into several small loops. An involvement of DNA triplexes in the chromosomal gene loci compactization may be associated with the gene functioning. The analogous analysis carried out for nucleotide sequences of long (LINE) and short (SINE) repeats dispersed over the genome as well as of satellite DNA has demonstrated a fundamental identity between mechanisms of chromosomal encoding and non-coding regions' compactization.

  11. Separation of Y-chromosomal haplotypes from male DNA mixtures via multiplex haplotype-specific extraction.

    Science.gov (United States)

    Rothe, Jessica; Nagy, Marion

    2015-11-01

    In forensic analysis, the interpretation of DNA mixtures is the subject of ongoing debate and requires expertise knowledge. Haplotype-specific extraction (HSE) is an alternative method that enables the separation of large chromosome fragments or haplotypes by using magnetic beads in conjunction with allele-specific probes. HSE thus allows physical separation of the components of a DNA mixture. Here, we present the first multiplex HSE separation of a Y-chromosomal haplotype consisting of six Yfiler short tandem repeat markers from a mixture of male DNA.

  12. Association of Chromosomal Telomere DNA With Nuclear Matrix in HeLa Cell

    Institute of Scientific and Technical Information of China (English)

    汪国顺; 罗文捷; 潘惟钧; 丁明孝; 翟中和

    1994-01-01

    Using Electron Spectroscopic Imaging (ESI), we visualized the in situ binding of nucleic acids to nuclear matrix and 3H-thymidine incorporation which indicates that a small partial DNA bound to nuclear matrix tightly. Furthermore we found that chromosomal telomere DNA could bind to nuclear matrix specifically by the dot and Southern hybridization. The result of the Southwestern blot suggests that telomere DNA has high affinity to lamin B, vimentin and some nuclear matrix proteins. Therefore, the nuclear matrix and lamina of HeLa cell are possibly associated with spatial organization and action of chromosome.

  13. Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability.

    Science.gov (United States)

    Doerfler, Lillian; Harris, Lorena; Viebranz, Emilie; Schmidt, Kristina H

    2011-10-31

    Genome instability is associated with human cancers and chromosome breakage syndromes, including Bloom's syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood. We show that spontaneous translocations that arise by nonallelic homologous recombination in DNA-damage-checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ) are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1's roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents. Our findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells.

  14. A screen for suppressors of gross chromosomal rearrangements identifies a conserved role for PLP in preventing DNA lesions.

    Directory of Open Access Journals (Sweden)

    Pamela Kanellis

    2007-08-01

    Full Text Available Genome instability is a hallmark of cancer cells. One class of genome aberrations prevalent in tumor cells is termed gross chromosomal rearrangements (GCRs. GCRs comprise chromosome translocations, amplifications, inversions, deletion of whole chromosome arms, and interstitial deletions. Here, we report the results of a genome-wide screen in Saccharomyces cerevisiae aimed at identifying novel suppressors of GCR formation. The most potent novel GCR suppressor identified is BUD16, the gene coding for yeast pyridoxal kinase (Pdxk, a key enzyme in the metabolism of pyridoxal 5' phosphate (PLP, the biologically active form of vitamin B6. We show that Pdxk potently suppresses GCR events by curtailing the appearance of DNA lesions during the cell cycle. We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint. Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase. Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

  15. Unique and universal features of Epsilonproteobacterial origins of chromosome replication and DnaA-DnaA box interactions

    Directory of Open Access Journals (Sweden)

    Pawel Jaworski

    2016-09-01

    Full Text Available In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts (oriC. While DnaA proteins share significant homology regardless of phylogeny, oriC regions exhibit more variable structures. The general architecture of oriCs is universal, i.e. they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of oriCs are shared by related species while being significantly different in unrelated bacteria. In this work, we characterised Epsilonproteobacterial oriC regions. Helicobacter pylori was the only species of the class for which oriC was characterised. A few unique features were found such as bipartite oriC structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual H. pylori oriC features raised questions of whether oriC structure and DnaA-DNA interactions are unique to this bacterium or they are common to related species. By in silico and in vitro analyses we identified putative oriCs in three Epsilonproteobacterial species: pathogenic Arcobacter butzleri, symbiotic Wolinella succinogenes and free-living Sulfurimonas denitrificans. We propose that oriCs typically co-localize with ruvC-dnaA-dnaN in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream (oriC1 and downstream (oriC2 of dnaA, and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in oriC2. Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial oriCs, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5

  16. Unique and Universal Features of Epsilonproteobacterial Origins of Chromosome Replication and DnaA-DnaA Box Interactions

    Science.gov (United States)

    Jaworski, Pawel; Donczew, Rafal; Mielke, Thorsten; Thiel, Marcel; Oldziej, Stanislaw; Weigel, Christoph; Zawilak-Pawlik, Anna

    2016-01-01

    In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts (oriC). While DnaA proteins share significant homology regardless of phylogeny, oriC regions exhibit more variable structures. The general architecture of oriCs is universal, i.e., they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of oriCs are shared by related species while being significantly different in unrelated bacteria. In this work, we characterized Epsilonproteobacterial oriC regions. Helicobacter pylori was the only species of the class for which oriC was characterized. A few unique features were found such as bipartite oriC structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual H. pylori oriC features raised questions of whether oriC structure and DnaA-DNA interactions are unique to this bacterium or whether they are common to related species. By in silico and in vitro analyses we identified putative oriCs in three Epsilonproteobacterial species: pathogenic Arcobacter butzleri, symbiotic Wolinella succinogenes, and free-living Sulfurimonas denitrificans. We propose that oriCs typically co-localize with ruvC-dnaA-dnaN in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream (oriC1) and downstream (oriC2) of dnaA, and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in oriC2. Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial oriCs, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5′-TTCAC-3

  17. Structural comparison and chromosomal localization of the human and mouse IL-13 genes

    Energy Technology Data Exchange (ETDEWEB)

    McKenzie, A.N.J.; Sato, A.; Doyle, E.L.; Zurawski, G. (DNAX Research Institute of Cellular and Molecular Biology, Palo Alto, CA (United States)); Li, X.; Milatovich, A.; Francke, U. (Stanford Univ. Medical School, CA (United States)); Largaespada, D.A.; Copeland, N.G.; Jenkins, N.A. (National Cancer Institute, Frederick, MD (United States))

    1993-06-15

    The genomic structure of the recently described cytokine IL-13 has been determined for both human and mouse genes. The nucleotide sequence of a 4.6-kb DNA segment of the human gene is described. The human IL-13 gene (IL 13) occurs as a single copy in the haploid genome and maps to human chromosome 5. A 4.3-kb DNA fragment of the mouse IL-13 gene (Il 13) has been sequenced and found to occur as a single copy, mapping to mouse chromosome 11. Intrachromosomal mapping studies revealed that both genes contain four exons and three introns and show a high degree of sequence identify throughout their length. Potential recognition sequences for transcription factors that are present in the 5'-flanking region and are conserved between both genes include IFN-responsive elements, binding sites for AP-1, AP-2, and AP-3, an NF-lL 6 site, and a TATA-like sequence. Both genes map to chromosomal locations adjacent to genes encoding other cytokines, including IL-3, GM-CSF, IL-5, and IL-4 suggesting that IL-13 is another member of this cytokine gene family that may have arisen by gene duplication. 26 refs., 5 figs., 3 tabs.

  18. Detection of Y chromosome microdeletions and mitochondrial DNA mutations in male infertility patients.

    Science.gov (United States)

    Güney, A I; Javadova, D; Kırac, D; Ulucan, K; Koc, G; Ergec, D; Tavukcu, H; Tarcan, T

    2012-04-27

    Infertility affects about 10-15% of all couples attempting pregnancy with infertility attributed to the male partner in approximately half of the cases. Proposed causes of male infertility include sperm motility disturbances, Y chromosome microdeletions, chromosomal abnormalities, single gene mutations, and sperm mitochondrial DNA (mtDNA) rearrangements. To investigate the etiology of decreased sperm fertility and motility of sperm and to develop an appropriate therapeutic strategy, the molecular basis of these defects must be elucidated. In this study, we aimed to reveal the relationships between the genetic factors including sperm mtDNA mutations, Y chromosome microdeletions, and sperm parameters that can be regarded as candidate factors for male infertility. Thirty men with a history of infertility and 30 fertile men were recruited to the study. Y chromosome microdeletions were analyzed by multiplex PCR. Mitochondrial genes ATPase6, Cytb, and ND1, were amplified by PCR and then analyzed by direct sequencing. No Y chromosome microdeletions were detected in either group. However, a total of 38 different nucleotide substitutions were identified in the examined mitochondrial genes in both groups, all of which are statistically non-significant. Fifteen substitutions caused an amino acid change and 12 were considered novel mutations. As a conclusion, mtDNA mutations and Y chromosome microdeletions in male infertility should be examined in larger numbers in order to clarify the effect of genetic factors.

  19. Chromosomal mapping of specific DNA gains and losses in solid tumors using comparative genomic hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Schrock, E.; Manoir, S. du; Speicher, M. [National Center for Human Genome Research, Bethesda, MD (United States)] [and others

    1994-09-01

    Comparative genomic hybridization (CGH) is a new molecular cytogenetic technique that is based on two color FISH and quantitative digital imaging microscopy. CGH is used to comprehensively survey tumor genomes for copy number changes and to determine the map position of amplification sites on normal reference chromosomes. CGH was used to analyze 107 different solid tumors, including 16 low grade astrocytomas, 15 recurrent astrocytic tumors, 13 high grade astrocytomas, 13 small cell lung cancers (SCLC), 14 breast cancer samples (7 diploid and 7 aneupoid tumors), 18 chromophobe renal cell carcinomas and 5 seminomas. Tumor DNA was extracted from frozen tissue, autopic material and formalin fixed, paraffin-embedded tissue samples. Our results revealed tumor specific gains and losses of certain chromosomes or chromosomal subregions (e.g., chromosomes 7 and 10 in glioblastomas, chromosomes 3 and 5 in SCLC). Numerous DNA-amplifications were mapped on reference metaphase and prometaphase chromosomes. The frequent amplification of the EGFR gene (malignant gliomas), protooncogenes of the myc family (SCLC) and of c-myc, int-2 and c-erbB2 (breast cancer) was confirmed. Many additional amplification sites, however, were mapped that were not described before. The results of CGH analysis were independently confirmed by means of cytogenetic banding analysis, interphase cytogenetics with region specific DNA-clones, Southern-Blot analysis, DNA-cytometry and studies of loss of heterozygosity.

  20. Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

    DEFF Research Database (Denmark)

    de Souza, S J; Camargo, A A; Briones, M R;

    2000-01-01

    by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48......Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central...... coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes...

  1. Unique signatures of natural background radiation on human Y chromosomes from Kerala, India.

    Directory of Open Access Journals (Sweden)

    Sanjay Premi

    Full Text Available BACKGROUND: The most frequently observed major consequences of ionizing radiation are chromosomal lesions and cancers, although the entire genome may be affected. Owing to its haploid status and absence of recombination, the human Y chromosome is an ideal candidate to be assessed for possible genetic alterations induced by ionizing radiation. We studied the human Y chromosome in 390 males from the South Indian state of Kerala, where the level of natural background radiation (NBR is ten-fold higher than the worldwide average, and that from 790 unexposed males as control. RESULTS: We observed random microdeletions in the Azoospermia factor (AZF a, b and c regions in >90%, and tandem duplication and copy number polymorphism (CNP of 11 different Y-linked genes in about 80% of males exposed to NBR. The autosomal homologues of Y-linked CDY genes largely remained unaffected. Multiple polymorphic copies of the Y-linked genes showing single Y-specific signals suggested their tandem duplication. Some exposed males showed unilocus duplication of DAZ genes resulting in six copies. Notably, in the AZFa region, approximately 25% of exposed males showed deletion of the DBY gene, whereas flanking genes USP9Y and UTY remained unaffected. All these alterations were detected in blood samples but not in the germline (sperm samples. CONCLUSIONS: Exposure to high levels of NBR correlated with several interstitial polymorphisms of the human Y chromosome. CNPs and enhanced transcription of the SRY gene after duplication are envisaged to compensate for the loss of Y chromosome in some cells. The aforesaid changes, confined to peripheral blood lymphocytes, suggest a possible innate mechanism protecting the germline DNA from the NBR. Genome analysis of a larger population focusing on greater numbers of genes may provide new insights into the mechanisms and risks of the resultant genetic damages. The present work demonstrates unique signatures of NBR on human Y chromosomes

  2. Positioning of human chromosomes in murine cell hybrids according to synteny.

    Science.gov (United States)

    Meaburn, Karen J; Newbold, Robert F; Bridger, Joanna M

    2008-12-01

    Chromosomes occupy non-random spatial positions in interphase nuclei. It remains unclear what orchestrates this high level of organisation. To determine how the nuclear environment influences the spatial positioning of chromosomes, we utilised a panel of stable mouse hybrid cell lines carrying a single, intact human chromosome. Eleven of 22 human chromosomes revealed an alternative location in hybrid nuclei compared to that of human fibroblasts, with the majority becoming more internally localised. Human chromosomes in mouse nuclei position according to neither their gene density nor size, but rather the position of human chromosomes in hybrid nuclei appears to mimic that of syntenic mouse chromosomes. These results suggest that chromosomes adopt the behaviour of their host species chromosomes and that the nuclear environment is an important determinant of the interphase positioning of chromosomes.

  3. Human phenol sulfotransferase STP2 gene: Molecular cloning, structural characterization, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, C.; Raftogianis, R.; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States)

    1996-05-01

    Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of {open_quotes}simple{close_quotes} planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs, STP2, as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans. STP2 spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of most STP2 exon-intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans, STM; a rat PST gene; a human estrogen ST (EST) gene, STE; and a guinea pig EST gene. The two initial STP2 exons, IA and IB, were identified by performing 5{prime}-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5{prime}untranslated region sequences. The two apparent 5{prime}-ons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements. STP2 was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization of STP2 will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissues. 63 refs., 7 figs., 1 tab.

  4. Repeat Sequences and Base Correlations in Human Y Chromosome Palindromes

    Institute of Scientific and Technical Information of China (English)

    Neng-zhi Jin; Zi-xian Liu; Yan-jiao Qi; Wen-yuan Qiu

    2009-01-01

    On the basis of information theory and statistical methods, we use mutual information, n-tuple entropy and conditional entropy, combined with biological characteristics, to analyze the long range correlation and short range correlation in human Y chromosome palindromes. The magnitude distribution of the long range correlation which can be reflected by the mutual information is P5>P5a>P5b (P5a and P5b are the sequences that replace solely Alu repeats and all interspersed repeats with random uncorrelated sequences in human Y chromosome palindrome 5, respectively); and the magnitude distribution of the short range correlation which can be reflected by the n-tuple entropy and the conditional entropy is P5>P5a>P5b>random uncorrelated sequence. In other words, when the Alu repeats and all interspersed repeats replace with random uncorrelated sequence, the long range and short range correlation decrease gradually. However, the random uncorrelated sequence has no correlation. This research indicates that more repeat sequences result in stronger correlation between bases in human Y chromosome. The analyses may be helpful to understand the special structures of human Y chromosome palindromes profoundly.

  5. Assignment of the human gene for the water channel of renal collecting duct Aquaporin 2 (AQP2) to chromosome 12 region q12-->q13

    NARCIS (Netherlands)

    Deen, P M; Weghuis, D O; Sinke, R J; Geurts van Kessel, A; Wieringa, B; van Os, C H

    1994-01-01

    The chromosomal localization of the gene encoding Aquaporin 2 (previously called WCH-CD), which acts as a water channel in the collecting tubules of the kidney, was determined. Southern blot hybridizations of chromosomal DNA from a panel of 25 different human-rodent hybrid cell lines assigned AQP2 t

  6. Distribution of 5S and 45S rDNA sites in plants with holokinetic chromosomes and the "chromosome field" hypothesis.

    Science.gov (United States)

    Sousa, A; Barros e Silva, A E; Cuadrado, A; Loarce, Y; Alves, M V; Guerra, M

    2011-08-01

    Secondary constrictions or 45S rDNA sites are commonly reported to be located mainly in the terminal regions of the chromosomes. This distribution has been assumed to be related to the existence of a "chromosome field" lying between the centromere and the telomere, an area in which certain cytogenetic events may predominantly occur. If this hypothesis is true this distribution should not be observed in holokinetic chromosomes, as they do not have a localized centromere. In order to evaluate this hypothesis, a comparative study was made of the distributions of 5S and 45S rDNA sites using fluorescence in situ hybridization in representatives of the genera Eleocharis, Diplacrum, Fimbristylis, Kyllinga and Rhynchospora, all of which belong to the family Cyperaceae. The numbers of sites per diploid chromosome complement varied from 2 to ∼10 for 5S rDNA, and from 2 to ∼45 for 45S rDNA. All of the 11 species analyzed had terminally located 45S rDNA sites on the chromosomes whereas the 5S rDNA sites also generally had terminal distributions, except for the Rhynchospora species, where their position was almost always interstitial. These results, together with other previously published data, suggest that the variation in the number and position of the rDNA sites in species with holokinetic chromosomes is non-random and similar to that reported for species with monocentric chromosomes. Therefore, the predominant terminal position of the 45S rDNA sites does not appear to be influenced by the centromere-telomere polarization as suggested by the "chromosome field" hypothesis. Additionally, the hybridization of 5S and 45S rDNA sites provides interesting markers to distinguish several chromosomes on the rather symmetrical karyotypes of Cyperaceae.

  7. Characterization of human PGD blastocysts with unbalanced chromosomal translocations and human embryonic stem cell line derivation?

    Science.gov (United States)

    Frydman, N; Féraud, O; Bas, C; Amit, M; Frydman, R; Bennaceur-Griscelli, A; Tachdjian, G

    2009-01-01

    Novel embryonic stem cell lines derived from embryos carrying structural chromosomal abnormalities obtained after preimplantation genetic diagnosis (PGD) are of interest to study in terms of the influence of abnormalities on further development. A total of 22 unbalanced blastocysts obtained after PGD were analysed for structural chromosomal defects. Morphological description and chromosomal status of these blastocysts was established and they were used to derive human embryonic stem cell (ESC) lines. An outgrowth of cells was observed for six blastocysts (6/22; 27%). For two blastocysts, the exact morphology was unknown since they were at early stage, and for four blastocysts, the inner cell mass was clearly visible. Fifteen blastocysts carried an unbalanced chromosomal defect linked to a reciprocal translocation, resulting in a positive outgrowth of cells for five blastocysts. One human ESC line was obtained from a blastocyst carrying a partial chromosome-21 monosomy and a partial chromosome-1 trisomy. Six blastocysts carried an unbalanced chromosomal defect linked to a Robertsonian translocation, and one showed a positive outgrowth of cells. One blastocyst carried an unbalanced chromosomal defect linked to an insertion and no outgrowth was observed. The efficiency of deriving human ESC lines with constitutional chromosomal disorders was low and probably depends on the initial morphological aspect of the blastocysts and/or the type of the chromosomal disorders.

  8. 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.

  9. Construction of a genetic map of human chromosome 17 by use of chromosome-mediated gene transfer

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Weiming; Gorman, P.A.; Rider, S.H.; Hedge, P.J.; Moore, G.; Prichard, C.; Sheer, D.; Solomon, E. (Imperial Cancer Research Fund, London (England))

    1988-11-01

    The authors used somatic-cell hybrids, containing as their only human genetic contribution part or all of chromosome 17, as donors for chromosome-mediated gene transfer. A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for >20 loci located on chromosome 17. By combining the data from this chromosome-mediated gene transfer transfectant panel, conventional somatic-cell hybrids containing well-defined breaks on chromosome 17, and in situ hybridization they propose the following order for these loci; pter-(TP53-RNP2-D17S1)-(MYH2-MYH1)-D17Z1-CRYB1-(ERBA1-GCSF-NGL)-acute promyelocytic leukemia breakpoint-RNU2-HOX2-(NGFR-COLIAI-MPO)-GAA-UMPH-GHC-TK1-GALK-qter. Using chromosome-mediated gene transfer, they have also regionally localized the random probes D17S6 to D17S19 on chromosome 17.

  10. The Sequence and Analysis of Duplication Rich Human Chromosome 16

    Science.gov (United States)

    Martin, Joel; Han, Cliff; Gordon, Laurie A.; Terry, Astrid; Prabhakar, Shyam; She, Xinwei; Xie, Gary; Hellsten, Uffe; Man Chan, Yee; Altherr, Michael; Couronne, Olivier; Aerts, Andrea; Bajorek, Eva; Black, Stacey; Blumer, Heather; Branscomb, Elbert; Brown, Nancy C.; Bruno, William J.; Buckingham, Judith M.; Callen, David F.; Campbell, Connie S.; Campbell, Mary L.; Campbell, Evelyn W.; Caoile, Chenier; Challacombe, Jean F.; Chasteen, Leslie A.; Chertkov, Olga; Chi, Han C.; Christensen, Mari; Clark, Lynn M.; Cohn, Judith D.; Denys, Mirian; Detter, John C.; Dickson, Mark; Dimitrijevic-Bussod, Mira; Escobar, Julio; Fawcett, Joseph J.; Flowers, Dave; Fotopulos, Dea; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Goodstein, David; Goodwin, Lynne A.; Grady, Deborah L.; Grigoriev, Igor; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Hildebrand, Carl E.; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Jewett, Phillip E.; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Krawczyk, Marie-Claude; Leyba, Tina; Longmire, Jonathan L.; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Ludeman, Thom; Mark, Graham A.; Mcmurray, Kimberly L.; Meincke, Linda J.; Morgan, Jenna; Moyzis, Robert K.; Mundt, Mark O.; Munk, A. Christine; Nandkeshwar, Richard D.; Pitluck, Sam; Pollard, Martin; Predki, Paul; Parson-Quintana, Beverly; Ramirez, Lucia; Rash, Sam; Retterer, James; Ricke, Darryl O.; Robinson, Donna L.; Rodriguez, Alex; Salamov, Asaf; Saunders, Elizabeth H.; Scott, Duncan; Shough, Timothy; Stallings, Raymond L.; Stalvey, Malinda; Sutherland, Robert D.; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Torney, David C.; Tran-Gyamfi, Mary; Tsai, Ming; Ulanovsky, Levy E.; Ustaszewska, Anna; Vo, Nu; White, P. Scott; Williams, Albert L.; Wills, Patricia L.; Wu, Jung-Rung; Wu, Kevin; Yang, Joan; DeJong, Pieter; Bruce, David; Doggett, Norman; Deaven, Larry; Schmutz, Jeremy; Grimwood, Jane; Richardson, Paul; et al.

    2004-01-01

    We report here the 78,884,754 base pairs of finished human chromosome 16 sequence, representing over 99.9 percent of its euchromatin. Manual annotation revealed 880 protein coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes and 3 RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobasepairs were identified and result in gene content differences across humans. One of the unique features of chromosome 16 is its high level of segmental duplication, ranked among the highest of the human autosomes. While the segmental duplications are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events which are likely to have had an impact on the evolution of primates and human disease susceptibility.

  11. The sequence and analysis of duplication rich human chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Joel; Han, Cliff; Gordon, Laurie A.; Terry, Astrid; Prabhakar, Shyam; She, Xinwei; Xie, Gary; Hellsten, Uffe; Man Chan, Yee; Altherr, Michael; Couronne, Olivier; Aerts, Andrea; Bajorek, Eva; Black, Stacey; Blumer, Heather; Branscomb, Elbert; Brown, Nancy C.; Bruno, William J.; Buckingham, Judith M.; Callen, David F.; Campbell, Connie S.; Campbell, Mary L.; Campbell, Evelyn W.; Caoile, Chenier; Challacombe, Jean F.; Chasteen, Leslie A.; Chertkov, Olga; Chi, Han C.; Christensen, Mari; Clark, Lynn M.; Cohn, Judith D.; Denys, Mirian; Detter, John C.; Dickson, Mark; Dimitrijevic-Bussod, Mira; Escobar, Julio; Fawcett, Joseph J.; Flowers, Dave; Fotopulos, Dea; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Goodstein, David; Goodwin, Lynne A.; Grady, Deborah L.; Grigoriev, Igor; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Hildebrand, Carl E.; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Jewett, Phillip E.; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Krawczyk, Marie-Claude; Leyba, Tina; Longmire, Jonathan L.; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Ludeman, Thom; Mark, Graham A.; Mcmurray, Kimberly L.; Meincke, Linda J.; Morgan, Jenna; Moyzis, Robert K.; Mundt, Mark O.; Munk, A. Christine; Nandkeshwar, Richard D.; Pitluck, Sam; Pollard, Martin; Predki, Paul; Parson-Quintana, Beverly; Ramirez, Lucia; Rash, Sam; Retterer, James; Ricke, Darryl O.; Robinson, Donna L.; Rodriguez, Alex; Salamov, Asaf; Saunders, Elizabeth H.; Scott, Duncan; Shough, Timothy; Stallings, Raymond L.; Stalvey, Malinda; Sutherland, Robert D.; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Torney, David C.; Tran-Gyamfi, Mary; Tsai, Ming; Ulanovsky, Levy E.; Ustaszewska, Anna; Vo, Nu; White, P. Scott; Williams, Albert L.; Wills, Patricia L.; Wu, Jung-Rung; Wu, Kevin; Yang, Joan; DeJong, Pieter; Bruce, David; Doggett, Norman; Deaven, Larry; Schmutz, Jeremy; Grimwood, Jane; Richardson, Paul; et al.

    2004-08-01

    We report here the 78,884,754 base pairs of finished human chromosome 16 sequence, representing over 99.9 percent of its euchromatin. Manual annotation revealed 880 protein coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes and 3 RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobasepairs were identified and result in gene content differences across humans. One of the unique features of chromosome 16 is its high level of segmental duplication, ranked among the highest of the human autosomes. While the segmental duplications are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events which are likely to have had an impact on the evolution of primates and human disease susceptibility.

  12. Report on the Second International Workshop on Human Chromosome 9

    Energy Technology Data Exchange (ETDEWEB)

    Kwiatkowski, D.J. [Brigham and Women`s Hospital, Boston, MA (United States); Armour, J. [Univ. of Leicester (England). Dept. of Genetics; Bale, A.E. [Yale Univ., New Haven, CT (United States). Dept. of Genetics] [and others

    1993-12-31

    The Second International Workshop on Human Chromosome 9 was held in Chatham, Massachusetts on April 18--20, 1993. Fifty-three abstracts were received and the data presented on posters. The purpose of the meeting was to bring together all interested investigators working on the map of chromosome 9, many of whom had disease-specific interests. After a brief presentation of interests and highlighted results, the meeting broke up into the following subgroups for production of consensus maps: 9p; 9cen-q32; 9q32 ter. A global mapping group also met. Reports of each of these working groups is presented in the summary.

  13. Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

    2007-02-07

    The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

  14. Reactivation of chromosomally integrated human herpesvirus-6 by telomeric circle formation.

    Directory of Open Access Journals (Sweden)

    Bhupesh K Prusty

    Full Text Available More than 95% of the human population is infected with human herpesvirus-6 (HHV-6 during early childhood and maintains latent HHV-6 genomes either in an extra-chromosomal form or as a chromosomally integrated HHV-6 (ciHHV-6. In addition, approximately 1% of humans are born with an inheritable form of ciHHV-6 integrated into the telomeres of chromosomes. Immunosuppression and stress conditions can reactivate latent HHV-6 replication, which is associated with clinical complications and even death. We have previously shown that Chlamydia trachomatis infection reactivates ciHHV-6 and induces the formation of extra-chromosomal viral DNA in ciHHV-6 cells. Here, we propose a model and provide experimental evidence for the mechanism of ciHHV-6 reactivation. Infection with Chlamydia induced a transient shortening of telomeric ends, which subsequently led to increased telomeric circle (t-circle formation and incomplete reconstitution of circular viral genomes containing single viral direct repeat (DR. Correspondingly, short t-circles containing parts of the HHV-6 DR were detected in cells from individuals with genetically inherited ciHHV-6. Furthermore, telomere shortening induced in the absence of Chlamydia infection also caused circularization of ciHHV-6, supporting a t-circle based mechanism for ciHHV-6 reactivation.

  15. DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage.

    OpenAIRE

    Holm, C.; Stearns, T.; Botstein, D

    1989-01-01

    The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These resul...

  16. Molecular structure and chromosomal mapping of the human homolog of the agouti gene

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, H.Y.; Woychik, R.P. [Oak Ridge National Lab., TN (United States); Bultman, S.J. [Oak Ridge National Lab., TN (United States)]|[Univ. of Tennessee, Oak Ridge, TN (United States); Loeffler, C.; Hansmann, I. [Universitaet Goettingen (Germany); Chen, W.J.; Furdon, P.J.; Wilkison, W. [Glaxo Research Institute, Research Triangle Park, NC (United States); Powell, J.G.; Usala, A.L. [Eastern Carolina Univ., Greenville, NC (United States)

    1994-10-11

    The agouti (a) locus in mouse chromosome 2 normally regulates coat color pigmentation. The mouse agouti gene was recently cloned and shown to encode a distinctive 131-amino acid protein with a consensus signal peptide. Here the authors describe the cloning of the human homolog of the mouse agouti gene using an interspecies DNA-hybridization approach. Sequence analysis revealed that the coding region of the human agouti gene is 85% identical to the mouse gene and has the potential to encode a protein of 132 amino acids with a consensus signal peptide. Chromosomal assignment using somatic-cell-hybrid mapping panels and fluorescence in situ hybridization demonstrated that the human agouti gene maps to chromosome band 20q11.2. This result revealed that the human agouti gene is closely linked to several traits, including a locus called MODY (for maturity onset diabetes of the young) and another region that is associated with the development of myeloid leukemia. Initial expression studies with RNA from several adult human tissues showed that the human agouti gene is expressed in adipose tissue and testis.

  17. Systematic analysis of human protein complexes identifies chromosome segregation proteins.

    Science.gov (United States)

    Hutchins, James R A; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A; Peters, Jan-Michael

    2010-04-30

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.

  18. Chromosomal localization of ribosomal DNA sequences in an apple rootstock using a digoxygenin detection system

    Institute of Scientific and Technical Information of China (English)

    ZHUJIMEI; SEGARDINER

    1995-01-01

    A 6kb rDNA probe comprising the 18S coding plus spacer sequences has been hybridized to the metaphase chromosomes of apple rootstock cultivar MM106 demonstrating the localization of ribosomal gene arrays in the vicinity of the telomeric regions of the short arms of chromosomes 6 and 14.The in situ results using digoxygenin labelling coupled to an alkaline phosphatase immunoassay were confirmed by silver staining for NORs and nucleoli.This study demonstrates the feasibility of molecular cytogenetic analysis of very small chromosomes(1.0-2.7μm) of apple.

  19. Chromosomal bands affected by acute oil exposure and DNA repair errors.

    Directory of Open Access Journals (Sweden)

    Gemma Monyarch

    Full Text Available BACKGROUND: In a previous study, we showed that individuals who had participated in oil clean-up tasks after the wreckage of the Prestige presented an increase of structural chromosomal alterations two years after the acute exposure had occurred. Other studies have also reported the presence of DNA damage during acute oil exposure, but little is known about the long term persistence of chromosomal alterations, which can be considered as a marker of cancer risk. OBJECTIVES: We analyzed whether the breakpoints involved in chromosomal damage can help to assess the risk of cancer as well as to investigate their possible association with DNA repair efficiency. METHODS: Cytogenetic analyses were carried out on the same individuals of our previous study and DNA repair errors were assessed in cultures with aphidicolin. RESULTS: Three chromosomal bands, 2q21, 3q27 and 5q31, were most affected by acute oil exposure. The dysfunction in DNA repair mechanisms, expressed as chromosomal damage, was significantly higher in exposed-oil participants than in those not exposed (p= 0.016. CONCLUSION: The present study shows that breaks in 2q21, 3q27 and 5q31 chromosomal bands, which are commonly involved in hematological cancer, could be considered useful genotoxic oil biomarkers. Moreover, breakages in these bands could induce chromosomal instability, which can explain the increased risk of cancer (leukemia and lymphomas reported in chronically benzene-exposed individuals. In addition, it has been determined that the individuals who participated in clean-up of the oil spill presented an alteration of their DNA repair mechanisms two years after exposure.

  20. Biodosimetry of ionizing radiation by selective painting of prematurely condensed chromosomes in human lymphocytes

    Science.gov (United States)

    Durante, M.; George, K.; Yang, T. C.

    1997-01-01

    Painting of interphase chromosomes can be useful for biodosimetric purposes in particular cases such as radiation therapy, accidental exposure to very high radiation doses and exposure to densely ionizing radiation, for example during space missions. Biodosimetry of charged-particle radiation is analyzed in the present paper. Target cells were human peripheral blood lymphocytes irradiated in vitro with gamma rays, protons and iron ions. After exposure, lymphocytes were incubated for different times to allow repair of radiation-induced damage and then fused to mitotic hamster cells to promote premature condensation in the interphase chromosomes. Chromosome spreads were then hybridized with whole-chromosome DNA probes labeled with fluorescent stains. Dose-response curves for the induction of chromatin fragments shortly after exposure, as well as the kinetics of rejoining and misrejoining, were not markedly dependent on linear energy transfer. However, after exposure to heavy ions, more aberrations were scored in the interphase cells after incubation for repair than in metaphase samples harvested at the first postirradiation mitosis. On the other hand, no significant differences were observed in the two samples after exposure to sparsely ionizing radiation. These results suggest that interphase chromosome painting can be a useful tool for biodosimetry of particle radiation.

  1. v-Src Causes Chromosome Bridges in a Caffeine-Sensitive Manner by Generating DNA Damage.

    Science.gov (United States)

    Ikeuchi, Masayoshi; Fukumoto, Yasunori; Honda, Takuya; Kuga, Takahisa; Saito, Youhei; Yamaguchi, Naoto; Nakayama, Yuji

    2016-06-02

    An increase in Src activity is commonly observed in epithelial cancers. Aberrant activation of the kinase activity is associated with malignant progression. However, the mechanisms that underlie the Src-induced malignant progression of cancer are not completely understood. We show here that v-Src, an oncogene that was first identified from a Rous sarcoma virus and a mutant variant of c-Src, leads to an increase in the number of anaphase and telophase cells having chromosome bridges. v-Src increases the number of γH2AX foci, and this increase is inhibited by treatment with PP2, a Src kinase inhibitor. v-Src induces the phosphorylation of KAP1 at Ser824, Chk2 at Thr68, and Chk1 at Ser345, suggesting the activation of the ATM/ATR pathway. Caffeine decreases the number of cells having chromosome bridges at a concentration incapable of inhibiting Chk1 phosphorylation at Ser345. These results suggest that v-Src induces chromosome bridges via generation of DNA damage and the subsequent DNA damage response, possibly by homologous recombination. A chromosome bridge gives rise to the accumulation of DNA damage directly through chromosome breakage and indirectly through cytokinesis failure-induced multinucleation. We propose that v-Src-induced chromosome bridge formation is one of the causes of the v-Src-induced malignant progression of cancer cells.

  2. The cohesion stabilizer sororin favors DNA repair and chromosome segregation during mouse oocyte meiosis.

    Science.gov (United States)

    Huang, Chun-Jie; Yuan, Yi-Feng; Wu, Di; Khan, Faheem Ahmed; Jiao, Xiao-Fei; Huo, Li-Jun

    2017-03-01

    Maintenance and timely termination of cohesion on chromosomes ensures accurate chromosome segregation to guard against aneuploidy in mammalian oocytes and subsequent chromosomally abnormal pregnancies. Sororin, a cohesion stabilizer whose relevance in antagonizing the anti-cohesive property of Wings-apart like protein (Wapl), has been characterized in mitosis; however, the role of Sororin remains unclear during mammalian oocyte meiosis. Here, we show that Sororin is required for DNA damage repair and cohesion maintenance on chromosomes, and consequently, for mouse oocyte meiotic program. Sororin is constantly expressed throughout meiosis and accumulates on chromatins at germinal vesicle (GV) stage/G2 phase. It localizes onto centromeres from germinal vesicle breakdown (GVBD) to metaphase II stage. Inactivation of Sororin compromises the GVBD and first polar body extrusion (PBE). Furthermore, Sororin inactivation induces DNA damage indicated by positive γH2AX foci in GV oocytes and precocious chromatin segregation in MII oocytes. Finally, our data indicate that PlK1 and MPF dissociate Sororin from chromosome arms without affecting its centromeric localization. Our results define Sororin as a determinant during mouse oocyte meiotic maturation by favoring DNA damage repair and chromosome separation, and thereby, maintaining the genome stability and generating haploid gametes.

  3. Chromosomal aberrations related to metastasis of human solid tumors

    Institute of Scientific and Technical Information of China (English)

    Lun-Xiu Qin

    2002-01-01

    The central role of sequential accumulation of genetic alterations during the development of cancer has been firmly established since the pioneering cytogenetic studies successfully defined recurrent chromosome changes in spedfic types of tumor. In the course of carcinogenesis, cells experience several genetic alterations that are associated with the transition from a preneoplastic lesion to an invasive tumor and finally to the metastatic state. Tumor progression is characterized by stepwise accumulation of genetic alterations.So does the dominant metastatic clone. Modern molecular genetic analyses have clarified that genomic changes accumulate during the development and progression of cancers. In comparison with the corresponding primary tumor,additional events of chromosomal aberrations (including gains or allelic losses) are frequently found in metastases, and the incidence of combined chromosomal alterations in the primary tumor, plus the occurrence of additional aberrations inthe distant metastases, correlated significantly with decreased postmetastatic survival. The deletions at 3p, 4p, 6q, 8p, 10q,11p, 11q, 12p, 13q, 16q, 17p, 18q, 21q, and 22q, as well as the over-representations at 1q, 8q, 9q, 14q and 15q, have been found to associate preferentially with the metastatic phenotype of human cancers. Among of them, the deletions on chromosomes 8p, 17p, 11p and 13p seem to be more significant, and more detail fine regions of them, including 8p11, 8p21-12, 8p22, 8p23, 17p13.3, 11p15.5, and 13q12-13 have been suggested harboring metastasis-suppressor genes.During the past decade, several human chromosomes have been functionally tested through the use of microcell-mediated chromosome transfer (MMCT), and metastasis-suppressor activities have been reported on chromosomes 1, 6, 7, 8, 10,11, 12, 16, and 17. However, it is not actually known at what stage of the metastatic cascade these alterations have occurred.There is still controversial with the association

  4. Human interphase chromosomes: a review of available molecular cytogenetic technologies

    Directory of Open Access Journals (Sweden)

    Yurov Yuri B

    2010-01-01

    Full Text Available Abstract Human karyotype is usually studied by classical cytogenetic (banding techniques. To perform it, one has to obtain metaphase chromosomes of mitotic cells. This leads to the impossibility of analyzing all the cell types, to moderate cell scoring, and to the extrapolation of cytogenetic data retrieved from a couple of tens of mitotic cells to the whole organism, suggesting that all the remaining cells possess these genomes. However, this is far from being the case inasmuch as chromosome abnormalities can occur in any cell along ontogeny. Since somatic cells of eukaryotes are more likely to be in interphase, the solution of the problem concerning studying postmitotic cells and larger cell populations is interphase cytogenetics, which has become more or less applicable for specific biomedical tasks due to achievements in molecular cytogenetics (i.e. developments of fluorescence in situ hybridization -- FISH, and multicolor banding -- MCB. Numerous interphase molecular cytogenetic approaches are restricted to studying specific genomic loci (regions being, however, useful for identification of chromosome abnormalities (aneuploidy, polyploidy, deletions, inversions, duplications, translocations. Moreover, these techniques are the unique possibility to establish biological role and patterns of nuclear genome organization at suprachromosomal level in a given cell. Here, it is to note that this issue is incompletely worked out due to technical limitations. Nonetheless, a number of state-of-the-art molecular cytogenetic techniques (i.e multicolor interphase FISH or interpahase chromosome-specific MCB allow visualization of interphase chromosomes in their integrity at molecular resolutions. Thus, regardless numerous difficulties encountered during studying human interphase chromosomes, molecular cytogenetics does provide for high-resolution single-cell analysis of genome organization, structure and behavior at all stages of cell cycle.

  5. Arrested human embryos are more likely to have abnormal chromosomes than developing embryos from women of advanced maternal age.

    Science.gov (United States)

    Qi, Shu-Tao; Liang, Li-Feng; Xian, Ye-Xing; Liu, Jian-Qiao; Wang, Weihua

    2014-01-01

    Aneuploidy is one of the major factors that result in low efficiency in human infertility treatment by in vitro fertilization (IVF). The development of DNA microarray technology allows for aneuploidy screening by analyzing all 23 pairs of chromosomes in human embryos. All chromosome screening for aneuploidy is more accurate than partial chromosome screening, as errors can occur in any chromosome. Currently, chromosome screening for aneuploidy is performed in developing embryos, mainly blastocysts. It has not been performed in arrested embryos and/or compared between developing embryos and arrested embryos from the same IVF cycle. The present study was designed to examine all chromosomes in blastocysts and arrested embryos from the same cycle in patients of advanced maternal ages. Embryos were produced by routine IVF procedures. A total of 90 embryos (45 blastocysts and 45 arrested embryos) from 17 patients were biopsied and analyzed by the Agilent DNA array platform. It was found that 50% of the embryos developed to blastocyst stage; however, only 15.6% of the embryos (both blastocyst and arrested) were euploid, and most (84.4%) of the embryos had chromosomal abnormalities. Further analysis indicated that 28.9% of blastocysts were euploid and 71.1% were aneuploid. By contrast, only one (2.2%) arrested embryo was euploid while others (97.8%) were aneuploid. The prevalence of multiple chromosomal abnormalities in the aneuploid embryos was also higher in the arrested embryos than in the blastocysts. These results indicate that high proportions of human embryos from patients of advanced maternal age are aneuploid, and the arrested embryos are more likely to have abnormal chromosomes than developing embryos.

  6. Synteny mapping of five human chromosome 7 genes on bovine chromosomes 4 and 21.

    Science.gov (United States)

    Antoniou, E; Womack, J E; Grosz, M D

    1999-01-01

    Five genes on human chromosome 7 (HSA 7) were assigned to bovine chromosome 21 (BTA 21) and 4 (BTA 4) using a bovine-rodent somatic hybrid cell panel. These five genes were alpha-I subunit of adenylate cyclase-inhibiting G-protein (GNAI1), alpha/beta preprotachykinin (TAC1), reelin (RELN), c-AMP dependant protein kinase type II beta regulatory chain (PRKAR2B) and apolipoprotein A1 regulatory protein 1 (TFCOUP2). Four genes mapped to BTA 4 (GNAI1, TAC1, RELN, PRKAR2B) while one gene mapped to BTA 21 (TFCOUP2). This study confirms the synteny conservation between HSA 7 and BTA 4, finely maps the breakpoints of conserved synteny on HSA 7 and defines a new synteny conservation between HSA 7 and BTA 21.

  7. Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos

    Directory of Open Access Journals (Sweden)

    Remi Sonneville

    2015-07-01

    Full Text Available During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2–7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication.

  8. FISH-mapped CEPH YACs spanning 0 to 46 cM on human chromosome 6

    Energy Technology Data Exchange (ETDEWEB)

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

    1996-08-15

    Seventy-six CEPH YACs were mapped by fluorescence in situ hybridization (FISH) to human metaphase chromosomes. These clones have been ordered from pter to 46 cM by combining the results of FISH with sequence-tagged site content mapping using data from the public databases. This created a minimal tiling path containing at least 37 Mb of human genomic DNA from 0 to 46 cM on chromosome 6 that contains up to four gaps not greater than 200 kb. These data provide an integration of the FLpter physical map values with cytogenetic band localization and markers on the genetic and radiation hybrid maps. We also assessed YAC chimerism and placed three additional Whitehead contigs within the integrated map. 27 refs., 1 fig., 1 tab.

  9. Characterization of cDNAs encoding human leukosialin and localization of the leukosialin gene to chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Pallant, A.; Eskenazi, A.; Frelinger, J.G. (Univ. of Rochester Medical Center, NY (USA)); Mattei, M.G. (Hopital d' Enfants de la Timone, Marseille (France)); Fournier, R.E.K. (Fred Hutchinson Cancer Research Center, Seattle, WA (USA)); Carlsson, S.R.; Fukuda, M. (La Jolla Center Research Foundation, CA (USA))

    1989-02-01

    The authors describe the isolation and characterization of cDNA clones encoding human leukosialin, a major sialoglycoprotein of human leukocytes. Leukosialin is very closely related or identical to the sialophorin molecule, which is involved in T-cell proliferation and whose expression is altered in Wiskott-Aldrich syndrome (WAS), an X-chromosome-linked immunodeficiency disease. Using a rabbit antiserum to leukosialin, a cDNA clone was isolated from a {lambda}gt11 cDNA library constructed from human peripheral blood cells. The {lambda}gt11 clone was used to isolate longer cDNA clones that correspond to the entire coding sequence of leukosialin. DNA sequence analysis reveals three domains in the predicted mature protein. The extracellular domain is enriched for Ser, Thr, and Pro and contains four contiguous 18-amino acid repeats. The transmembrane and intracellular domains of the human leukosialin molecule are highly homologous to the rat W3/13 molecule. RNA gel blot analysis reveals two polyadenylylated species of 2.3 and 8 kilobases. Southern blot analysis suggests that human leukosialin is a single-copy gene. Analysis of monochromosomal cell hybrids indicates that the leukosialin gene is not X chromosome linked and in situ hybridization shows leukosialin is located on chromosome 16. These findings demonstrate that the primary mutation in WAS is not a defect in the structural gene for leukosialin.

  10. Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli

    DEFF Research Database (Denmark)

    Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid

    2016-01-01

    Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. Dna...... replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology...... in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on ori...

  11. Genetic variation of the East Balkan Swine (Sus scrofa) in Bulgaria, revealed by mitochondrial DNA and Y chromosomal DNA.

    Science.gov (United States)

    Hirata, D; Doichev, V D; Raichev, E G; Palova, N A; Nakev, J L; Yordanov, Y M; Kaneko, Y; Masuda, R

    2015-04-01

    East Balkan Swine (EBS) Sus scrofa is the only aboriginal domesticated pig breed in Bulgaria and is distributed on the western coast of the Black Sea in Bulgaria. To reveal the breed's genetic characteristics, we analysed mitochondrial DNA (mtDNA) and Y chromosomal DNA sequences of EBS in Bulgaria. Nucleotide diversity (πn ) of the mtDNA control region, including two newly found haplotypes, in 54 EBS was higher (0.014 ± 0.007) compared with that of European (0.005 ± 0.003) and Asian (0.006 ± 0.003) domestic pigs and wild boar. The median-joining network based on the mtDNA control region showed that the EBS and wild boar in Bulgaria comprised mainly two major mtDNA clades, European clade E1 (61.3%) and Asian clade A (38.7%). The coexistence of two mtDNA clades in EBS in Bulgaria may be the relict of historical pig translocation. Among the Bulgarian EBS colonies, the geographical differences in distribution of two mtDNA clades (E1 and A) could be attributed to the source pig populations and/or historical crossbreeding with imported pigs. In addition, analysis of the Y chromosomal DNA sequences for the EBS revealed that all of the EBS had haplotype HY1, which is dominant in European domestic pigs.

  12. 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.

  13. DNA marker mining of ILSTS035 microsatellite locus on chromosome 6 of Hanwoo cattle

    Indian Academy of Sciences (India)

    Jung-Sou Yeo; Jea-Young Lee; Jae-Woo Kim

    2004-12-01

    We describe tests for detecting and locating quantitative trait loci (QTL) for traits in Hanwoo cattle. From results of a permutation test to detect QTL for marbling, we selected the microsatellite locus ILSTS035 on chromosome 6 for further analysis. -means clustering analysis applied to five traits and nine DNA markers in ILSTS035 resulted in three cluster groups. Finally we employed the bootstrap test method to calculate confidence intervals using the resampling method to find major DNA markers. We conclude that the major markers of ILSTS035 locus on chromosome 6 of Hanwoo cattle are markers 235 bp and 266 bp.

  14. Genetic integrity of the human Y chromosome exposed to groundwater arsenic

    Directory of Open Access Journals (Sweden)

    Ali Sher

    2010-08-01

    Full Text Available Abstract Background Arsenic is a known human carcinogen reported to cause chromosomal deletions and genetic anomalies in cultured cells. The vast human population inhabiting the Ganges delta in West Bengal, India and Bangladesh is exposed to critical levels of arsenic present in the groundwater. The genetic and physiological mechanism of arsenic toxicity in the human body is yet to be fully established. In addition, lack of animal models has made work on this line even more challenging. Methods Human male blood samples were collected with their informed consent from 5 districts in West Bengal having groundwater arsenic level more than 50 μg/L. Isolation of genomic DNA and preparation of metaphase chromosomes was done using standard protocols. End point PCR was performed for established sequence tagged sites to ascertain the status of recombination events. Single nucleotide variants of candidate genes and amplicons were carried out using appropriate restriction enzymes. The copy number of DYZ1 array per haploid genome was calculated using real time PCR and its chromosomal localization was done by fluorescence in-situ hybridization (FISH. Results We studied effects of arsenic exposure on the human Y chromosome in males from different areas of West Bengal focusing on known recombination events (P5-P1 proximal; P5-P1 distal; gr/gr; TSPY-TSPY, b1/b3 and b2/b3, single nucleotide variants (SNVs of a few candidate Y-linked genes (DAZ, TTY4, BPY2, GOLGA2LY and the amplicons of AZFc region. Also, possible chromosomal reorganization of DYZ1 repeat arrays was analyzed. Barring a few microdeletions, no major changes were detected in blood DNA samples. SNV analysis showed a difference in some alleles. Similarly, DYZ1 arrays signals detected by FISH were found to be affected in some males. Conclusions Our Y chromosome analysis suggests that the same is protected from the effects of arsenic by some unknown mechanisms maintaining its structural and functional

  15. High similarity of U2 snDNA sequence between A and B chromosomes in the grasshopper Abracris flavolineata.

    Science.gov (United States)

    Menezes-de-Carvalho, Nahanna Zimmermann; Palacios-Gimenez, Octavio Manuel; Milani, Diogo; Cabral-de-Mello, Diogo Cavalcanti

    2015-10-01

    B chromosomes are frequently enriched for a wide variety of repetitive DNAs. Among grasshoppers in the species Abracris flavolineata (Ommatolampidinae) the B chromosomes are submetacentric, C-negative and harbor repetitive DNAs such as, U2 snDNA, C 0 t-1 DNA, two Mariner-like elements and some microsatellites. Here, we provide evidence showing the intragenome similarity between the B chromosome and the A complement in A. flavolineata, combining analysis of microdissection and chromosome painting and B chromosome-specific amplification through polymerase chain reaction (PCR) of U2 snDNA. Chromosome painting revealed signals spread through the C-negative regions, including the A and B chromosomes. Moreover, significant clustered signals forming bands were observed in some A chromosomes, and for the B chromosome, significant signals were located on both arms, which could be caused by accumulation of repetitive DNA sequences. The C-positive regions did not reveal any signals. Sequence comparison of U2 snDNA between that obtained from a genome without the B chromosome and that from µB-DNA revealed high similarity with the occurrence of four shared haplotypes, one of them (i.e., Hap1) being highly prevalent and putatively ancestral. The highest divergence from Hap1 was observed for Hap3, which was caused by only six mutational steps. These data support an intraspecific origin of the B chromosome in A. flavolineata that is highly similar with the A complement, and the low U2 snDNA sequence diversity observed in the B chromosome could be related to its recent origin, besides intrachromosomal concerted evolution for U2 snDNA repeats in the B chromosome.

  16. Chromosome alterations in breast carcinomas: frequent involvement of DNA losses including chromosomes 4q and 21q.

    OpenAIRE

    Schwendel, A.; Richard, F.; Langreck, H.; Kaufmann, O.; Lage, H.; Winzer, K.J.; Petersen, I; Dietel, M

    1998-01-01

    Comparative genomic hybridization was applied to map DNA gains and losses in 39 invasive ductal breast carcinomas. Frequent abnormalities included gains on chromosomal regions 1q, 8q, 11q12-13, 16p, 19, 20q and X as well as frequent losses on 1p, 5q, 6q, 9p, 11q, 13q and 16q. Furthermore, frequent losses on 4q (20 cases) and 21q (14 cases) were found for the first time in this tumour type. High copy number amplifications were observed at 8q12-24, 11q11-13 and 20q13-ter. Highly differentiated ...

  17. DNA and bone structure preservation in medieval human skeletons.

    Science.gov (United States)

    Coulson-Thomas, Yvette M; Norton, Andrew L; Coulson-Thomas, Vivien J; Florencio-Silva, Rinaldo; Ali, Nadir; Elmrghni, Samir; Gil, Cristiane D; Sasso, Gisela R S; Dixon, Ronald A; Nader, Helena B

    2015-06-01

    Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified.

  18. Evaluating the relationship between spermatogenic silencing of the X chromosome and evolution of the Y chromosome in chimpanzee and human

    NARCIS (Netherlands)

    E.M. Achame; W.M. Baarends (Willy); J.H. Gribnau (Joost); J.A. Grootegoed (Anton)

    2010-01-01

    textabstractChimpanzees and humans are genetically very similar, with the striking exception of their Y chromosomes, which have diverged tremendously. The male-specific region (MSY), representing the greater part of the Y chromosome, is inherited from father to son in a clonal fashion, with natural

  19. Prognostic value of DNA alterations on chromosome 17p13.2 for intrahepatic cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)

    Ubol Chuensumran; Sopit Wongkham; Chawalit Pairojkul; Siri Chauin; Songsak Petmitr

    2007-01-01

    AIM: To characterize and evaluate DNA alterations among intrahepatic cholangiocarcinoma (ICC) patients.METHODS: DNA from tumor and corresponding normal tissues of 52 patients was amplified with 33 arbitrary primers. The DNA fragment that alters most frequently in ICC was cloned, sequenced, and identified by comparison with known nucleotide sequences in the genome database (www.ncbi.nlm.nih.gov). The DNA copy numbers of the allelic alterations in cholangiocarcinoma were determined by quantitative real-time PCR and interpreted as allelic loss or DNA amplification by comparison with the reference gene. Associations between allelic imbalance and clinicopathological parameters of ICC patients were evaluated by x2-test.The Kaplan-Meier method was used to analyze survival rates.RESULTS: From 33 primers, an altered DNA fragment (518 bp) amplified from BC17 random primer was found frequently in the tumors analyzed and mapped to chromosome 17p13.2. Sixteen of 52 (31%) cases showed DNA amplification, while 7 (13%) showed allelic loss. Interestingly, DNA amplification on chromosome 17p13.2 was associated with a good prognosis, median survival time (wk) of amp vs no amp was 44.14 vs 24.14,P = 0.002; whereas allelic loss of this DNA sequence corresponded with a poor prognosis, median survival time (wk) of loss vs no loss was 18.00 vs 28.71, P =0.019). Moreover, Kaplan-Meier curves comparing the DNA alterations with survival depicted highly significant separation that the median survival time equal to DNA amplification, allelic loss, and normal was 44.14 wk,18.00 wk, and 24.29 wk, respectively (P = 0.005).CONCLUSION: Alterations in the DNA sequence on chromosome 17p13.2 may be involved in cholangiocarcinogenesis, and could be used as a prognostic marker in the treatment of ICC patients.

  20. Molecular organization and chromosomal localization of 5S rDNA in Amazonian Engystomops (Anura, Leiuperidae).

    Science.gov (United States)

    Rodrigues, Débora Silva; Rivera, Miryan; Lourenço, Luciana Bolsoni

    2012-03-20

    For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA

  1. Genetic diversity within Streptococcus mutans evident from chromosomal DNA restriction fragment polymorphisms.

    Science.gov (United States)

    Caufield, P W; Walker, T M

    1989-02-01

    Attempts to study the acquisition, transmission, and other aspects of the natural history of Streptococcus mutans infections in humans have been hampered by limitations and inconsistencies in methods by which phenotypic characteristics of individual isolates are examined. Because most mutans streptococci associated with human dental caries fall within the biotype I (serotypes c and f) grouping, designated S. mutans, these typing methods are of little value in distinguishing individual isolates. Here we show that strains of S. mutans obtained from over 30 individuals demonstrate unique "fingerprints" of chromosomal DNA digested with restriction endonuclease HaeIII. To demonstrate that this polymorphism in restriction fragments can be used to study the acquisition and transmission of this organism, we examined isolates of S. mutans from three mother-infant pairs obtained at the time the infant first became colonized by this organism. Results indicate that strains of S. mutans found in infants exhibit restriction fragment profiles identical to those of their mothers, strongly supporting the notion that mothers transmit this organism to their infants. Also, we show that strains of S. mutans with the same restriction fragment profile were stably maintained over a 3-year interval in the one mother-infant pair studied. Moreover, we found that mothers and their infants harbored only a few individual strains, suggesting that transmission of this organism is probably confined within discrete family cohorts. Collectively, these findings demonstrate the potential utility of genomic fingerprinting in studying the natural history of S. mutans infections in humans.

  2. The chromosomal constitution of fish hybrid lineage revealed by 5S rDNA FISH.

    Science.gov (United States)

    Zhang, Chun; Ye, Lihai; Chen, Yiyi; Xiao, Jun; Wu, Yanhong; Tao, Min; Xiao, Yamei; Liu, Shaojun

    2015-12-03

    The establishment of the bisexual fertile fish hybrid lineage including the allodiploid and allotetraploid hybrids, from interspecific hybridization of red crucian carp (Carassius auratus red var. 2n = 100, 2n = AA) (♀) × common carp (Cyprinus carpio L. 2n = 100, 2n = BB) (♂), provided a good platform to investigate genetic relationship between the parents and their hybrid progenies. The chromosomal inheritance of diploid and allotetraploid hybrid progenies in successive generations, was studied by applying 5S rDNA fluorescence in situ hybridization. Signals of 5S rDNA distinguished the chromosomal constitution of common carp (B-genome) from red crucian carp (A-genome), in which two strong signals were observed on the first submetacentric chromosome, while no major signal was found in common carp. After fish hybridization, one strong signal of 5S rDNA was detected in the same locus on the chromosome of diploid hybrids. As expected, two strong signals were observed in 4nF3 tetraploid hybrids offspring and it is worth mentioning that two strong signals were detected in a separating bivalent of a primary spermatocyte in 4nF3. Furthermore, the mitosis of heterozygous chromosomes was shown normal and stable with blastular tissue histological studies. We revealed that 5S rDNA signal can be applied to discern A-genome from B-genome, and that 5S rDNA bearing chromosomes can be stably passed down in successive generations. Our work provided a significant method in fish breeding and this is important for studies in fish evolutionary biology.

  3. Separation of DNA replication from the assembly of break-competent meiotic chromosomes.

    Directory of Open Access Journals (Sweden)

    Hannah G Blitzblau

    Full Text Available The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms.

  4. The combined evidential value of autosomal and Y-chromosomal DNA profiles obtained from the same sample

    NARCIS (Netherlands)

    de Zoete, J.; Sjerps, M.; Meester, R.; Cator, E.

    2014-01-01

    When a Y-chromosomal and a (partial) autosomal DNA profile are obtained from one crime sample, and both profiles match the suspect's profiles, we would like to know the combined evidential value. To calculate the likelihood ratio of observing the autosomal and Y-chromosomal DNA profiles combined, we

  5. Chromosome conformation elucidates regulatory relationships in developing human brain.

    Science.gov (United States)

    Won, Hyejung; de la Torre-Ubieta, Luis; Stein, Jason L; Parikshak, Neelroop N; Huang, Jerry; Opland, Carli K; Gandal, Michael J; Sutton, Gavin J; Hormozdiari, Farhad; Lu, Daning; Lee, Changhoon; Eskin, Eleazar; Voineagu, Irina; Ernst, Jason; Geschwind, Daniel H

    2016-10-27

    Three-dimensional physical interactions within chromosomes dynamically regulate gene expression in a tissue-specific manner. However, the 3D organization of chromosomes during human brain development and its role in regulating gene networks dysregulated in neurodevelopmental disorders, such as autism or schizophrenia, are unknown. Here we generate high-resolution 3D maps of chromatin contacts during human corticogenesis, permitting large-scale annotation of previously uncharacterized regulatory relationships relevant to the evolution of human cognition and disease. Our analyses identify hundreds of genes that physically interact with enhancers gained on the human lineage, many of which are under purifying selection and associated with human cognitive function. We integrate chromatin contacts with non-coding variants identified in schizophrenia genome-wide association studies (GWAS), highlighting multiple candidate schizophrenia risk genes and pathways, including transcription factors involved in neurogenesis, and cholinergic signalling molecules, several of which are supported by independent expression quantitative trait loci and gene expression analyses. Genome editing in human neural progenitors suggests that one of these distal schizophrenia GWAS loci regulates FOXG1 expression, supporting its potential role as a schizophrenia risk gene. This work provides a framework for understanding the effect of non-coding regulatory elements on human brain development and the evolution of cognition, and highlights novel mechanisms underlying neuropsychiatric disorders.

  6. Chromosomal inversions between human and chimpanzee lineages caused by retrotransposons.

    Directory of Open Access Journals (Sweden)

    Jungnam Lee

    Full Text Available The long interspersed element-1 (LINE-1 or L1 and Alu elements are the most abundant mobile elements comprising 21% and 11% of the human genome, respectively. Since the divergence of human and chimpanzee lineages, these elements have vigorously created chromosomal rearrangements causing genomic difference between humans and chimpanzees by either increasing or decreasing the size of genome. Here, we report an exotic mechanism, retrotransposon recombination-mediated inversion (RRMI, that usually does not alter the amount of genomic material present. Through the comparison of the human and chimpanzee draft genome sequences, we identified 252 inversions whose respective inversion junctions can clearly be characterized. Our results suggest that L1 and Alu elements cause chromosomal inversions by either forming a secondary structure or providing a fragile site for double-strand breaks. The detailed analysis of the inversion breakpoints showed that L1 and Alu elements are responsible for at least 44% of the 252 inversion loci between human and chimpanzee lineages, including 49 RRMI loci. Among them, three RRMI loci inverted exonic regions in known genes, which implicates this mechanism in generating the genomic and phenotypic differences between human and chimpanzee lineages. This study is the first comprehensive analysis of mobile element bases inversion breakpoints between human and chimpanzee lineages, and highlights their role in primate genome evolution.

  7. Y-chromosome haplotype distribution in Han Chinese populations and modern human origin in East Asians

    Institute of Scientific and Technical Information of China (English)

    KE; Yuehai

    2001-01-01

    [1]Cann, R. L., Stoneking, M., Wilson, A. C., Mitochondria DNA and human evolution, Nature, 1987, 325: 31-36.[2]Vigilant, L., Stoneking, M., Harpending, H. et al., African populations and the evolution of human mitochondrial DNA, Science, 1997, 253: 1503-1507.[3]Cavalli-Sforza, L. L., Piazza, M. P., The History and Geography of Human Genes, Princeton: Princeton University Press, 1994.[4]Brooks, A. S., Wood, B., Paleoanthropology, The Chinese side of the story, Nature, 1990, 344: 288-289.[5]Li, T., Etler, D. A., New middle Pleistocene hominid crania from Yunxian in China, Nature, 1992, 357: 404-407.[6]Wu, X. Z., Poirier, F. E., Human Evolution in China, Oxford: Oxford University Press, 1995.[7]Etler, D. A., The fossil evidence for human evolution in Asia, Annu. Rev. Anthropol., 1996, 25: 275-301.[8]Wolpoff, M. H., Interpretations of multiregional evolution, Science, 1996, 274: 704-707.[9]Stringer, C. B., Andrew, P., Genetic and fossil evidence for the origin of modern humans, Science ,1988, 239: 1263-1268.[10]Wilson, A. C.,Cann, R. L., The recent African genesis of humans, Scientific American, 1992, (4): 68-75.[11]Weng, Z., Yuan, Y., Du, R., Analysis of the genetic structure of human populations in China, Acta Anthropol. Sin. (in Chi-nese)1989, 8: 261-268.[12]Zhao, T., Zhang, G., Zhu, Y. et al., The distribution of immunoglobulin Gm allotypes in forty Chinese populations, Acta Anthropol. Sin. (in Chinese), 1986, 6: 1-8.[13]Chu, J. Y., Huang, W., Kuang, S. Q. et al., Genetic relationship of populations in China, Proc. Natl. Acad. Sci., 1998, 95: 11763-11768.[14]Jobling, M. A., Tyler-Smith, C., Fathers and sons: the Y chromosome and human evolution, Trends in Genetics,1995, 11: 449-455.[15]Oefner, P. J., Underhill, P. A., Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC), Am. J. Hum. Genet., 1995, 57: A266.[16]Oefner, P. J., Underhill, P. A., DNA mutation detection

  8. Recruiting a microtubule-binding complex to DNA directs chromosome segregation in budding yeast

    OpenAIRE

    Murray, Andrew W.; Lacefield, Soni; Lau, Tsz Cham Derek

    2009-01-01

    Accurate chromosome segregation depends on the kinetochore, the complex of proteins that link microtubules to centromeric DNA1. The budding yeast kinetochore consists of more than 80 proteins assembled on a 125bp region of DNA1. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve the segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting...

  9. Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation.

    Science.gov (United States)

    Taylor, James A; Pastrana, Cesar L; Butterer, Annika; Pernstich, Christian; Gwynn, Emma J; Sobott, Frank; Moreno-Herrero, Fernando; Dillingham, Mark S

    2015-01-01

    The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed.

  10. Ribosomal DNA is active in different B chromosome variants of the grasshopper Eyprepocnemis plorans.

    Science.gov (United States)

    Ruíz-Estévez, Mercedes; López-León, M Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2013-09-01

    B chromosomes are considered to be genetically inert elements. However, some of them are able to show nucleolus organizer region (NOR) activity, as detected by both cytological and molecular means. The grasshopper Eyprepocnemis plorans shows a B chromosome polymorphism characterized by the existence of many B variants. One of them, B24, shows NOR activity in about half of B-carrying males in the Torrox population. Molecular data have suggested the recent origin for B chromosomes in this species, and on this basis it would be expected that NOR activity was widespread among the different B variants. Here we test this hypothesis in four different B chromosome variants (B1, B2, B5, and B24) from 11 natural populations of the grasshopper E. plorans covering the south and east of the Iberian Peninsula plus the Balearic Islands. We used two different approaches: (1) the cytological observation of nucleoli attached to the distal region of the B chromosome (where the rDNA is located), and (2) the molecular detection of the rDNA transcripts carrying an adenine insertion characteristic of B chromosome ITS2 sequences. The results showed NOR expression not only for B24 but also for the B1 and B2 variants. However, the level of B-NOR expression in these latter variants, measured by the proportion of cells showing nucleoli attached to the B chromosomes, was much lower than that previously reported for B24. This suggests the possibility that structural or genetic background conditions are enhancing the expressivity of the rDNA in the B24 variant.

  11. Gyramides prevent bacterial growth by inhibiting DNA gyrase and altering chromosome topology.

    Science.gov (United States)

    Rajendram, Manohary; Hurley, Katherine A; Foss, Marie H; Thornton, Kelsey M; Moore, Jared T; Shaw, Jared T; Weibel, Douglas B

    2014-06-20

    Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division.

  12. Chromosome segregation regulation in human zygotes : Altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex

    NARCIS (Netherlands)

    Van De Werken, C.; Avo Santos, M.; Laven, J. S E; Eleveld, C.; Fauser, B. C J M; Lens, S. M A; Baart, E. B.

    2015-01-01

    STUDY QUESTION Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? SUMMARY ANSWER Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal

  13. Chromosome segregation regulation in human zygotes : Altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex

    NARCIS (Netherlands)

    Van De Werken, C.; Avo Santos, M.; Laven, J. S E; Eleveld, C.; Fauser, B. C J M; Lens, S. M A; Baart, E. B.

    2015-01-01

    STUDY QUESTION Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? SUMMARY ANSWER Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal

  14. [Analysis of sperm chromosomal abnormalities and sperm DNA fragmentation in infertile males].

    Science.gov (United States)

    Qiu, Yi; Wang, Leiguang; Zhang, Lihong; Yang, Dantong; Zhang, Aidong; Yu, Jianchun

    2008-12-01

    To investigate changes in sperm chromosome and sperm DNA integrity of infertile males. The level of DNA fragmentation was determined by Sperm Chromatin Dispersion (SCD) test in infertile males with idiopathic severe oligoasthenozoospermia (ISOA, n= 19), couples with unexplained recurrent miscarriage (URM, n= 38) and adult healthy fertile men (control group, n= 32). Multi-color fluorescence in situ hybridization (FISH) was performed with probes specific for chromosomes 13, 18, 21, X and Y in the control group (n= 5), the ISOA (n= 10) and the URM (n= 12). Patients with ISOA and URM showed a significantly higher abnormality with total rate of 4.02% (n= 19) and 3.91%(n= 38) for chromosomes 13, 18 and 21, and 2.03%, 1.98% for chromosomes X and Y, respectively, in their spermatozoa compared to control (1.29% and 0.61%, Pchromosomal aberration and the rate of sperm DNA fragmentation (gamma = 0.874, Pabnormal sperm (gamma = - 0.571, gamma = - 0.616 and gamma = 0.637, respectively, Pchromosomal aneuploidy and may lead to male infertility. Screening for sperm DNA damage may provide useful information in the diagnosis of male idiopathic infertility.

  15. The mutation rate of the human mtDNA deletion mtDNA4977.

    Science.gov (United States)

    Shenkar, R; Navidi, W; Tavaré, S; Dang, M H; Chomyn, A; Attardi, G; Cortopassi, G; Arnheim, N

    1996-10-01

    The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.

  16. The mutation rate of the human mtDNA deletion mtDNA{sup 4977}

    Energy Technology Data Exchange (ETDEWEB)

    Shenkar, R. [Univ. of Colorado Health Science Center, Denver, CO (United States); Navidi, W. [Colorado School of Mines, Golden, CO (United States); Tavare, S. [Univ. of California, Los Angeles, CA (United States)] [and others

    1996-10-01

    The human mitochondrial mutation mtDNA{sup 4977} is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation rate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA{sup 4977} in cultured human cells is 5.95 x 10{sup {minus}8} per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations. 17 refs., 1 fig., 1 tab.

  17. Isolation and mapping of a polymorphic DNA sequence (HBI18P1) on chromosome 11 (D11S147)

    Energy Technology Data Exchange (ETDEWEB)

    Julier, C.; Nakamura, Y.; Lanthrop, G.M.; Lalouel, J.M.; White, R. (Univ. of Utah School of Medicine, Salt Lake City (USA))

    1989-11-25

    A 4.7 kb PstI fragment, isolated from cosmid HBI18, was subcloned. MspI identifies a 2 allele polymorphism with bands at 5.2 kb (M2) and 4.8 kb (M2). HBI18P1 polymorphic system is derived from the same cosmid as pHBI18P2. Both systems have been assigned to chromosome 11 by multilocus linkage analysis with markers known to map on this chromosome. Co-dominant segregation of the MspI RFLP has been observed in 41 three generation families. This probe may contain repetitive sequences, and should be used in the presence of an excess of total human DNA in the prehybridization and hybridization mixtures.

  18. Super-resolution fluorescence imaging of chromosomal DNA.

    Science.gov (United States)

    Zessin, Patrick J M; Finan, Kieran; Heilemann, Mike

    2012-02-01

    Super-resolution microscopy is a powerful tool for understanding cellular function. However one of the most important biomolecules - DNA - remains somewhat inaccessible because it cannot be effectively and appropriately labeled. Here, we demonstrate that robust and detailed super-resolution images of DNA can be produced by combining 5-ethynyl-2'-deoxyuridine (EdU) labeling using the 'click chemistry' approach and direct stochastic optical reconstruction microscopy (dSTORM). This method can resolve fine chromatin structure, and - when used in conjunction with pulse labeling - can reveal the paths taken by individual fibers through the nucleus. This technique should provide a useful tool for the study of nuclear structure and function.

  19. Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene

    Directory of Open Access Journals (Sweden)

    Huxley Clare

    2001-02-01

    Full Text Available Abstract Background Rab GTPases are regulators of intracellular membrane traffic. The Rab27 subfamily consists of Rab27a and Rab27b. Rab27a has been recently implicated in Griscelli Disease, a disease combining partial albinism with severe immunodeficiency. Rab27a plays a key role in the function of lysosomal-like organelles such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Little is known about Rab27b. Results The human RAB27B gene is organised in six exons, spanning about 69 kb in the chromosome 18q21.1 region. Exon 1 is non-coding and is separated from the others by 49 kb of DNA and exon 6 contains a long 3' untranslated sequence (6.4 kb. The mouse Rab27b cDNA shows 95% identity with the human cDNA at the protein level and maps to mouse chromosome 18. The mouse mRNA was detected in stomach, large intestine, spleen and eye by RT-PCR, and in heart, brain, spleen and kidney by Northern blot. Transient over-expression of EGF-Rab27b fusion protein in cultured melanocytes revealed that Rab27b is associated with melanosomes, as observed for EGF-Rab27a. Conclusions Our results indicate that the Rab27 subfamily of Ras-like GTPases is highly conserved in mammals. There is high degree of conservation in sequence and gene structure between RAB27A and RAB27B genes. Exogenous expression of Rab27b in melanocytes results in melanosomal association as observed for Rab27a, suggesting the two Rab27 proteins are functional homologues. As with RAB27A in Griscelli Disease, RAB27B may be also associated with human disease mapping to chromosome 18.

  20. Chromosome surveys of human populations: between epidemiology and anthropology.

    Science.gov (United States)

    de Chadarevian, Soraya

    2014-09-01

    It is commonly held that after 1945 human genetics turned medical and focussed on the individual rather than on the study of human populations that had become discredited. However, a closer look at the research practices at the time quickly reveals that human population studies, using old and new tools, prospered in this period. The essay focuses on the rise of chromosome analysis as a new tool for the study of human populations. It reviews a broad array of population studies ranging from newborn screening programmes to studies of isolated or 'primitive' people. Throughout, it highlights the continuing role of concerns and opportunities raised by the propagation of atomic energy for civilian and military uses, the collection of large data bases and computers, and the role of international organisations like the World Health Organisation and the International Biological Programme in shaping research agendas and carving out a space for human heredity in the postwar era.

  1. Chromosomes of human sperm: variability among normal individuals

    Energy Technology Data Exchange (ETDEWEB)

    Brandriff, B.; Gordon, L.; Ashworth, L.; Watchmaker, G.; Moore, D. II; Wyrobek, A.J.; Carrano, A.V.

    1985-01-01

    The chromosomal constitution of 2468 human sperm cells has been investigated by fusion of human sperm with hamster eggs. The overall frequency of cells with structural aberrations was 7.7%, ranging from 1.9% to 15.8%, and varying significantly among individuals. The highest frequency occurred in sperm from the oldest donor (49 years), who also had had a vasectomy reversal three years prior to sampling. The overall aneuploidy frequency was 1.7%, ranging from 0.6% to 3.1%. In nine out of ten donors from whom blood samples were available the frequency of sperm cells with structural aberrations was higher than that for lymphocytes. Two previously reported donors were resampled after an interval of 14 and 16 months respectively, and were each found to have similar frequencies of sperm chromosome abnormalities at both sampling times. A father-son pair included in the study had several chromosome breakpoints in common, although no more frequency than unrelated individuals. 32 references, 2 figures, 5 tables.

  2. Non-meiotic chromosome instability in human immature oocytes.

    Science.gov (United States)

    Daina, Gemma; Ramos, Laia; Rius, Mariona; Obradors, Albert; Del Rey, Javier; Giralt, Magda; Campillo, Mercedes; Velilla, Esther; Pujol, Aïda; Martinez-Pasarell, Olga; Benet, Jordi; Navarro, Joaquima

    2014-02-01

    Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25-45 years of age) and 24 IVF oocyte donors (18-33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.

  3. Seeing the wood for the trees: a minimal reference phylogeny for the human Y chromosome.

    Science.gov (United States)

    van Oven, Mannis; Van Geystelen, Anneleen; Kayser, Manfred; Decorte, Ronny; Larmuseau, Maarten H D

    2014-02-01

    During the last few decades, a wealth of studies dedicated to the human Y chromosome and its DNA variation, in particular Y-chromosome single-nucleotide polymorphisms (Y-SNPs), has led to the construction of a well-established Y-chromosome phylogeny. Since the recent advent of new sequencing technologies, the discovery of additional Y-SNPs is exploding and their continuous incorporation in the phylogenetic tree is leading to an ever higher resolution. However, the large and increasing amount of information included in the "complete" Y-chromosome phylogeny, which now already includes many thousands of identified Y-SNPs, can be overwhelming and complicates its understanding as well as the task of selecting suitable markers for genotyping purposes in evolutionary, demographic, anthropological, genealogical, medical, and forensic studies. As a solution, we introduce a concise reference phylogeny whereby we do not aim to provide an exhaustive tree that includes all known Y-SNPs but, rather, a quite stable reference tree aiming for optimal global discrimination capacity based on a strongly reduced set that includes only the most resolving Y-SNPs. Furthermore, with this reference tree, we wish to propose a common standard for Y-marker as well as Y-haplogroup nomenclature. The current version of our tree is based on a core set of 417 branch-defining Y-SNPs and is available online at http://www.phylotree.org/Y.

  4. The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

    Science.gov (United States)

    Farr, Christine J; Antoniou-Kourounioti, Melissa; Mimmack, Michael L; Volkov, Arsen; Porter, Andrew C G

    2014-04-01

    As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.

  5. Fine mapping of the human pentraxin gene region on chromosome 1q23

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, M.T.; Whitehead, A.S. [Univ. of Dublin (Ireland); Divane, A. [Univ. of Cambridge (United Kingdom)

    1996-12-31

    The 1q21 to 25 region of human chromosome 1 contains genes which encode proteins with immune- and inflammation-associated functions. These include the pentraxin genes, for C-reactive protein (CRP), serum amyloid P(SAP) protein (APCS), and a CRP pseudogene (CRPP1). The region of chromosome 1 containing this cluster is syntenic with distal mouse chromosome 1. We constructed an approximately 1.4 megabase yeast artificial chromosome (YAC) contig with the pentraxin genes at its core. This four-YAC contig includes other genes with immune functions including the FCER1A gene, which encodes the {alpha}-subunit of the IgE high-affinity Fc receptor and the 1F1-16 gene, an interferon-{gamma}-induced gene. In addition, it contains the histone H3F2 and H4F2 genes and the gene for erythroid {alpha}-spectrin (SPTA1). The gene order is cen.-SPTA1-H4F2-H3F2-1F1-16-CRP-CRPP1-APCS-FCERIA-tel. The contig thus consists of a cluster of genes whose products either have immunological importance, bind DNA, or both. 68 refs., 3 figs., 2 tabs.

  6. 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.

  7. Identification of the human chromosomal region containing the iridogoniodysgenesis anomaly locus by genomic-mismatch scanning.

    Science.gov (United States)

    Mirzayans, F; Mears, A J; Guo, S W; Pearce, W G; Walter, M A

    1997-01-01

    Genome-mismatch scanning (GMS) is a new method of linkage analysis that rapidly isolates regions of identity between two genomes. DNA molecules from regions of identity by descent from two relatives are isolated based on their ability to form extended mismatch-free heteroduplexes. We have applied this rapid technology to identify the chromosomal region shared by two fifth-degree cousins with autosomal dominant iridogoniodysgenesis anomaly (IGDA), a rare ocular neurocristopathy. Markers on the short arm of human chromosome 6p were recovered, consistent with the results of conventional linkage analysis conducted in parallel, indicating linkage of IGDA to 6p25. Control markers tested on a second human chromosome were not recovered. A GMS error rate of approximately 11% was observed, well within an acceptable range for a rapid, first screening approach, especially since GMS results would be confirmed by family analysis with selected markers from the putative region of identity by descent. These results demonstrate not only the value of this technique in the rapid mapping of human genetic traits, but the first application of GMS to a multicellular organism. Images Figure 2 PMID:9245991

  8. 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.

  9. Preleptotene chromosome condensation stage in human foetal and neonatal testes.

    Science.gov (United States)

    Luciani, J M; Devictor, M; Stahl, A

    1977-04-01

    A preleptotene stage of chromosome condensation analogous to that already described in various plants and in the oocytes of several animal species has been observed in the human foetal testis. Contrary to what has been previously described, this stage in the testis is not followed by decondensation leading to leptotene filaments. This observation underlines the problem of the precise significance of this stage and its relation to initiation of meiosis. It is suggested that meiosis may be initiated during this condensation phase and that the male germ cell, despite its XY chromosome constitution, tends to evolve towards meiosis. This proposal pleads in favour of both the role of somatic cells in the inhibition of meiosis in the male foetus and the role of environmental factors rather than genetic constitution of the germ cell in meiotic induction.

  10. Chromosome conformation capture uncovers potential genome-wide interactions between human conserved non-coding sequences.

    Directory of Open Access Journals (Sweden)

    Daniel Robyr

    Full Text Available Comparative analyses of various mammalian genomes have identified numerous conserved non-coding (CNC DNA elements that display striking conservation among species, suggesting that they have maintained specific functions throughout evolution. CNC function remains poorly understood, although recent studies have identified a role in gene regulation. We hypothesized that the identification of genomic loci that interact physically with CNCs would provide information on their functions. We have used circular chromosome conformation capture (4C to characterize interactions of 10 CNCs from human chromosome 21 in K562 cells. The data provide evidence that CNCs are capable of interacting with loci that are enriched for CNCs. The number of trans interactions varies among CNCs; some show interactions with many loci, while others interact with few. Some of the tested CNCs are capable of driving the expression of a reporter gene in the mouse embryo, and associate with the oligodendrocyte genes OLIG1 and OLIG2. Our results underscore the power of chromosome conformation capture for the identification of targets of functional DNA elements and raise the possibility that CNCs exert their functions by physical association with defined genomic regions enriched in CNCs. These CNC-CNC interactions may in part explain their stringent conservation as a group of regulatory sequences.

  11. 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.

  12. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

    Science.gov (United States)

    Biradar, Siddanagouda S; Nie, Xiaojun; Feng, Kewei; Weining, Song

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.

  13. Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

    Science.gov (United States)

    Tillman, Robert E.; Wooley, Andrea L.; Hughes, Maureen M.; Wehrly, Tara D.; Swat, Wojciech; Sleckman, Barry P.

    2002-01-01

    Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination. PMID:11828005

  14. Human imprinted chromosomal regions are historical hot-spots of recombination.

    Directory of Open Access Journals (Sweden)

    Ionel Sandovici

    2006-07-01

    Full Text Available Human recombination rates vary along the chromosomes as well as between the two sexes. There is growing evidence that epigenetic factors may have an important influence on recombination rates, as well as on crossover position. Using both public database analysis and wet-bench approaches, we revisited the relationship between increased rates of meiotic recombination and genome imprinting. We constructed metric linkage disequilibrium (LD maps for all human chromosomal regions known to contain one or more imprinted genes. We show that imprinted regions contain significantly more LD units (LDU and have significantly more haplotype blocks of smaller sizes than flanking nonimprinted regions. There is also an excess of hot-spots of recombination at imprinted regions, and this is likely to do with the presence of imprinted genes, per se. These findings indicate that imprinted chromosomal regions are historical "hot-spots" of recombination. We also demonstrate, by direct segregation analysis at the 11p15.5 imprinted region, that there is remarkable agreement between sites of meiotic recombination and steps in LD maps. Although the increase in LDU/Megabase at imprinted regions is not associated with any significant enrichment for any particular sequence class, major sequence determinants of recombination rates seem to differ between imprinted and control regions. Interestingly, fine-mapping of recombination events within the most male meiosis-specific recombination hot-spot of Chromosome 11p15.5 indicates that many events may occur within or directly adjacent to regions that are differentially methylated in somatic cells. Taken together, these findings support the involvement of a combination of specific DNA sequences and epigenetic factors as major determinants of hot-spots of recombination at imprinted chromosomal regions.

  15. Initiation of DNA replication from non-canonical sites on an origin-depleted chromosome.

    Directory of Open Access Journals (Sweden)

    Naomi L Bogenschutz

    Full Text Available Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism.

  16. Genotoxic and antigenotoxic effects of Fucus vesiculosus extract on cultured human lymphocytes using the chromosome aberration and Comet assays

    Directory of Open Access Journals (Sweden)

    Cleide Leite-Silva

    2007-01-01

    Full Text Available The brown seaweed Fucus vesiculosus (Fucales, Fucaceae was screened for its protective activity using doxorubicin-induced DNA damage in human lymphocytes. In this study, we assessed the genotoxic and antigenotoxic potential of three different concentrations (0.25, 0.5 and 1.0 mg mL-1 of F. vesiculosus aqueous extract using the chromosome aberration and Comet assays. Treatment of human lymphocyte cultures with 0.25, 0.5 and 1.0 mg mL-1 F. vesiculosus aqueous extract had no effect on the chromosome aberration frequency or on the extent of DNA damage detected by the Comet assay. The antigenotoxic effects of the extract were tested in human lymphocyte cultures treated with 15 µg mL-1 of doxorubicin, either alone or combined with the different concentrations of the extract, which was added to the cultures before, simultaneously with or after the doxorubicin. Only when lymphocytes were pre-treated with extract there was a reduction in doxorubicin-induced chromosome aberrations and DNA damage as detected by the Comet assay. These results demonstrate that F. vesiculosus aqueous extract is not genotoxic in cultured human lymphocytes and indicate that when added to lymphocyte cultures before doxorubicin it has antigenotoxic activity against doxorubicin-induced DNA damage.

  17. DNA integrity of human leukocytes after magnetic resonance imaging.

    Science.gov (United States)

    Szerencsi, Ágnes; Kubinyi, Györgyi; Váliczkó, Éva; Juhász, Péter; Rudas, Gábor; Mester, Ádám; Jánossy, Gábor; Bakos, József; Thuróczy, György

    2013-10-01

    This study focuses on the effects of high-field (3T) magnetic resonance imaging (MRI) scans on the DNA integrity of human leukocytes in vitro in order to validate the study where genotoxic effects were obtained and published by Lee et al. The scanning protocol and exposure situation were the same as those used under routine clinical brain MRI scan. Peripheral blood samples from healthy non-smoking male donors were exposed to electromagnetic fields (EMF) produced by 3T magnetic resonance imaging equipment for 0, 22, 45, 67, and 89 min during the scanning procedure. Samples of positive control were exposed to ionizing radiation (4 Gy of (60)Co-γ). Single breaks of DNA in leukocytes were detected by single-cell gel electrophoresis (Comet assay). Chromosome breakage, chromosome loss and micronuclei formations were detected by a micronucleus test (MN). Three independent experiments were performed. The data of comet tail DNA%, olive tail moment and micronucleus frequency showed no DNA damages due to MRI exposure. The results of the Comet assay and the micronucleus test indicate that the applied exposure of MRI does not appear to produce breaks in the DNA and has no significant effect on DNA integrity.

  18. Evidence for a DNA-relay mechanism in ParABS-mediated chromosome segregation.

    Science.gov (United States)

    Lim, Hoong Chuin; Surovtsev, Ivan Vladimirovich; Beltran, Bruno Gabriel; Huang, Fang; Bewersdorf, Jörg; Jacobs-Wagner, Christine

    2014-05-23

    The widely conserved ParABS system plays a major role in bacterial chromosome segregation. How the components of this system work together to generate translocation force and directional motion remains uncertain. Here, we combine biochemical approaches, quantitative imaging and mathematical modeling to examine the mechanism by which ParA drives the translocation of the ParB/parS partition complex in Caulobacter crescentus. Our experiments, together with simulations grounded on experimentally-determined biochemical and cellular parameters, suggest a novel 'DNA-relay' mechanism in which the chromosome plays a mechanical function. In this model, DNA-bound ParA-ATP dimers serve as transient tethers that harness the elastic dynamics of the chromosome to relay the partition complex from one DNA region to another across a ParA-ATP dimer gradient. Since ParA-like proteins are implicated in the partitioning of various cytoplasmic cargos, the conservation of their DNA-binding activity suggests that the DNA-relay mechanism may be a general form of intracellular transport in bacteria.DOI: http://dx.doi.org/10.7554/eLife.02758.001.

  19. Isolation and Quantitation of Topoisomerase Complexes Accumulated on Escherichia coli Chromosomal DNA

    Science.gov (United States)

    Aedo, Sandra

    2012-01-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA. PMID:22869559

  20. Isolation and quantitation of topoisomerase complexes accumulated on Escherichia coli chromosomal DNA.

    Science.gov (United States)

    Aedo, Sandra; Tse-Dinh, Yuk-Ching

    2012-11-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.

  1. Antibodies to left-handed Z-DNA bind to interband regions of Drosophila polytene chromosomes

    Science.gov (United States)

    Nordheim, Alfred; Pardue, Mary Lou; Lafer, Eileen M.; Möller, Achim; Stollar, B. David; Rich, Alexander

    1981-12-01

    Antibodies which are specific to the Z-DNA conformation have been purified and characterized on the basis of their binding to three different DNA polymers which can form this left-handed helix. These antibodies bind specifically to polytene chromosomes of Drosophila melanogaster as visualized by fluorescent staining. The staining is found in the interband regions and its intensity varies among different interbands in a reproducible manner. This is the first identification of the Z-DNA conformation in material of biological origin.

  2. Protocols for Pachytene Chromosome and DNA Fiber FISH in Cotton

    Institute of Scientific and Technical Information of China (English)

    PENG Ren-hai; LING Jian; WANG Kun-bo; WANG Chun-ying; SONG Guo-li; LIU Fang

    2008-01-01

    @@ Fluorescence in situ hybridization (FISH) has become the most important technique in plant molecular cytogenetics research.FISH-based physical mapping provides a valuable complementary approach in genome sequeneing to measure the physical distances between adjacent BAC contigs and delineating the structure and DNA composition of genomic regions of centromere and telomere.The accuracy and precision of the FISH-base physical maps depend on the resolution power of FISH.

  3. Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay

    Directory of Open Access Journals (Sweden)

    Gosalvez Jaime

    2009-04-01

    Full Text Available Abstract Background Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide. Results Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose. Conclusion This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.

  4. Localization of a human homolog of the mouse pericentrin gene (PCNT) to chromosome 21qter

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiming [Univ. of Geneva Medical School (Switzerland); Gos, A.; Morris, M.A. [Cantonal Hospital, Geneva (Switzerland)] [and others

    1996-08-01

    Exon trapping was used to identify portions of genes from cosmid DNA of a human chromosome 21-specific library LL21NC02-Q. More than 650 potential exons have been cloned and characterized to date. Among these, 3 trapped {open_quotes}exons{close_quotes} showed strong homology to different regions of the cDNA for the mouse pericentrin (Pcnt) gene, indicating that these 3 exons are portions of a human homolog of the mouse pericentrin gene. With PCR amplification, Southern blot analysis, and FISH, we have mapped this presumed human pericentrin gene (PCNT) to the long arm of chromosome 21 between marker PFKL and 21qter. Pericentrin is a conserved protein component of the filamentous matrix of the centrosome involved in the initial establishment of the organized microtubule array. No candidate hereditary disorder for pericentrin deficiency/abnormality has yet been mapped in the most distal region of 21q; in addition the role of triplication of the pericentrin gene in the pathophysiology or etiology of trisomy 21 is currently unknown. 16 refs., 3 figs.

  5. Applying Small-Scale DNA Signatures as an Aid in Assembling Soybean Chromosome Sequences

    Directory of Open Access Journals (Sweden)

    Myron Peto

    2010-01-01

    Full Text Available Previous work has established a genomic signature based on relative counts of the 16 possible dinucleotides. Until now, it has been generally accepted that the dinucleotide signature is characteristic of a genome and is relatively homogeneous across a genome. However, we found some local regions of the soybean genome with a signature differing widely from that of the rest of the genome. Those regions were mostly centromeric and pericentromeric, and enriched for repetitive sequences. We found that DNA binding energy also presented large-scale patterns across soybean chromosomes. These two patterns were helpful during assembly and quality control of soybean whole genome shotgun scaffold sequences into chromosome pseudomolecules.

  6. Cytomolecular discrimination of the A(m) chromosomes of Triticum monococcum and the A chromosomes of Triticum aestivum using microsatellite DNA repeats.

    Science.gov (United States)

    Megyeri, Mária; Mikó, Péter; Farkas, András; Molnár-Láng, Márta; Molnár, István

    2017-02-01

    The cytomolecular discrimination of the A(m)- and A-genome chromosomes facilitates the selection of wheat-Triticum monococcum introgression lines. Fluorescence in situ hybridisation (FISH) with the commonly used DNA probes Afa family, 18S rDNA and pSc119.2 showed that the more complex hybridisation pattern obtained in T. monococcum relative to bread wheat made it possible to differentiate the A(m) and A chromosomes within homoeologous groups 1, 4 and 5. In order to provide additional chromosomal landmarks to discriminate the A(m) and A chromosomes, the microsatellite repeats (GAA)n, (CAG)n, (CAC)n, (AAC)n, (AGG)n and (ACT)n were tested as FISH probes. These showed that T. monococcum chromosomes have fewer, generally weaker, simple sequence repeat (SSR) signals than the A-genome chromosomes of hexaploid wheat. A differential hybridisation pattern was observed on 6A(m) and 6A chromosomes with all the SSR probes tested except for the (ACT)n probe. The 2A(m) and 2A chromosomes were differentiated by the signals given by the (GAA)n, (CAG)n and (AAC)n repeats, while only (GAA)n discriminated the chromosomes 3A(m) and 3A. Chromosomes 7A(m) and 7A could be differentiated by the lack of (GAA)n and (AGG)n signals on 7A. As potential landmarks for identifying the A(m) chromosomes, SSR repeats will facilitate the introgression of T. monococcum chromatin into wheat.

  7. Mapping of guanylin to murine chromosome 4 and human chromosome 1p34-p35

    Energy Technology Data Exchange (ETDEWEB)

    Sciaky, D.; Cohen, M.B. [Univ. of Cincinnati, OH (United States); Jenkins, N.A. [Mammalian Genetics Lab., Frederick, MD (United States)] [and others

    1995-03-20

    Guanylin is a 15-amino-acid peptide similar in structure and in function to ST{sub a}, the heat stable enterotoxin of enterotoxigenic Escherichia coli (4). Both guanylin and ST{sub a} bind guanylyl cyclase-C (GC-C), resulting in increased levels of intracellular cGMP and induction of Cl- secretion (4) via the cystic fibrosis transmembrane regulator (CFM) (2). Guanylin is a highly regulated intestinal gene that is differentially expressed along the duodenal-to-colonic and villus-to-crypt axes. Guanylin mRNA abundance is maximal in the distal small intestine and proximal colon, where the mRNA is detected mainly in differentiated villus epithelial cells and superficial colonic epithelial cells, respectively. The murine guanylin gene (Guca2) has been isolated and sequenced; the gene is 1.7 kb and consists of 3 exons. We report here the mapping of Guca2 to mouse chromosome 4 by linkage analysis and to human chromosome region 1p34-p35 using fluorescence in situ hybridization (FISH). 20 refs., 2 figs.

  8. Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break

    Science.gov (United States)

    Qiu, Zhijun; Zhang, Zhenhua; Roschke, Anna; Varga, Tamas; Aplan, Peter D.

    2017-01-01

    Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX “knockdown” cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression. PMID:28225067

  9. Patterns of association in the human metaphase complement: ring analysis and estimation of associativity of specific chromosome regions.

    Science.gov (United States)

    Rodman, T C; Flehinger, B J; Squire, R D

    1978-02-23

    The pattern of metaphase chromosome association in the human complement was studied by two methods of statistical analysis of interchromosomal distances. Those methods included ring analysis in which a characteristic position of the centromere of each chromosome relative to the center of a two dimensional representation of a metaphase complement was defined, and estimation of the capacity for associativity of each of three regions of each chromosome: the centromere (c) and the ends of each arm (p, q). The following information was obtained: 1. In general, the distance from the center is directly related to chromosome size. 2. The most notable deviation from that size-related progression is displayed by the X chromosomes. The markedly peripheral position of the X is characteristic of both X's of the female and the single X of the male. 3. The relative associativity of each chromosome of the complement is, in general, inversely related to size with an additional preferential capacity of associativity displayed by the acrocentric chromosomes. Analyses of the different inter-regional classes established that the supplementary associativity factor of the acrocentric chromosomes was inherent in their pericentromeric and p-arm regions and excluded the ends of the q arms from participation in that factor. 4. Those analyses demonstrated that the specific morphology or 'geometry' of the acrocentric chromosomes contributes little to their high relative associativity. In addition to the tendency for the c/p regions of the acrocentric chromosomes to associate with each other, presumably because of their common function in nucleolar organization, those regions also displayed a propensity to associate with the distal regions of the arms of other chromosomes. A molecular basis for that propensity other than that of ribosomal DNA is postulated to be that of other fractions of highly reiterated DNA sequences. 5. Analysis of the relative associativities of each of the three regions

  10. The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites.

    Science.gov (United States)

    Taylor, James A; Ouimet, Marie-Claude; Wargachuk, Richard; Marczynski, Gregory T

    2011-10-01

    The Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNA-binding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control. © 2011 Blackwell Publishing Ltd.

  11. Human chromosome pellicle antibody recognizing centromere protein—C (CENP0C),the main component of the kinetochore

    Institute of Scientific and Technical Information of China (English)

    XIEYONG; ZUMEINI; 等

    1997-01-01

    Recently the antichromosome antisera from several sclerogerma patients have been found to recognize the pellicle of metaphase and anaphase chromosomes.In order to identify the pellicle components,we used these antichromosome antisera to screen a human embryonic cDNA library.The sequences of the positive clones are identical to the cDNA gene sequence of CENP-C (centromere protein C),a human centromere autoantigen.This result suggusts that CENP-C is a component of the pellicle of human metaphase and anaphase chromosomes.

  12. Random amplified polymorphic DNA markers of the Brassica alboglabra chromosome of a B. campestris-alboglabra addition line

    DEFF Research Database (Denmark)

    Bagger Jørgensen, Rikke; Chen, B.Y.; Cheng, B.F.

    1996-01-01

    The alien C-genome chromosome in a Brassica campestris-alboglabra monosomic addition line was characterized by random amplified polymorphic DNA (RAPD) analysis. The alien chromosome carried three loci, E(c), W-c and Lap-1C, controlling synthesis of erucic acid, white flower colour and a fast......-migrating band of leucine aminopeptidase (Lap-1C(c)) respectively. The RAPD analysis revealed 17 markers specific to the alien chromosome. Among 45 offspring plants from the selfed addition line the alien C-chromosome was transmitted to 15 plants, four plants had only parts of this chromosome and the remaining...

  13. Chromosomal localization of the parathyroid hormone/parathyroid hormone-related protein receptor gene to human chromosome 3p21.1-p24.2

    Energy Technology Data Exchange (ETDEWEB)

    Gelbert, L.; Heath, H. III [Univ. of Utah Health Sciences Center, Salt Lake City, UT (United States); Schipani, E. [Massachusetts General Hospital, Boston, MA (United States)] [and others

    1994-10-01

    The human PTH/PTH-related peptide (PTH/PTHrP) receptor could be involved in hereditary disorders of PTH or PTHrP action. Knowledge of the gene`s chromosomal location would allow studies linking it to specific disease traits. Therefore, we mapped the human PTH/PTHrP receptor gene by polymerase chain reaction of human/rodent somatic cell hybrid panels using oligonucleotide primers designed to amplify a portion of the gene from genomic DNA. The PTH/PTHrP gene was unambiguously assigned to the short arm of human chromosome 3, in the region designated 3p21.1-p24.2. Analysis of a second chromosome 3-specific mapping panel suggests that the gene is located near the 3p21.2-p21.3 boundary. The availability of highly polymorphic markers located in this region will permit exploration of the PTH/PTHrP receptor locus in genetic linkage searches for the causes of bone, calcium, and other potential disorders. 14 refs., 2 figs.

  14. Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes

    Science.gov (United States)

    Puterova, Janka; Razumova, Olga; Martinek, Tomas; Alexandrov, Oleg; Divashuk, Mikhail; Kubat, Zdenek; Hobza, Roman; Karlov, Gennady

    2017-01-01

    Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes. PMID:28057732

  15. Y-chromosome and mtDNA genetics reveal significant contrasts in affinities of modern Middle Eastern populations with European and African populations.

    Directory of Open Access Journals (Sweden)

    Danielle A Badro

    Full Text Available The Middle East was a funnel of human expansion out of Africa, a staging area for the Neolithic Agricultural Revolution, and the home to some of the earliest world empires. Post LGM expansions into the region and subsequent population movements created a striking genetic mosaic with distinct sex-based genetic differentiation. While prior studies have examined the mtDNA and Y-chromosome contrast in focal populations in the Middle East, none have undertaken a broad-spectrum survey including North and sub-Saharan Africa, Europe, and Middle Eastern populations. In this study 5,174 mtDNA and 4,658 Y-chromosome samples were investigated using PCA, MDS, mean-linkage clustering, AMOVA, and Fisher exact tests of F(ST's, R(ST's, and haplogroup frequencies. Geographic differentiation in affinities of Middle Eastern populations with Africa and Europe showed distinct contrasts between mtDNA and Y-chromosome data. Specifically, Lebanon's mtDNA shows a very strong association to Europe, while Yemen shows very strong affinity with Egypt and North and East Africa. Previous Y-chromosome results showed a Levantine coastal-inland contrast marked by J1 and J2, and a very strong North African component was evident throughout the Middle East. Neither of these patterns were observed in the mtDNA. While J2 has penetrated into Europe, the pattern of Y-chromosome diversity in Lebanon does not show the widespread affinities with Europe indicated by the mtDNA data. Lastly, while each population shows evidence of connections with expansions that now define the Middle East, Africa, and Europe, many of the populations in the Middle East show distinctive mtDNA and Y-haplogroup characteristics that indicate long standing settlement with relatively little impact from and movement into other populations.

  16. Functional gene groups are concentrated within chromosomes, among chromosomes and in the nuclear space of the human genome.

    Science.gov (United States)

    Thévenin, Annelyse; Ein-Dor, Liat; Ozery-Flato, Michal; Shamir, Ron

    2014-09-01

    Genomes undergo changes in organization as a result of gene duplications, chromosomal rearrangements and local mutations, among other mechanisms. In contrast to prokaryotes, in which genes of a common function are often organized in operons and reside contiguously along the genome, most eukaryotes show much weaker clustering of genes by function, except for few concrete functional groups. We set out to check systematically if there is a relation between gene function and gene organization in the human genome. We test this question for three types of functional groups: pairs of interacting proteins, complexes and pathways. We find a significant concentration of functional groups both in terms of their distance within the same chromosome and in terms of their dispersal over several chromosomes. Moreover, using Hi-C contact map of the tendency of chromosomal segments to appear close in the 3D space of the nucleus, we show that members of the same functional group that reside on distinct chromosomes tend to co-localize in space. The result holds for all three types of functional groups that we tested. Hence, the human genome shows substantial concentration of functional groups within chromosomes and across chromosomes in space.

  17. Chromosome aberrations in human blood lymphocytes exposed to energetic protons

    Science.gov (United States)

    Hada, Megumi; George, Ms Kerry; Cucinotta, Francis A.

    During space flight, astronauts are exposed to space radiation consisting of high-energy protons, high charge and energy (HZE) nuclei, as well as secondary particles that are generated when the primary particles penetrate the spacecraft shielding. Secondary particles have a higher LET value than primary protons and are therefore expected to have a higher relative biological effectiveness (RBE). To investigate this theory, we exposed human peripheral blood lymphocytes to protons with energies of 250 MeV, 800MeV, 2 GeV, or 2.5 GeV. LET values for these protons ranged from 0.4 to 0.2 keV/µm. and doses ranged from 0.2 to 3 Gy. Over this energy range the probability of nuclear reaction leading to secondary radiation, and the multiplicity of reaction products such as neutrons and mesons increases substantially. The effect of aluminum and polyethylene shielding was also assessed using the 2 GeV and 2.5GeV proton beams. After exposure lymphocytes were stimulated to divide and chromosomes were collected from cells in the first G2 and metaphase cell cycle after exposure using a chemical induced premature chromosome condensation (PCC) technique. Dose response data for chromosome damage was analyzed using the fluorescence in situ hybridization (FISH) chromosome painting technique. Selected samples were also analyzed with multicolor FISH (mFISH) and multicolor banding FISH (mBAND) techniques. Data indicates that the dose response for simple-type exchanges is similar for proton and gamma exposure, whereas protons induce higher yields of complex exchanges that are energy dependent. RBE values will be presented for each proton energy, and the effects of shielding and possible cytogenetic signatures of proton exposure will be discussed.

  18. Transillumination spatially modulated illumination microscopy for human chromosome imaging

    Science.gov (United States)

    Pitris, Costas; Heracleous, Peter; Patsalis, Philippos

    2005-03-01

    Human chromosome analysis is an essential task in cytogenetics, especially in prenatal screening, genetic syndrome diagnosis, cancer pathology research and mutagen dosimetry. Chromosomal analysis begins with the creation of a karyotype, which is a layout of chromosome images organized by decreasing size in pairs. Both manual and automatic classification of chromosomes are limited by the resolution of the microscope and imaging system used. One way to improve the results of classification and even detect subtleties now remaining undetected, is to enhance the resolution of the images. It is possible to achieve lateral resolution beyond the classical limit, by using spatially modulated illumination (SMI) in a wide-field, non-confocal microscope. In this case, the sample is illuminated with spatially modulated light, which makes normally inaccessible high-resolution information visible in the observed image by shifting higher frequencies within the OTF limits of the microscope. Although, SMI microscopes have been reported in the past, this manuscript reports the development of a transillumination microscope for opaque, non-fluorescent samples. The illumination path consisted of a light source illuminating a ruled grating which was subsequently imaged on the sample. The grating was mounted on a rotating and translating stage so that the magnification and rotation of the pattern could be adjusted. The imaging lens was a 1.25 NA oil immersion objective. Test samples showed resolution improvement, as judged from a comparison of the experimentally obtained FWHM. Further studies using smaller fringe distance or laser interference pattern illumination will be evaluated to further optimize the SMI results.

  19. Chromosome Aberration in Human Blood Lymphocytes Exposed to Energetic Protons

    Science.gov (United States)

    Hada, M.; George, Kerry A.; Cucinotta, F. A.

    2008-01-01

    During space flight, astronauts are exposed to a space radiation consisting of high-energy protons, high charge and energy (HZE) nuclei, as well as secondary particles that are generated when the primary particles penetrate the spacecraft shielding. Secondary particles have a higher LET value than primary protons and therefore expected to have a higher relative biological effectiveness (RBE). To investigate this theory, we exposed human peripheral blood lymphocytes to protons with energies of 250 MeV, 800MeV, 2 GeV, or 2.5 GeV. LET values for these protons ranged from 0.4 to 0.2 keV/micrometer. and doses ranged from 0.2 to 3 Gy. Over this energy the probability of nuclear reaction leading to secondary radiation, and the multiplicity of reaction produces such as neutrons and mesons increases substantially. The effect of aluminum and polyethylene shielding was also assessed using the 2 GeV and 2.5GeV proton beams. After exposure lymphocytes were stimulated to divide and chromosomes were collected from cells in the first G2 and metaphase cell cycle after exposure using a chemical induced premature chromosome condensation (PCC) technique. Dose response data for chromosome damage was analyzed using the fluorescence in situ hybridization (FISH) chromosome painting technique. Selected samples were also analyzed with multicolor FISH (mFISH) and multicolor banding FISH (mBAND) techniques. Data indicates that the dose response for simple-type exchanges is similar for proton and gamma exposure, whereas protons induce higher yields of complex exchanges that are LET dependent. RBE values will be presented for each proton energy, and the effects of shielding and possible cytogenetic signatures of proton exposure will be discussed.

  20. Progress towards construction of a total restriction fragment map of a human chromosome.

    NARCIS (Netherlands)

    H. Vissing; F.G. Grosveld (Frank); E. Solomon; G. Moore; N. Lench; N. Shennan; R. Williamson

    1987-01-01

    textabstractWe present an approach to the construction of an overlapping restriction fragment map of a single human chromosome. A genomic cosmid library genome was constructed from a mouse-human hybrid cell line containing chromosome 17 as its only human genetic component. Cosmids containing human i

  1. High-speed AFM of human chromosomes in liquid

    Science.gov (United States)

    Picco, L. M.; Dunton, P. G.; Ulcinas, A.; Engledew, D. J.; Hoshi, O.; Ushiki, T.; Miles, M. J.

    2008-09-01

    Further developments of the previously reported high-speed contact-mode AFM are described. The technique is applied to the imaging of human chromosomes at video rate both in air and in water. These are the largest structures to have been imaged with high-speed AFM and the first imaging in liquid to be reported. A possible mechanism that allows such high-speed contact-mode imaging without significant damage to the sample is discussed in the context of the velocity dependence of the measured lateral force on the AFM tip.

  2. Chromosomal aberrations, Yq microdeletion, and sperm DNA fragmentation in infertile men opting for assisted reproduction.

    Science.gov (United States)

    Shamsi, Monis B; Kumar, Rajeev; Malhotra, Neena; Singh, Nita; Mittal, Suneeta; Upadhyay, Ashish D; Dada, Rima

    2012-09-01

    Male infertility is a multi-factorial disorder, and identification of its etiology in an individual is critical for treatment. Systematically elucidating the underlying genetic causes (chromosomal and Yq microdeletion) and factors, such as reactive oxygen species (ROS) levels and total antioxidant capacity (TAC), which contribute to sperm DNA damage, may help to reduce the number of men with idiopathic infertility and provide them with the most suitable therapeutics and counseling. This study was done to comprehensively investigate genetic and oxidative stress factors that might be the etiology of a large percentage of men with idiopathic infertility. One hundred twelve infertile men and 76 fertile controls were screened for chromosomal aberrations and Yq microdeletions. ROS, TAC, and sperm DNA damage were assessed in cytogenetically normal, non-azoospermic men with intact Y chromosome (n = 93). ROS was assessed in neat and washed semen by chemiluminescence; seminal TAC with a commercially available kit; and sperm DNA damage by the comet assay. Two men had cytogenetic abnormalities and seven men harbored Yq microdeletions. ROS levels in neat and washed semen of infertile men were significantly higher (P fragmentation in infertile men was significantly higher (P < 0.01) than controls. Genetic factors and oxidative stress cumulatively account for large number of idiopathic infertile cases. Unlike, genetic causes, which cannot be cured, timely identification and management of oxidative stress may help to reverse/reduce the effects on induced DNA damage, and improve the outcomes for infertile males.

  3. The use of premature chromosome condensation to study in interphase cells the influence of environmental factors on human genetic material

    Directory of Open Access Journals (Sweden)

    Vasiliki I. Hatzi

    2006-01-01

    Full Text Available Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental factors to which humans are exposed in their natural and anthropogenic environment. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics and occupational environment that constitute part of the anthropogenic environment. It is well known that genetic damage due to these factors has dramatic implications for human health. Since most of the environmental genotoxic factors induce arrest or delay in cell cycle progression, the conventional analysis of chromosomes at metaphase may underestimate their genotoxic potential. Premature Chromosome Condensation (PCC induced either by means of cell fusion or specific chemicals, enables the microscopic visualization of interphase chromosomes whose morphology depends on the cell cycle stage, as well as the analysis of structural and numerical aberrations at the G1 and G2 phases of the cell cycle. The PCC has been successfully used in problems involving cell cycle analysis, diagnosis and prognosis of human leukaemia, assessment of interphase chromosome malformations resulting from exposure to radiation or chemicals, as well as elucidation of the mechanisms underlying the conversion of DNA damage into chromosomal damage. In this report, particular emphasis is given to the advantages of the PCC methodology used as an alternative to conventional metaphase analysis in answering questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics and experimental therapeutics.

  4. Small supernumerary marker chromosomes (sSMC) in humans; are there B chromosomes hidden among them

    OpenAIRE

    Ogilvie Caroline; Kosyakova Nadezda; Mrasek Kristin; Liehr Thomas; Vermeesch Joris; Trifonov Vladimir; Rubtsov Nikolai

    2008-01-01

    Abstract Background Small supernumerary marker chromosomes (sSMC) and B-chromosomes represent a heterogeneous collection of chromosomes added to the typical karyotype, and which are both small in size. They may consist of heterochromatic and/or euchromatic material. Also a predominance of maternal transmission was reported for both groups. Even though sSMC and B-chromosomes show some similarity it is still an open question if B-chromosomes are present among the heterogeneous group of sSMC. Ac...

  5. Assignment of ALDH3 to human chromosome 17p11.2 and ALDH5 to human chromosome 9p13

    Energy Technology Data Exchange (ETDEWEB)

    Hiraoka, L.R. [Stanford Univ. Medical Center, CA (United States); Hsu, L. [City of Hope National Medical Center, Duarte, CA (United States); Hsieh, C.L. [Washington Univ. School of Medicine, St. Louis, MO (United States)

    1995-01-01

    Aldehyde dehydrogenases are a group of enzymes responsible for catalyzing the conversion of numerous aldehydes to their corresponding acids. These enzymes play a major role in the detoxification of alcohol-derived acetaldehydes and in the metabolism of corticosteroids, biogenic amines, neurotransmitters, and lipid peroxidation. ALDH activities are found in most tissues, with the highest activity in the liver. Five human liver ALDH isozymes, ALDH1, 2, 3, 4, and {gamma}-butyrylaldehyde dehydrogenase ({gamma}-ABDH), have been purified and characterized, and one isozyme, ALDH5, has been identified by using a 29-nucleotide probe that is conserved in ALDH1 and ALDH2 for cDNA library screening. Several other isozymes from other tissues have also been reported. Genes for ALDH3 and ALDH5 have been previously localized to chromosomes 17 and 9, respectively. Here we report the regional assignment of these two loci. 17 refs., 1 fig.

  6. Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

    Directory of Open Access Journals (Sweden)

    Greicy H Goto

    2015-08-01

    Full Text Available Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

  7. Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

    Science.gov (United States)

    Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

    2016-09-19

    Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

  8. Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

    OpenAIRE

    Keith A. Maggert

    2014-01-01

    The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “co...

  9. Cloning and characterization of dispersed repetitive DNA derived from microdissected sex chromosomes of Rumex acetosa.

    Science.gov (United States)

    Mariotti, Beatrice; Navajas-Pérez, Rafael; Lozano, Rafael; Parker, John S; de la Herrán, Roberto; Rejón, Carmelo Ruiz; Rejón, Manuel Ruiz; Garrido-Ramos, Manuel; Jamilena, Manuel

    2006-02-01

    Rumex acetosa is characterized by a multiple chromosome system (2n = 12 + XX for females, and 2n = 12 + XY1Y2 for males), in which sex is determined by the ratio between the number of X chromosomes and autosome sets. For a better understanding of the molecular structure and evolution of plant sex chromosomes, we have generated a sex chromosome specific library of R. acetosa by microdissection. The screening of this library has allowed us to identify 5 repetitive DNA families that have been characterized in detail. One of these families, DOP-20, has shown no homology with other sequences in databases. Nevertheless, the putative proteins encoded by the other 4 families, DOP-8, DOP-47, DOP-60, and DOP-61, show homology with proteins from different plant retroelements, including poly proteins from Ty3-gypsy- and Ty1-copia-like long terminal repeat (LTR) retroelements, and reverse transcriptase from non-LTR retro elements. Results indicate that sequences from these 5 families are dispersed throughout the genome of both males and females, but no appreciable accumulation or differentiation of these types of sequences have been found in the Y chromosomes. These repetitive DNA sequences are more conserved in the genome of other dioecious species such as Rumex papillaris, Rumex intermedius, Rumex thyrsoides, Rumex hastatulus, and Rumex suffruticosus, than in the polygamous, gynodioecious, or hermaphrodite species Rumex induratus, Rumex lunaria, Rumex con glom er atus, Rumex crispus, and Rumex bucephalo phorus, which supports a single origin of dioecious species in this genus. The implication of these transposable elements in the origin and evolution of the heteromorphic sex chromosomes of R. acetosa is discussed.

  10. The active gene that encodes human High Mobility Group 1 protein (HMG1) contains introns and maps to chromosome 13

    Energy Technology Data Exchange (ETDEWEB)

    Ferrari, S. [Dipartimento di Genetica e di Biologia dei Microrganismi, Milan (Italy); Finelli, P.; Rocchi, M. [Istituto di Genetica, Bari (Italy)] [and others

    1996-07-15

    The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene. We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes. We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene. The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 and q12, as determined by in situ hybridization. The mouse Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band. 18 refs., 3 figs.

  11. Mapping a gene that determines erythrocytic GTP concentration to a region of mouse chromosome 9 which is syntenic to human chromosome 3p

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, F.F.; Jenuth, J.P.; Noy, J.L. [Univ. of Calgary, Alberta (Canada)] [and others

    1994-09-01

    Inbred mouse strains were surveyed for erythrocytic GTP concentration by high performance liquid chromatography and found to fall into two groups. Strains having low GTP levels between 1.4-3.4 nmole/10{sup 9} cells are represented by C3H/HeJ. Strains having high GTP levels between 11.0 - 14.8 nmole/10{sup 9} cells are represented by C57BL/6J. Erythocytic ATP levels did not vary significantly among these strains (63-87 nmole/10{sup 9} cells). Crosses between low and high GTP strains gave F{sub 1} progeny having intermediate levels of GTP. The progeny of F{sub 1}`s backcrossed to paternal strains segregated in a 1:1 ratio for GTP concentration characteristic of the F{sub 1} and parental strain. We designated the GTP concentration-determining trait Gtpc. Typing of the twelve BXH recombinant inbred strains revealed 0/12 strain distribution pattern differences with Gtpc for loci on both chromosomes 5 and 9. Backcross analysis did not provide evidence for linkage of Gtpc to W (dominant white spotting) on chromosome 5 with 15/45 recombinants. Backcross analysis testing for linkage of Gtpc to transferrin (Trf) on chromosome 9 gave evidence for linkage with a recombination frequency of 9.68 {plus_minus} 3.07. DNA-based typing of repeat length polymorphic markers on chromosome 9 gave a map distance of 10.7 {plus_minus} 3.6 between D9 MITl4 and Gtpc and placed Gtpc on the telomeric side of Trf. This region of mouse chromosome 9 is syntenic to human chromosome 3p and encompasses a cluster of G-protein loci.

  12. The prevalence of chromosomal deletions relating to developmental delay and/or intellectual disability in human euploid blastocysts.

    Directory of Open Access Journals (Sweden)

    Wenyin He

    Full Text Available Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy and structural (deletion, duplication or others anomalies. Our previous study indicated that chromosomal deletion(s is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34-149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision.

  13. The prevalence of chromosomal deletions relating to developmental delay and/or intellectual disability in human euploid blastocysts.

    Science.gov (United States)

    He, Wenyin; Sun, Xiaofang; Liu, Lian; Li, Man; Jin, Hua; Wang, Wei-Hua

    2014-01-01

    Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy) and structural (deletion, duplication or others) anomalies. Our previous study indicated that chromosomal deletion(s) is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s) using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34-149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s) are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision.

  14. Admixture estimates for Caracas, Venezuela, based on autosomal, Y-chromosome, and mtDNA markers.

    Science.gov (United States)

    Martínez, Helios; Rodríguez-Larralde, Alvaro; Izaguirre, Mary Helen; De Guerra, Dinorah Castro

    2007-04-01

    The present Venezuelan population is the product of admixture of Amerindians, Europeans, and Africans, a process that was not homogeneous throughout the country. Blood groups, short tandem repeats (STRs), mtDNA, and Y-chromosome markers have been used successfully in admixture studies, but few such studies have been conducted in Venezuela. In this study we aim to estimate the admixture components of samples from two different socioeconomic levels from Caracas, Venezuela's capital city, compare their differences, and infer sexual asymmetry in the European Amerindian union patterns. Gene frequencies for blood groups ABO and Rh (CDE) and for the STRs VWA, F13A01, and FES/FPS and mtDNA and Y-chromosome haplogroups were studied in a sample of 60 individuals living in Caracas, taken from a private clinic (high socioeconomic level), and 50 individuals, also living in Caracas, drawn from a public maternity clinic (low socioeconomic level). The admixture analysis for the five autosomal markers gives a high European component (0.78) and an almost negligible African sub-Saharan component (0.06) for the high socioeconomic level, whereas for the low socioeconomic level the sub-Saharan, European, and Amerindian components were 0.21, 0.42, and 0.36, respectively. Estimates of admixture based on mtDNA and Y-chromosome markers reveal that the Amerindian contribution to these Caracas samples is almost entirely through females, because the Y-chromosome Amerindian and African sub-Saharan chromosomes found in this study were scarce. Our study reveals that the identification of the grandparents' geographic origin is an important methodological aspect to take into account in genetic studies related to the reconstruction of historical events.

  15. DNA and chromosome breaks induced by {sup 123}I-estrogen in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, J.L. [Argonne National Lab., IL (United States). Center for Mechanistic Biology and Biotechnology]|[Univ. of Chicago, IL (United States). Dept. of Radiation and Cellular Oncology; Mustafi, R.; Hughes, A.; DeSombre, E.R. [Univ. of Chicago, IL (United States)

    1997-07-01

    The effects of the Auger electron-emitting isotope I-123, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen positive Chinese hamster ovary (CHO-ER) cells. Exposure to the {sup 123}I-estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.2. The corresponding ratio with {sup 60}Co gamma rays was 15.6. The dose-response was biphasic suggesting that either receptor sites are saturated at high does, or that there is a nonrandom distribution of breaks induced by the {sup 123}I-estrogen. The {sup 123}I-estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1,000 disintegrations per cell. This corresponds to the mean lethal dose of {sup 123}I-estrogen for these cells suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that {sup 123}I-estrogen-induced chromosome breaks are rejoined. The F-ratio, the ratio of dicentrics to centric rings, was 5.8 {plus_minus} 1.7, which is similar to that seen with high LET radiations. Their results suggest that I-123 bound to estrogen is an efficient clastogenic agent, that the cytotoxic damage produced by I-123 bound to estrogen is very like high LET-induced damage, and the I-123 in the estrogen-receptor-DNA complex is probably in close proximity to the sugar-phosphate backbone of the DNA.

  16. Molecular cytogenetics of human germ cell tumours : i(12p) and related chromosomal anomalies

    NARCIS (Netherlands)

    Geurts van Kessel, A; Suijkerbuijk, R F; Sinke, R J; Looijenga, L; Oosterhuis, J W; de Jong, B

    1993-01-01

    Human testicular germ cell tumours (TGCTs) comprise a heterogeneous group of solid neoplasms. These tumours are characterized by a highly specific chromosomal anomaly, i.e. an isochromosome of the short arm of chromosome 12. At present, this i(12p) chromosome has been observed in about 80% of TGCTs.

  17. Characterization of a gene from the EDM1-PSACH region of human chromosome 19p

    Energy Technology Data Exchange (ETDEWEB)

    Lennon, G.G.; Giorgi, D.; Martin, J.R. [Lawrence Livermore National Lab., CA (United States)] [and others

    1994-09-01

    Genetic linkage mapping has indicated that both multiple epiphyseal dysplasia (EDM1), a dominantly inherited chondrodysplasia, and pseudoachondroplasia (PSACH), a skeletal disorder associated with dwarfism, map to a 2-3 Mb region of human chromosome 19p. We have isolated a partial cDNA from this region using hybrid selection, and report on progress towards the characterization of the genomic structure and transcription of the corresponding gene. Sequence analysis of the cDNA to date indicates that this gene is likely to be expressed within extracellular matrix tissues. Defects in this gene or neighboring gene family members may therefore lead to EDM1, PSACH, or other connective tissue and skeletal disorders.

  18. Chromosomal localization of the [delta] opioid receptor gene to human 1p34. 3-36. 1 and mouse 4D bands by in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Befort, K.; Kieffer, B. (Ecole Superieure de Biotechnologie de Strasbourg (France)); Mattei, M.G.; Roeckel, N. (Hopital d' Enfants de la Timone, Marseille (France))

    1994-03-01

    The aim of the present study is to determine the precise localization of this gene in the murine as well as human genome by in situ hybridization. Southern analysis, using the noncoding part of the cDNA as a probe (NotI-BamHI fragment, 1040 bp) under high-stringency conditions, shows the existence of a single-copy gene in the murine genome. In a similar analysis performed on human genomic DNA, the coding part of the cDNA (PstI-NotI fragment, 976 bp) clearly detected a single gene in the human genome (not shown). The authors therefore used these two probes for the chromosomal localization of the murine gene and its human counterpart, respectively. Chromosome spread preparations from concanavalin A (mouse) or phytohemagglutinin (human)-stimulated lymphocytes were hybridized with tritium-labeled cDNAs, exposed for 15 days, and developed, as described previously. For the murine assignment, a total of 100 metaphase cells were examined, and 149 silver grains were found. Forty-two grains were associated with chromosome 4, and 73.8% of them mapped to the D1-D3 region of the chromosome. In the 120 metaphase human cells analyzed, 197 silver grains were found, 25.8% of which were located on chromosome 1. The distribution of grains allowed mapping of the human [delta] opioid receptor gene to the p34.3-p36.1 region of the short arm with a maximum in the p35 band.

  19. Noc protein binds to specific DNA sequences to coordinate cell division with chromosome segregation.

    Science.gov (United States)

    Wu, Ling Juan; Ishikawa, Shu; Kawai, Yoshikazu; Oshima, Taku; Ogasawara, Naotake; Errington, Jeff

    2009-07-08

    Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis, the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo. We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division.

  20. Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-strand break repair model for T-DNA integration.

    Science.gov (United States)

    Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

    2017-02-28

    Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-strand break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-strand break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosome 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. This article is protected by copyright. All rights reserved.

  1. In situ hybridization of bat chromosomes with human (TTAGGGn probe, after previous digestion with Alu I

    Directory of Open Access Journals (Sweden)

    Karina de Cassia Faria

    2002-01-01

    Full Text Available The purpose of this work was to verify the ability of the enzyme Alu I to cleave and/or remove satellite DNA sequences from heterochromatic regions in chromosomes of bats, by identifying the occurrence of modifications in the pattern of fluorescence in situ hybridization with telomeric DNA. The localization and fluorescence intensity of the telomeric DNA sites of the Alu-digested and undigested chromosomes of species Eumops glaucinus, Carollia perspicillata, and Platyrrhinus lineatus were analyzed. Telomeric sequences were detected at the termini of chromosomes of all three species, although, in C. perspicillata, the signals were very faint or absent in most chromosomes. This finding was interpreted as being due to a reduced number of copies of the telomeric repeat, resulting from extensive telomeric association and/or rearrangements undergone by the chromosomes of Carollia. Fluorescent signals were also observed in centromeric and pericentromeric regions in several two-arm chromosomes of E. glaucinus and C. perspicillata. In E. glaucinus and P. lineatus, some interstitial and terminal telomeric sites were observed to be in association with regions of constitutive heterochromatin and ribosomal DNA (NORs. After digestion, these telomeric sites showed a significant decrease in signal intensity, indicating that enzyme Alu I cleaves and/or removes part of the satellite DNA present in these regions. These results suggest that the telomeric sequence is a component of the heterochromatin, and that the C-band- positive regions of bat chromosomes have a different DNA composition.

  2. ASSIGNMENT OF THE GENE CODING FOR HUMAN CYTOCHROME-C-OXIDASE SUBUNIT-VIB TO CHROMOSOME-19, BAND-Q13.1, BY FLUORESCENCE INSITU HYBRIDIZATION

    NARCIS (Netherlands)

    TAANMAN, JW; VANDERVEEN, AY; SCHRAGE, C; DEVRIES, H; BUYS, CHCM

    1991-01-01

    A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence

  3. Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae.

    Science.gov (United States)

    Hizume, Masahiro; Shibata, Fukashi; Matsumoto, Ayako; Maruyama, Yukie; Hayashi, Eiji; Kondo, Teiji; Kondo, Katsuhiko; Zhang, Shozo; Hong, Deyuan

    2002-08-01

    Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0-3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.

  4. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.

    Science.gov (United States)

    Simsek, Deniz; Brunet, Erika; Wong, Sunnie Yan-Wai; Katyal, Sachin; Gao, Yankun; McKinnon, Peter J; Lou, Jacqueline; Zhang, Lei; Li, James; Rebar, Edward J; Gregory, Philip D; Holmes, Michael C; Jasin, Maria

    2011-06-01

    Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

  5. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.

    Directory of Open Access Journals (Sweden)

    Deniz Simsek

    2011-06-01

    Full Text Available Nonhomologous end-joining (NHEJ is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4, suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

  6. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.

    Directory of Open Access Journals (Sweden)

    Deniz Simsek

    2011-06-01

    Full Text Available Nonhomologous end-joining (NHEJ is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4, suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

  7. A linkage map of mouse chromosome 8: further definition of homologous linkage relationships between mouse chromosome 8 and human chromosomes 8, 16, and 19.

    Science.gov (United States)

    Howard, T A; Rochelle, J M; Saunders, A M; Seldin, M F

    1991-05-01

    Using an interspecific cross, a mouse chromosome 8 linkage map spanning 72 cM has been defined by the segregation of restriction fragment length variants. Linkage and genetic distance were established for 10 loci by analysis of 114 meiotic events and indicated the following gene order: (centromere)-Insr-3.5 cM-Plat-26.3 cM-Crryps/Mel/Jund-3.5 cM-Junb/Ucp-10.5 cM-Mt-1-27.2 cM-Acta2-0.9 cM-Aprt. These data provide further definition of mouse chromosome 8 linkage relationships and the relationship between segments of this chromosome and human chromosomes 8, 16, and 19.

  8. Localization of a human homolog of the mouse Tiam-1 gene to chromosome 21q22.1

    Energy Technology Data Exchange (ETDEWEB)

    Haiming Chen; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)

    1995-11-01

    Exon trapping was applied to genomic DNA from a chromosome 21-specific cosmid library (LL21NC02-Q) to clone portions of genes from this chromosome. Among a large number of trapped exons, three showed striking homology to different regions of the cDNA for the mouse T-lymphoma invasion and metastasis gene (Tiam-1) at both nucleotide and predicted amino acid sequence levels, suggesting that these three exons are part of a human homolog of the mouse Tiam-1 gene. We mapped this presumed human TIAM1 gene to chromosome 21 by using appropriate somatic cell hybrids, YACs, and cosmids. The TIAM1 gene localizes to YAC 760H5 of the I. Chumakov et al. YAC contig between markers D21S298 and D21S404 in band 21q22.1. This human gene (which is a member of the group of guanine nucleotide-dissociation stimulators that modulate the activity of Rho-like proteins) may be important in the development or metastasis of malignancies that are associated with abnormalities on chromosome 21, including the various forms of leukemia frequent in trisomy 21. 25 refs., 2 figs.

  9. [Analysis of DNA and chromosome damage by the methods of molecular cytogenetics].

    Science.gov (United States)

    Arutiunian, R M; Oganesian, G G

    2011-01-01

    The main hybrid techniques of molecular cytogenetics are described. Methodological aspects of combination of fluorescence in situ hybridization (FISH) with the comet assay and micronuclei (MN) test are discussed along with results of their application to evaluate and locate DNA and chromosome damage in the genome. The experience of the authors with the application of FISH in combination with the comet assay and MN test are reported.

  10. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

    NARCIS (Netherlands)

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H. F.; Fousteri, Maria I.

    2007-01-01

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase III alpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is indispensa

  11. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

    NARCIS (Netherlands)

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H. F.; Fousteri, Maria I.

    2007-01-01

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase III alpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is

  12. Identification and genetic mapping of a homeobox gene to the 4p16. 1 region of human chromosome 4

    Energy Technology Data Exchange (ETDEWEB)

    Stadler, H.S.; Padanilam, B.J.; Solursh, M. (Univ. of Iowa, Iowa City (United States)); Buetow, K. (Fox Chase Cancer Center, Philadelphia, PA (United States)); Murray, J.C. (Univ. of Iowa Hospitals and Clinics, Iowa City (United States))

    1992-12-01

    A human craniofacial cDNA library was screened with a degenerate oligonucleotide probe based on the conserved third helix of homeobox genes. From this screening, we identified a homeobox gene, H6, which shared only 57-65% amino acid identity to previously reported homeodomains. H6 was physically mapped to the 4P16.1 region by using somatic cell hybrids containing specific deletions of human chromosome 4. Linkage data from a single-stranded conformational polymorphism derived from the 3[prime] untranslated region of the H6 cDNA placed this homeobox gene more than 20 centimorgans proximal of the previously mapped HOX7 gene on chromosome 4. Identity comparisons of the H6 Homeodomain with previously reported homeodomains reveal the highest identities to be with the Nk class of homeobox genes in Drosophila melanogaster. 53 refs., 5 figs., 2 tabs.

  13. Identification and genetic mapping of a homeobox gene to the 4p16.1 region of human chromosome 4.

    Science.gov (United States)

    Stadler, H S; Padanilam, B J; Buetow, K; Murray, J C; Solursh, M

    1992-12-01

    A human craniofacial cDNA library was screened with a degenerate oligonucleotide probe based on the conserved third helix of homeobox genes. From this screening, we identified a homeobox gene, H6, which shared only 57-65% amino acid identity to previously reported homeodomains. H6 was physically mapped to the 4p16.1 region by using somatic cell hybrids containing specific deletions of human chromosome 4. Linkage data from a single-stranded conformational polymorphism derived from the 3' untranslated region of the H6 cDNA placed this homeobox gene more than 20 centimorgans proximal of the previously mapped HOX7 gene on chromosome 4. Identity comparisons of the H6 homeodomain with previously reported homeodomains reveal the highest identities to be with the Nk class of homeobox genes in Drosophila melanogaster.

  14. Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca).

    Science.gov (United States)

    Wang, Yongping; Xu, Zhe; Guo, Ximing

    2004-02-01

    Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of CRASSOSTREA: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)--the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)--the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of CRASSOSTREA: This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.

  15. Bacterial chromosome segregation: structure and DNA binding of the Soj dimer — a conserved biological switch

    OpenAIRE

    Leonard, Thomas A.; Butler, P Jonathan; Löwe, Jan

    2005-01-01

    Soj and Spo0J of the Gram-negative hyperthermophile Thermus thermophilus belong to the conserved ParAB family of bacterial proteins implicated in plasmid and chromosome partitioning. Spo0J binds to DNA near the replication origin and localises at the poles following initiation of replication. Soj oscillates in the nucleoid region in an ATP- and Spo0J-dependent fashion. Here, we show that Soj undergoes ATP-dependent dimerisation in solution and forms nucleoprotein filaments with DNA. Crystal s...

  16. SCAI promotes DNA double-strand break repair in distinct chromosomal contexts

    DEFF Research Database (Denmark)

    Hansen, Rebecca Kring; Mund, Andreas; Poulsen, Sara Lund

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer...... in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI...

  17. DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

    Directory of Open Access Journals (Sweden)

    Gaëlle Demarre

    2010-05-01

    Full Text Available DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

  18. The future of human DNA vaccines.

    Science.gov (United States)

    Li, Lei; Saade, Fadi; Petrovsky, Nikolai

    2012-12-31

    DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including "epigenetics" and "omics" approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans.

  19. Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast.

    Directory of Open Access Journals (Sweden)

    Gloria Palou

    2015-09-01

    Full Text Available A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism Saccharomyces cerevisiae. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint