YAC cloning of telomeres

Human telomeres have been successfully cloned in Saccharomyces cerevisiae by complementing deficient yeast artificial chromosomes (YACs). This technique allows cloning of DNA sequences that can recognize particular chromosomal ends, and therefore facilitates the mapping of eukaryotic genomes. Although the biology of adopting foreign telomeres in yeast is not fully understood, the cloning system itself seems to be a useful tool for constructing telomeric DNA libraries from higher eukaryotes. Here we describe the techniques that are currently being used in cloning of telomeric DNA.     

YAC cloning of DNA embedded in an agarose matrix

Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions. It minimizes any shearing that may result from handling of high molecular weight DNA and can be done with nanogram to microgram amounts of material, which facilitates construction of YACs from sources of DNA other than genomic DNA isolated from cells. The average size of the YACs recovered (200-1000 kb) and efficiency of transformation of ligation products (200-1000 cfu/micrograms) are similar to those reported using aqueous protocols. This method has been used to construct chromosome specific YACs, and it should be possible to apply the technique to the construction of chromosome specific libraries using flow sorted chromosomes as source material, and the cloning of restriction fragments isolated by preparative pulsed field gel electrophoresis.     

Analysis of a YAC with human telomeres and oriP from epstein-barr virus in yeast and 293 cells.

One approach to the construction and propagation of a mammalian artificial chromosome is to build it up in Saccharomyces cerevisiae, using a yeast artificial chromosome (YAC) base. We have demonstrated that circular YACs carrying the Epstein-Barr virus origin of plasmid replication ( oriP ) are maintained as stable, episomal elements in human cells. We wished to determine whether this technology could be extended, to generate linear extrachromosomal elements. Here, we describe the generation of retrofitting constructs, which permit the addition of human telomeres and the oriP domain to YACs. The constructs contain 0.8 kb of human telomere sequence separated by a unique Not I site from 0.7 kb of Tetrahymena telomere sequence. These constructs seed telomere formation with approximately 40-60% efficiency in human 293-EBNA and HT1080 cells whether or not the Tetrahymena sequence is removed by Not I digestion. A detailed analysis demonstrates that YACs carrying the human telomere cassettes on both arms show instability of the telomere sequences in S.cerevisiae at a frequency of approximately 50%. Introduction of correctly retrofitted, linear oriP YACs into human 293-EBNA cells by lipofection resulted in the generation of circular extrachromosomal elements varying in size from 8 to 300 kb. However, no apparently linear YACs could be detected, suggesting that extrachromosomal maintenance of DNA with the oriP /EBNA-1 system is not compatible with linear molecules capped by telomeres.     

An improved method of partially digesting plant megabase DNA suitable for YAC cloning: application to the construction of a 5.5 genome equivalent YAC library of tomato

An improved method for preparing partially digested tomato DNA has been developed, that is suitable for YAC cloning. It involves (i) isolation of high molecular-weight DNA from agarose-embedded leaf protoplasts, (ii) controlled partial digestion in situ using Eco RI endonuclease in the presence of Eco RI methylase (M. Eco RI), and (iii) fractionation of the partial digest on a Clamped Homogeneous Electric Fields (CHEF) gel. Unlike methods commonly used for generating partial digests, the present method allows one to produce digests in which the bulk of restriction fragments are of the desired size. Use of these partial digests in constructing YAC libraries of the tomato lines Moneymaker- Cf4 and VFNT Cherry resulted in libraries (total 21 060 clones, 5.5 genome equivalents) in which 80% of the YACs have inserts between 200 and 600 kb. Both libraries have been screened with selected RFLP markers linked to the Cladosporium fulvum Cf4 locus on chromosome 1, using a three-dimensional PCR-based screening technique. To this end, the RFLP markers have been sequenced to allow for the synthesis of specific primers. Thus, for each marker tested several YAC clones have been isolated, including a family of clones that carry leucine-rich repeat sequences located around the Cf4/ Cf9 locus.     

An improved method of plant megabase DNA isolation in agarose microbeads suitable for physical mapping and YAC cloning

The isolation of high quality megabase DNA from plant cells that is susceptible to a variety of molecular reagents is a critical first step in the physical analysis of complex genomes. A method for the isolation of such DNA by encapsulating plant protoplasts in agarose microbeads is presented. In comparison with the conventional agarose plug method, microbeads provide a dramatic increase in the surface area yielding megabase DNA that can be treated essentially as an aqueous DNA solution. Examples of the utility of DNA prepared by this technique for physical mapping, partial restriction enzyme digestion and cloning of large inserts as YACs are presented.     

An improved general approach for cloning and characterizing telomeres: the protozoan parasite Trypanosoma cruzi as model organism

We here describe a general strategy for cloning and characterizing telomeric and sub-telomeric regions of the human protozoan parasite Trypanosoma cruzi. The use of a bacterial artificial chromosome vector and a telomeric adaptor produced stable telomeric recombinant clones with inserts ranging from 5 to 25 kb. Analysis of these recombinants provided unique landmarks for chromosomal mapping and sequencing and enabled us to derive a more accurate picture of T. cruzi telomeric organization.     

Women and telomeres

Last year''s Nobel Prizes for Carol Greider and Elizabeth Blackburn should be encouraging for all female scientists with childrenCarol Greider, a molecular biologist at Johns Hopkins University (Baltimore, MD, USA), recalled that when she received a phone call from the Nobel Foundation early in October last year, she was staring down a large pile of laundry. The caller informed her that she had won the 2009 Nobel Prize in Physiology or Medicine along with Elizabeth Blackburn, her mentor and co-discoverer of the enzyme telomerase, and Jack Szostak. The Prize was not only the ultimate reward for her own achievements, but it also highlighted a research field in biology that, unlike most others, is renowned for attracting a significant number of women.Indeed, the 2009 awards stood out in particular, as five women received Nobel prizes. In addition to the Prize for Greider and Blackburn, Ada E. Yonath received one in chemistry, Elinor Ostrom became the first female Prize-winner in economics, and Herta Müller won for literature (Fig 1).Open in a separate windowFigure 1The 2009 Nobel Laureates assembled for a photo during their visit to the Nobel Foundation on 12 December 2009. Back row, left to right: Nobel Laureates in Chemistry Ada E. Yonath and Venkatraman Ramakrishnan, Nobel Laureates in Physiology or Medicine Jack W. Szostak and Carol W. Greider, Nobel Laureate in Chemistry Thomas A. Steitz, Nobel Laureate in Physiology or Medicine Elizabeth H. Blackburn, and Nobel Laureate in Physics George E. Smith. Front row, left to right: Nobel Laureate in Physics Willard S. Boyle, Nobel Laureate in Economic Sciences Elinor Ostrom, Nobel Laureate in Literature Herta Müller, and Nobel Laureate in Economic Sciences Oliver E. Williamson. © The Nobel Foundation 2009. Photo: Orasis.Greider, the daughter of scientists, has overcome many obstacles during her career. She had dyslexia that placed her in remedial classes; “I thought I was stupid,” she told The New York Times (Dreifus, 2009). Yet, by far the biggest challenge she has tackled is being a woman with children in a man''s world. When she attended a press conference at Johns Hopkins to announce the Prize, she brought her children Gwendolyn and Charles with her (Fig 2). “How many men have won the Nobel in the last few years, and they have kids the same age as mine, and their kids aren''t in the picture? That''s a big difference, right? And that makes a statement,” she said.The Prize […] highlighted a research field in biology that, unlike most others, is renowned for attracting a significant number of womenOpen in a separate windowFigure 2Mother, scientist and Nobel Prize-winner: Carol Greider is greeted by her lab and her children. © Johns Hopkins Medicine 2009. Photo: Keith Weller.Marie Curie (1867–1934), the Polish–French physicist and chemist, was the first woman to win the Prize in 1903 for physics, together with her husband Pierre, and again for chemistry in 1911—the only woman to twice achieve such recognition. Curie''s daughter Irène Joliot-Curie (1897–1956), a French chemist, also won the Prize with her husband Frédéric in 1935. Since Curie''s 1911 prize, 347 Nobel Prizes in Physiology or Medicine and Chemistry (the fields in which biologists are recognized) have been awarded, but only 14—just 4%—have gone to women, with 9 of these awarded since 1979. That is a far cry from women holding up half the sky.Yet, despite the dominance of men in biology and the other natural sciences, telomere research has a reputation as a field dominated by women. Daniela Rhodes, a structural biologist and senior scientist at the MRC Laboratory of Molecular Biology (Cambridge, UK) recalls joining the field in 1993. “When I went to my first meeting, my world changed because I was used to being one of the few female speakers,” she said. “Most of the speakers there were female.” She estimated that 80% of the speakers at meetings at Cold Spring Harbour Laboratory in those early days were women, while the ratio in the audience was more balanced.Since Curie''s 1911 prize, 347 Nobel Prizes in Physiology or Medicine and Chemistry […] have been awarded, but only 14—just 4%—have gone to women…“There''s nothing particularly interesting about telomeres to women,” Rhodes explained. “[The] field covers some people like me who do structural biology, to cell biology, to people interested in cancers […] It could be any other field in biology. I think it''s [a result of] having women start it and [including] other women.” Greider comes to a similar conclusion: “I really see it as a founder effect. It started with Joe Gall [who originally recruited Blackburn to work in his lab].”Gall, a cell biologist, […] welcomed women to his lab at a time when the overall situation for women in science was “reasonably glum”…Gall, a cell biologist, earned a reputation for being gender neutral while working at Yale University in the 1950s and 1960s; he welcomed women to his lab at a time when the overall situation for women in science was “reasonably glum,” as he put it. “It wasn''t that women were not accepted into PhD programs. It''s just that the opportunities for them afterwards were pretty slim,” he explained.“Very early on he was very supportive to a number of women who went on and then had their own labs and […] many of those women [went] out in the world [to] train other women,” Greider commented. “A whole tree that then grows up that in the end there are many more women in that particular field simply because of that historical event.Thomas Cech, who won the Nobel Prize for Chemistry in 1989 and who worked in Gall''s lab with Blackburn, agreed: “In biochemistry and metabolism, we talk about positive feedback loops. This was a positive feedback loop. Joe Gall''s lab at Yale was an environment that was free of bias against women, and it was scientifically supportive.”Gall, now 81 and working at the Carnegie Institution of Washington (Baltimore, MD, USA), is somewhat dismissive about his positive role. “It never occurred to me that I was doing anything unusual. It literally, really did not. And it''s only been in the last 10 or 20 years that anyone made much of it,” he said. “If you look back, […] my laboratory [was] very close to [half] men and [half] women.”During the 1970s and 1980s; “[w]hen I entered graduate school,” Greider recalled, “it was a time when the number of graduate students [who] were women was about 50%. And it wasn''t unusual at all.” What has changed, though, is the number of women choosing to pursue a scientific career further. According to the US National Science Foundation (Arlington, VA, USA), women received 51.8% of doctorates in the life sciences in 2006, compared with 43.8% in 1996, 34.6% in 1986, 20.7% in 1976 and 11.9% in 1966 (www.nsf.gov/statistics).In fact, Gall suspects that biology tends to attract more women than the other sciences. “I think if you look in biology departments that you would find a higher percentage [of women] than you would in physics and chemistry,” he said. “I think […] it''s hard to dissociate societal effects from specific effects, but probably fewer women are inclined to go into chemistry [or] physics. Certainly, there is no lack of women going into biology.” However, the representation of women falls off at each level, from postdoc to assistant professor and tenured professor. Cech estimated that only about 20% of the biology faculty in the USA are women.“[It] is a leaky pipeline,” Greider explained. “People exit the system. Women exit at a much higher proportion than do men. I don''t see it as a [supply] pipeline issue at all, getting the trainees in, because for 25 years there have been a great number of women trainees.“We all thought that with civil rights and affirmative action you''d open the doors and women would come in and everything would just follow. And it turned out that was not true.”Nancy Hopkins, a molecular biologist and long-time advocate on issues affecting women faculty members at the Massachusetts Institute of Technology (Cambridge, MA, USA), said that the situation in the USA has improved because of civil rights laws and affirmative action. “I was hired—almost every woman of my generation was hired—as a result of affirmative action. Without it, there wouldn''t have been any women on the faculty,” she said, but added that: “We all thought that with civil rights and affirmative action you''d open the doors and women would come in and everything would just follow. And it turned out that was not true.”Indeed, in a speech at an academic conference in 2005, Harvard President Lawrence Summers said that innate differences between males and females might be one reason why fewer women than men succeeded in science and mathematics. The economist, who served as Secretary of Treasury under President William Clinton, told The Boston Globe that “[r]esearch in behavioural genetics is showing that things people previously attributed to socialization weren''t [due to socialization after all]” (Bombardieri, 2005).Some attendees of the meeting were angered by Summers''s remarks that women do not have the same ‘innate ability'' as men in some fields. Hopkins said she left the meeting as a protest and in “a state of shock and rage”. “It isn''t a question of political correctness, it''s about making unscientific, unfounded and damaging comments. It''s what discrimination is,” she said, adding that Summers''s views reflect the problems women face in moving up the ladder in academia. “To have the president of Harvard say that the second most important reason for their not being equal was really their intrinsic genetic inferiority is so shocking that no matter how many times I think back to his comments, I''m still shocked. These women were not asking to be considered better or special. They were just asking to have their gender be invisible.”Nonetheless, women are making inroads into academia, despite lingering prejudice and discrimination. One field of biology that counts a relatively high number of successful women among its upper ranks is developmental biology. Christiane Nüsslein-Volhard, for example, is Director of the Max Planck Institute for Developmental Biology in Tübingen, Germany, and won the Nobel Prize for Physiology or Medicine in 1995 for her work on the development of Drosophila embryos. She estimated that about 30% of speakers at conferences in her field are women.…for many women, the recent Nobel Prize for Greider […] and Blackburn […] therefore comes as much needed reassurance that it is possible to combine family life and a career in scienceHowever, she also noted that women have never been the majority in her own lab owing to the social constraints of German society. She explained that in Germany, Switzerland and Austria, family issues pose barriers for many women who want to have children and advance professionally because the pressure for women to not use day care is extremely strong. As such, “[w]omen want to stay home because they want to be an ideal mother, and then at the same time they want to go to work and do an ideal job and somehow this is really very difficult,” she said. “I don''t know a single case where the husband stays at home and takes care of the kids and the household. This doesn''t happen. So women are now in an unequal situation because if they want to do the job, they cannot; they don''t have a chance to find someone to do the work for them. […] The wives need wives.” In response to this situation, Nüsslein-Volhard has established the CNV Foundation to financially support young women scientists with children in Germany, to help pay for assistance with household chores and child care.Rhodes, an Italian native who grew up in Sweden, agreed with Nüsslein-Volhard''s assessment of the situation for many European female scientists with children. “Some European countries are very old-fashioned. If you look at the Protestant countries like Holland, women still do not really go out and have a career. It tends to be the man,” she said. “What I find depressing is [that in] a country like Sweden where I grew up, which is a very liberated country, there has been equality between men and women for a couple of generations, and if you look at the percentage of female professors at the universities, it''s still only 10%.” In fact, studies both from Europe and the USA show that academic science is not a welcoming environment for women with children; less so than for childless women and fathers, who are more likely to succeed in academic research (Ledin et al, 2007; Martinez et al, 2007).For Hopkins, her divorce at the age of 30 made a choice between children or a career unavoidable. Offered a position at MIT, she recalled that she very deliberately chose science. She said that she thought to herself: “Okay, I''m going to take the job, not have children and not even get married again because I couldn''t imagine combining that career with any kind of decent family life.” As such, for many women, the recent Nobel Prize for Greider, who raised two children, and Blackburn (Fig 3), who raised one, therefore comes as much needed reassurance that it is possible to combine family life and a career in science. Hopkins said the appearance of Greider and her children at the press conference sent “the message to young women that they can do it, even though very few women in my generation could do it. The ways in which some women are managing to do it are going to become the role models for the women who follow them.”Open in a separate windowFigure 3Elizabeth Blackburn greets colleagues and the media at a reception held in Genentech Hall at UCSF Mission Bay to celebrate her award of the Nobel Prize in Physiology or Medicine. © University of California, San Francisco 2009. Photo: Susan Merrell.     

Translating the telomeres

Telomeres are transcribed into long noncoding telomeric repeat-containing RNA (TERRA). Or so we thought. Recently, Al-Turki and Griffith provided evidence that TERRA can code for valine-arginine (VR) or glycine-leucine (GL) dipeptide repeat proteins by undergoing repeat-associated non-ATG (RAN) translation. This finding uncovers a new mechanism by which telomeres can impact cellular function.     

Composition of plant telomeres

Telomeres are essential elements of eukaryotic chromosomes that differentiate native chromosome ends from deleterious DNA double-strand breaks (DSBs). This is achieved by assembling chromosome termini in elaborate high-order nucleoprotein structures that in most organisms encompass telomeric DNA, specific telomere-associated proteins as well as general chromatin and DNA repair factors. Although the individual components of telomeric chromatin are evolutionary highly conserved, cross species comparisons have revealed a remarkable flexibility in their utilization at telomeres. This review outlines the strategies used for chromosome end protection and maintenance in mammals, yeast and flies and discusses current progress in deciphering telomere structure in plants.     

人基因组百万碱基级巨型YAC的构建

人基因组百万碱基级巨型ＹＡＣ的构建戴长虹,柴建华（复旦大学遗传学研究所,上海２００４３３）关键词人基因组;酵母人工染色体（ＹＡＣ）;百万碱基;巨型ＹＡＣ人类的全部遗传信息包含在３ｘ１０＇核着酸对（ｈｐ）中,共编码５００００～１０００００个基因。人类基...     

Chinese hamster telomeres are comparable in size to mouse telomeres.

We report here the results of a telomere length analysis in four male Chinese hamsters by quantitative fluorescence in situ hybridization (Q-FISH). We were able to measure telomere length of 64 (73%) of 88 Chinese hamster telomeres. We could not measure telomere length in chromosome 10 or in the short arms of chromosomes 5, 6, 7 and 8 because of the overlaps between the interstitial and terminal telomeric signals. Our analysis in the 73% of Chinese hamster telomeres indicate that their average length is approximately 38 kb. Therefore, Chinese hamster telomeres are comparable in length to mouse telomeres, but are much longer than human telomeres. Similar to previous Q-FISH studies on human and mouse chromosomes, our results indicate that individual Chinese hamster chromosomes may have specific telomere lengths, suggesting that chromosome-specific factors may be involved in telomere length regulation.     

Rapid screening of a YAC library by pulsed-field gel Southern blot analysis of pooled YAC clones

A new method for screening of YAC libraries is described. Individual YACs were pooled into groups of 384 clones and prepared as samples suitable for pulsed-field gel electrophoresis. A five hit human YAC library (Brownstein et al., 1989) containing approximately 60,000 clones was condensed into 150 such pools and chromosomal DNAs in each sample were separated on three pulsed field gels containing 50 samples each. Southern blots prepared from these gels were hybridized with probes of interest to identify pools containing homologous YACs. Further purification was performed using standard colony hybridization procedures. Twenty-one probes used thus far have identified 47 positive pools and corresponding YACs have been purified from 28 of these. Some significant advantages of this method include avoidance of DNA sequence analysis and primer generation prior to YAC screening and the ability to handle the entire library on three filters. The screening approach described here permits rapid isolation of YACs corresponding to unsequenced loci and will accelerate establishment of YAC contigs for large chromosomal segments.