Budding yeast with human telomeres: a puzzling structure

Telomeres share some common features among eukaryotes, with few exceptions such as the fruit fly Drosophila that uses transposons as telomeres, they consist of G-rich repetitive DNA that is elongated by telomerase and/or alternative pathways depending on recombination. Telomere structure comprises both cis-acting satellite DNA (telomeric DNA) and proteins that interact directly and/or indirectly with the underlying DNA. Telomeric DNAs are surprisingly conserved among the vertebrates and very similar in most eukaryotes, but present some differences in yeast such as Saccharomyces cerevisiae. The telomeric proteins are more variable although the basic mechanisms which control telomere lengthening and capping are very similar, in fact orthologues of the yeast telomeric proteins, which have been studied first, have been identified in other organisms. Here we describe the structure of human telomeres in budding yeast as compared to canonical yeast and mammalian telomeres taking into consideration the more recent findings highlighting the mechanisms that are responsible for chromosome end protection and lengthening, and the role of chromatin organization in telomere function. This yeast represents a model for the study of mammalian telomeres that could be reconstituted step-by-step in all their components, moreover it could be useful for the assembly of mammalian artificial chromosome.     

C3-Activating proteases on human lymphoblastoid cells superinfected with epstein-barr virus

The role played by macrophages in feedback inhibition of the immune response during murine brucellosis, allowing establishment of chronic infection, was investigated using a number of approaches. First, it was shown that the degree of splenomegaly (a measure of macrophage influx) following infection with Brucella abortus strain 19 did not correlate with the course of bacterial numbers in the spleen of CBA, BALB/c, and C57B1/10 mice. Second, it was shown that a rough, mucoid mutant, B. abortus strain 19R, although causing a chronic infection, did not induce splenomegaly. Nor could “suppressor macrophages” be demonstrated in these spleens. Delayed-type hypersensitivity during this infection was lower than with B. abortus strain 19. Third, no nonspecific suppression of unrelated immune responses in CBA mice infected with B. abortus strain 19 could be demonstrated, despite the very large numbers of macrophages in the spleen. The responses tested included delayed-type hypersensitivity and cell-mediated resistance to Listeria monocytogenes, antibody response to sheep erythrocytes (both serum antibody and plaque-forming cells in the spleen) and skin-graft rejection.     

Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin

A hypomorphic mutation made in the ORC2 gene of a human cancer cell line through homologous recombination decreased Orc2 protein levels by 90%. The G1 phase of the cell cycle was prolonged, but there was no effect on the utilization of either the c-Myc or beta-globin cellular origins of replication. Cells carrying this mutation failed to support the replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV), and this defect was rescued by reintroduction of Orc2. Orc2 specifically associates with oriP in cells, most likely through its interaction with EBNA1. Geminin, an inhibitor of the mammalian replication initiation complex, inhibits replication from oriP. Therefore, ORC and the human replication initiation apparatus is required for replication from a viral origin of replication.     

Application of in situ PCR to yeast cells for screening YAC libraries.

We have used in situ PCR technology in yeast cells with the ultimate goal of cloning and screening genomic yeast artificial chromosome (YAC) libraries. The target sequences in YAC clones were amplified "in situ" in yeast cells by the same set of microsatellite primers used in solution-based PCR screening. The method is fast and sensitive and obviates the steps required for individual isolation of DNAs from hundreds to thousands of YAC clones and thus has an advantage over conventional solution-based PCR screening. This approach can conceivably be applied to the products of automated robotic workstations.     

Morphology of a human-derived YAC in yeast meiosis

In meiosis of human males DNA is packaged along pachytene chromosomes about 20 time more compactly than in meiosis of yeast. Nevertheless, a human-derived yeast artificial chromosome (YAC) shows the same degree of compaction of DNA as endogenous chromosomes in meiotic prophase nuclei of yeast. This suggests that in yeast meiosis, human and yeast DNA adopt a similar organization of chromatin along the pachytene chromosome cores. Therefore meiotic chromatin organization does not seem to be an inherent chromosomal property but is governed by the host-specific cellular environment. We suggest that there is a correlation between the less dense DNA packaging and the increased rate of recombination that has been reported for human-derived YACs as compared with human DNA in its natural environment.     

Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells.

Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.     

Humanized telomeres and an attempt to express a functional human telomerase in yeast

The maintenance of telomeric repeat DNA depends on an evolutionarily conserved reverse trans criptase called telomerase. In vitro, only the catalytic subunit and a telomerase-associated RNA are required for the synthesis of species-specific repeat DNA. In an attempt to establish a heterologous system for the study of the human telomerase enzyme, we expressed the two core components and predicted regulatory subunits in the yeast Saccharomyces cerevisiae. We show that adequate substrates for human telomerase can be generated; the expressed enzyme was localized in the nucleus and it had the capacity to synthesize human-specific repeats in vitro. However, there was no evidence for human telomerase activity at yeast telomeres in vivo. Therefore functional replacement of the yeast telomerase by the human enzyme may require additional human-specific components. We also replaced the template region of the yeast telomerase RNA with one that dictates the synthesis of vertebrate repeats and performed a detailed molecular analysis of the composition of the telomeres upon outgrowth of such strains. The results suggest that vertebrate repeats on yeast telomeres are subject to a very high degree of repeat turnover and show that an innermost tract of 50 bp of yeast repeats are resistant to replacement.     

Immunoblotting reactivity of human sera from various sources against purified epstein-barr virus

The immunoblotting technique was used to analyse polypeptides of purified Epstein-Barr virus reacting with antibodies present in sera from clinically healthy individuals, from patients with infectious mononucleosis (IM) or AIDS, and from renal transplant recipients with molecular sizes in the range of 40–290 kDa were detected.The 47- and 160-kDa nucleocapsid polypeptides, as well as the 72-, 74-, 140-, 220- and 290-kDa membrane polypeptides were the major viral proteins detected in the sera. Sera from clinically healthy individuals contained antibodies directed against all EBV membrane and nucleocapsid antigens. Sera from renal transplant recipients, from patients with IM and from patients with AIDS failed to react with certain nucleocapsid and membrane antigens; in particular, sera from AIDS patients and renal transplant recipients did not react with the 220-kDa polypeptide, one of the major membrane antigens, while sera from subjects with IM and from healthy individuals did.A high proportion of sera from patients with IM (38% vs 5% of clinically healthy individuals and 0–5% of the AIDS patients and renal transplant recipients) reacted with a 42-kDa polypeptide, suggesting its possible role in acute EBV infection.     

A dicistronic construct allows easy detection of human CFTR expression from YAC DNA in human cells.

We have made a dicistronic construct where the picornaviral internal ribosome-entry site (IRES) driving the expression of the beta-geo gene has been inserted into the 3'untranslated region of the human CFTR gene present in a YAC. When introduced into the human cell line Caco-2 expressing the CFTR gene, the expression of the dicistronic gene can be detected by lacZ staining and follows the accumulation of the endogenous CFTR mRNA upon differentiation of the cells. These data demonstrate that this IRES-based approach presents an alternative to mRNA in situ hybridisation and allows detection of expression in an autologous system.     

Analysis of chromosome 21 yeast artificial chromosome (YAC) clones.

Chromosome 21 contains genes relevant to several important diseases. Yeast artificial chromosome (YAC) clones, because they span > 100 kbp, will provide attractive material for initiating searches for such genes. Twenty-two YAC clones, each of which maps to a region of potential relevance either to aspects of the Down syndrome phenotype or to one of the other chromosome 21-associated genetic diseases, have been analyzed in detail. Clones total approximately 6,000 kb and derive from all parts of the long arm. Rare restriction-site maps have been constructed for each clone and have been used to determine regional variations in clonability, methylation frequency, CpG island density, and CpG island frequency versus gene density. This information will be useful for the isolation and mapping of new genes to chromosome 21 and for walking in YAC libraries.     

Buildup from birth onward of short telomeres in human hematopoietic cells

Telomere length (TL) limits somatic cell replication. However, the shortest among the telomeres in each nucleus, not mean TL, is thought to induce replicative senescence. Researchers have relied on Southern blotting (SB), and techniques calibrated by SB, for precise measurements of TL in epidemiological studies. However, SB provides little information on the shortest telomeres among the 92 telomeres in the nucleus of human somatic cells. Therefore, little is known about the accumulation of short telomeres with age, or whether it limits the human lifespan. To fill this knowledge void, we used the Telomere-Shortest-Length-Assay (TeSLA), a method that tallies and measures single telomeres of all chromosomes. We charted the age-dependent buildup of short telomeres (<3 kb) in human hematopoietic cells from 334 individuals (birth-89 years) from the general population, and 18 patients with dyskeratosis congenita-telomere biology disorders (DC/TBDs), whose hematopoietic cells have presumably reached or are close to their replicative limit. For comparison, we also measured TL with SB. We found that in hematopoietic cells, the buildup of short telomeres occurs in parallel with the shortening with age of mean TL. However, the proportion of short telomeres was lower in octogenarians from the general population than in patients with DC/TBDs. At any age, mean TL was longer and the proportion of short telomeres lower in females than in males. We conclude that though converging to the TL-mediated replicative limit, hematopoietic cell telomeres are unlikely to reach this limit during the lifespan of most contemporary humans.     

Recombination can either help maintain very short telomeres or generate longer telomeres in yeast cells with weak telomerase activity

Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.     

A double-strand break within a yeast artificial chromosome (YAC) containing human DNA can result in YAC loss, deletion or cell lethality.

Human chromosomal DNA contains many repeats which might provide opportunities for DNA repair. We have examined the consequences of a single double-strand break (DSB) within a 360-kb dispensable yeast artificial chromosome (YAC) containing human DNA (YAC12). An Alu-URA3-YZ sequence was targeted to several Alu sites within the YAC in strains of the yeast Saccharomyces cerevisiae; the strains contained a galactose-inducible HO endonuclease that cut the YAC at the YZ site. The presence of a DSB in most YACs led to deletion of the URA3 cassette, with retention of the telomeric markers, through recombination between surrounding Alus. For two YACs, the DSBs were not repaired and there was a G2 delay associated with the persistent DSBs. The presence of persistent DSBs resulted in cell death even though the YACs were dispensable. Among the survivors of the persistent DSBs, most had lost the YAC. By a pullback procedure, cell death was observed to begin at least 6 h after induction of a break. For YACs in which the DSB was rapidly repaired, the breaks did not cause cell cycle delay or lead to cell death. These results are consistent with our previous conclusion that a persistent DSB in a plasmid (YZ-CEN) also caused lethality (C. B. Bennett, A. L. Lewis, K. K. Baldwin, and M. A. Resnick, Proc. Natl. Acad. Sci. USA 90:5613-5617, 1993). However, a break in the YZ-CEN plasmid did not induce lethality in the strain (CBY) background used in the present study. The differences in survival levels appear to be due to the rapid degradation of the plasmid in the CBY strain. We, therefore, propose that for a DSB to cause cell cycle delay and death by means other than the loss of essential genetic material, it must remain unrepaired and be long-lived.     

Characterization of Arabidopsis thaliana telomeres isolated in yeast.

In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome.     

Isolation and characterization of a yeast artificial chromosome (YAC) contig around the human steroid sulfatase gene.

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.     

Complementation of null CF mice with a human CFTR YAC transgene.

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.