Mitotic Recombination among Subtelomeric Y'' Repeats in Saccharomyces Cerevisiae

Y's are a dispersed family of repeats that vary in copy number, location and restriction fragment lengths between strains but exhibit within-strain homogeneity. We have studied mitotic recombination between members of the subtelomeric Y' repeated sequence family of Saccharomyces cerevisiae. Individual copies of Y's were marked with SUP11 and URA3 which allowed for the selection of duplications and losses of the marked Y's. Duplications occurred by ectopic recombinational interactions between Y's at different chromosome ends as well as by unequal sister chromatid exchange. Several of the ectopic duplications resulted in an originally Y'-less chromosome end acquiring a marked Y'. Among losses, most resulted from ectopic exchange or conversion in which only the marker sequence was lost. In some losses, the chromosome end became Y'-less. Although the two subsets of Y's, Y'-longs (6.7 kb) and Y'-shorts (5.2 kb), share extensive sequence homology, a marked Y' recombines highly preferentially within its own subset. These mitotic interactions can in part explain the maintenance of Y's and their subsets, the homogeneity among Y's within a strain, as well as diversity between strains.     

The Structure and Evolution of Subtelomeric Y'' Repeats in Saccharomyces Cerevisiae

The subtelomeric Y' family of repeated DNA sequences in the yeast Saccharomyces cerevisiae is of unknown origin and function. Y's vary in copy number and location among strains. Eight Y's, from two strains, were cloned and sequenced over the same 3.2-kb interval in order to assess the within- and between-strain variation as well as address their origin and function. One entire Y' sequence was reconstructed from two clones presented here and a previously sequenced 833-bp region. It contains two large overlapping open reading frames (ORFs). The putative protein sequences have no strong homologies to any known proteins except for one region that has 27% identity with RNA helicases. RNA homologous to each ORF was detected. Comparison of the sequences revealed that the known long (Y'-L) and short (Y'-S) size classes, which coexist within cells, differ by several insertions and/or deletions within this region. The Y'-Ls from strain Y55 also differ from those of strain YP1 by several short deletions in the same region. Most of these deletions appear to have occurred between short (2-10 bp) direct repeats. The single base pair polymorphisms and the deletions are clustered in the first half of the interval compared. There is 0.30-1.13% divergence among Y'-Ls within a strain and 1.15-1.75% divergence between strains in the interval. This is similar to known unique sequence variation but contrasts with the 8-18% divergence among the adjacent subtelomeric repeats, X. Subsets of Y's exhibit concerted evolution; however, more than one variant appears to be maintained within strains. The observed sequence variation disrupts the first ORF in many Y's while most of the second ORF including the putative helicase region is unaffected. The structure and distribution of the Y' elements are consistent with having originated as a mobile element. However, they now appear to move via recombination. Recombination can account for the homogenization within subsets of Y's but does not account for the maintenance of different variants.     

Identification of the telomere in Trypanosoma cruzi reveals highly heterogeneous telomere lengths in different parasite strains.

Here we describe the cloning and characterisation of the Trypanosoma cruzi telomere. In the Y strain, it is formed by typical GGGTTA repeats with a mean size of approximately 500 bp. Adjacent to the telomere repeats we found a DNA sequence with significant homology to the T.cruzi 85 kDa surface antigen (gp85). Examination of the telomere in nine T.cruzi strains reveals differences in the organisation of chromosome ends. In one group of strains the size of the telomere repeat is relatively homogeneous and short (0.5-1.5 kb) as in the Y strain, while in the other, the length of the repeat is very heterogeneous and significantly longer, ranging in size from 1 to >10 kb. These different strains can be grouped similarly to previously existing classifications based on isoenzyme loci, rRNA genes, mini-exon gene sequences, randomly amplified polymorphic DNA and rRNA promoter sequences, suggesting that differential control of telomere length and organisation appeared as an early event in T. cruzi evolution. Two-dimensional pulsed field gel electrophoresis analysis shows that some chromosomes carry telomeres which are significantly larger than the mean telomere length. Importantly, the T.cruzi telomeres are organised in nucleosomal and non-nucleosomal chromatin.     

The Chromosome End in Yeast: Its Mosaic Nature and Influence on Recombinational Dynamics

Yeast chromosome ends are composed of several different repeated elements. Among six clones of chromosome ends from two strains of Saccharomyces cerevisiae, at least seven different repeated sequence families were found. These included the previously identified Y'' and X elements. Some families are highly variable in copy number and location between strains of S. cerevisiae, while other elements appear constant in copy number and location. Three repeated sequence elements are specific to S. cerevisiae and are not found in its evolutionarily close relative, Saccharomyces paradoxus. Two other repeated sequences are found in both S. cerevisiae and S. paradoxus. None of those described here is found (by low stringency DNA hybridization) in the next closest species, Saccharomyces bayanus. The loosely characterized X element is now more precisely defined. X is a composite of at least four small (ca. 45-140 bp) sequences found at some, but not all, ends. There is also a potential ``core'''' X element of approximately 560 bp which may be found at all ends. Distal to X, only one of six clones had (TG(1-3))(n) telomere sequence at the junction between X and Y''. The presence of these internal (TG(1-3))(n) sequences correlates with the ability of a single Y'' to expand into a tandem array of Y''s by unequal sister chromatid exchange. The presence of shared repeated elements proximal to the X region can override the strong preference of Y''s to recombine ectopically with other Y''s of the same size class. The chromosome ends in yeast are evolutionarily dynamic in terms of subtelomeric repeat structure and variability.     

Dependence of the regulation of telomere length on the type of subtelomeric repeat in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, chromosomes terminate with approximately 400 bp of a simple repeat poly(TG(1-3)). Based on the arrangement of subtelomeric X and Y' repeats, two types of yeast telomeres exist, those with both X and Y' (Y' telomeres) and those with only X (X telomeres). Mutations that result in abnormally short or abnormally long poly(TG(1-3)) tracts have been previously identified. In this study, we investigated telomere length in strains with two classes of mutations, one that resulted in short poly(TG(1-3)) tracts (tel1) and one that resulted in elongated tracts (pif1, rap1-17, rif1, or rif2). In the tel1 pif1 strain, Y' telomeres had about the same length as those in tel1 strains and X telomeres had lengths intermediate between those in tel1 and pif1 strains. Strains with either the tel1 rap1-17 or tel1 rif2 genotypes had short tracts for all chromosome ends examined, demonstrating that the telomere elongation characteristic of rap1-17 and rif2 strains is Tel1p-dependent. In strains of the tel1 rif1 or tel1 rif1 rif2 genotypes, telomeres with Y' repeats had short terminal tracts, whereas most of the X telomeres had long terminal tracts. These results demonstrate that the regulation of telomere length is different for X and Y' telomeres.     

The acid phosphatase genes PHO10 and PHO11 in S. cerevisiae are located at the telomeres of chromosomes VIII and I.

Of the three regulated acid phosphatase genes in S. cerevisiae (PHO5, PHO10 and PHO11) two have previously been cloned (PHO5 and PHO11). We have now identified PHO10 and show by restriction mapping that it is highly homologous to PHO11. This homology includes not only the coding sequence but also a stretch of about 2 kb upstream and 2.2 kb downstream of the genes. Analysis of strains in which either gene had been disrupted shows that the two genes are located at the telomeres of two different chromosomes. PHO10 3.6 kb from the end of a chromosome I. This makes PHO11 the gene closest to the end of a chromosome that has been physically mapped so far in S. cerevisiae. The organization of the two genes varies strongly from strain to strain consistent with a high incidence of telomere rearrangement. In one of twenty transformants examined a conversion event could be directly demonstrated that resulted in a chromosome VIII which had acquired a copy of the telomere from chromosome I.     

Isolation and characterization of a repeated sequence (RPS1) of Candida albicans.

A repeated sequence, named RPS1, approximately 2 kb in size, is found mainly in chromosome 6, the second most variable chromosome among the eight chromosomes of Candida albicans. Most of the RPS1 units of chromosome 6 seem to be located within a single region of about 100 kb in strain FC18. In both strains FC18 and NUM812, a part of RPS1 is apparently tandemly repeated. A unit of RPS1 has been cloned and sequenced. It consists of 2114 bp and has a GC content of 40 mol%. The repeat unit contains smaller repeats of about 80-170 bp which are called REP1, REP2, REP3, REP4 and REP5; REP2 is duplicated. The small repeats are classified into two groups by their homology. One comprises REP1, REP2 and REP5, and the other REP3 and REP4. They are termed the REP1 and REP3 families, respectively. The two families both contain a common 29 bp sequence, called COM29. The dispersed repetitive sequence RPS1 may be involved in chromosomal rearrangements and may in part explain chromosome polymorphism in C. albicans. The origin of RPS1 was not determined.     

Organization and Mapping of a Sequence on the DROSOPHILA MELANOGASTER X and Y Chromosomes That Is Transcribed during Spermatogenesis

The D. melanogaster DNA segment in the recombinant phage lambda Dm2L1 contains at least eight copies of a tandemly repeated 1250-base pair (bp) sequence (henceforth called the 2L1 sequence). Testes from XO D. melanogaster males contain an abundant 800-base RNA species that is homologous to a 520-bp region of the 2L1 sequence. Blotting experiments show that the 2L1 sequence is repeated in the D. melanogaster genome and is present on both the X and Y chromosomes. With the use of X-Y translocations, the 2L1 sequence has been mapped to a region between kl-1 and kl-2 on the long arm of the Y chromosome. In Oregon-R wild type there are an estimated 200 copies of the 2L1 sequence on the X chromosome and probably at least 80 copies of the Y chromosome. In some other strains the repetition frequency on the Y chromosome is about the same, but the copy number on the X chromosome is much reduced. On the basis of the five strains investigated, there is a correlation between copy number of the 2L1 sequence on the X chromosome and the presence of a particular allele of the Stellate locus (Ste; 1-45.7). It seems that low copy number corresponds to Ste+ and high copy number corresponds to Ste. The Ste locus determines whether single or star-shaped crystals are observed in the spermatocytes of XO males. Studies using D. simulans and D. mauritiana DNA show that the 2L1 sequence is homologous to restriction fragments in male DNA but not female DNA, indicating that this sequence is present only on the Y chromosome in these two species. In DNA derived from D. erecta, D. teissieri and D. yakuba, there is very little, if any, hybridization with the 2L1 sequence probe.     

Structure and polymorphism of human telomere-associated DNA

We have analyzed the DNA sequences associated with four different human telomeres. Two are members of distinct repeated sequence families which are located mainly but not exclusively at telomeres. Two are unique in the genome, one deriving from the long arm telomere of chromosome 7 and the other from the pseudoautosomal telomere. One telomere-associated repeated sequence has a polymorphic distribution among the chromosome ends, being present at a different combination of ends in different individuals. These data thus identify a new source of human genetic variation and indicate that the canonical features of the organization of telomere-associated DNA are widely conserved in evolution.     

The Saccharomyces cerevisiae chromosome III left telomere has a type X, but not a type Y'', ARS region.

A yeast Saccharomyces cerevisiae telomeric region was isolated by chromosome walking from HML alpha, the most distal known gene on the chromosome III left (IIIL) end. The terminal heterodisperse 3.3-kilobase (kb) SalI fragment on chromosome IIIL, 8.6 kb distal to HML alpha, was cloned in a circular vector to generate a telomeric probe. Southern hybridization and DNA sequencing analyses indicated that 0.6 kb (+/- 200 base pairs) of 5'-C1-3A-3' simple tandem repeat sequence, adjacent to a 1.2-kb type X ARS region, constitutes the telomere on the chromosome IIIL end, and no type Y' ARS region homologies exist between HML alpha and the IIIL terminus.     

Structure of the major block of alphoid satellite DNA on the human Y chromosome

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.     

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location.     

Pneumocystis carinii telomere repeats are composed of TTAGGG and the subtelomeric sequence contains a gene encoding the major surface glycoprotein

We have cloned a telomere and adjacent sequences from rat-derived Pneumocystis carinii using the ability of foreign telomeres to complement a yeast artificial chromosome (YAC) deficient by one telomere in Saccharomyces cerevisiae . Characterization of the cloned DNA in the recombinant YAC demonstrated that it was a chimera of two P. carinii sequences, namely a 13.5 kb fragment of mitochondrial DNA and an 8.3 kb distal portion consisting of subtelomeric DNA. The P. carinii telomere repeat was demonstrated to be TTAGGG, the most common telomere repeat found in organisms from the animal and fungal kingdoms. Karyotype analysis confirmed that this sequence was present on all the P. carinii chromosomes. Sequence adjacent to the telomere repeats was shown by Bal 31 exonuclease digestion to be located at the chromosome ends. Analysis of the subtelomeric fragment revealed homology to the gene encoding the major surface glycoprotein of P. carinii     

Identification of autonomously replicating circular subtelomeric Y'' elements in Saccharomyces cerevisiae.

We marked a large number of yeast telomeres within their Y' regions by transforming strains with a fragment of Y' DNA into which the URA3 gene had been inserted. A few of the Ura+ transformants obtained were very unstable and were found to contain autonomously replicating URA3-marked circular Y' elements in high copy number. These marked extrachromosomal circles were capable of reintegrating into the chromosome at other telomeric locations. In contrast, most of the Ura+ transformants obtained were quite stable mitotically and were marked at bona fide chromosomal ends. These stable transformants gave rise to mitotically unstable URA3-marked circular Y' elements at a low frequency (up to 2.5%). The likelihood that such excisions and integrations represent a natural process in Saccharomyces cerevisiae is supported by our identification of putative Y' circles in untransformed strains. The transfer of Y' information among telomeres via a circular intermediate may be important for homogenizing the sequences at the ends of yeast chromosomes and for generating the frequent telomeric rearrangements that have been observed in S. cerevisiae.     

The U1 snRNA gene repeat from the sea urchin (Strongylocentrotus purpuratus): the 70 kilobase tandem repeat ends directly 3'' to a U1 gene.

Lambda phage clones containing multiple copies of the 1.1 kb tandemly repeated unit of the sea urchin (S. purpuratus) U1 RNA genes were isolated from a gene library. The 1.1 kb repeat unit encodes a single copy of the predominant U1 RNA expressed in oocytes and embryos prior to the blastula stage. The tandem repeat unit is about 80 kb in size and is probably present one time per haploid genome as judged by pulsed-field electrophoresis of sperm DNA digested with restriction enzymes which do not cut in the repeat unit. Two of the phage contained DNA flanking the repeat unit as well as several repeat units. The tandem repeat unit ends just 3' to the U1 coding region. There is only limited homology in the 5' flanking region with U1 snRNA genes from the sea urchin L. variegatus.     

Pinning down loose ends: mapping telomeres and factors affecting their length.

A degenerately repeated sequence, proximal to the telomere heptanucleotide repeat in maize, contains restriction enzyme sites that permit the separation of telomeres from the rest of the chromosomes. Probing with a telomere-specific oligonucleotide revealed genotype-dependent telomere lengths that vary more than 25-fold in maize among the 22 inbreds that have been surveyed. These lengths were found to segregate reproducibly in a recombinant inbred family where 50% of the variation can be accounted for by three loci. The dynamic control over telomere length in maize appears to act rapidly to achieve new genotypically determined telomere lengths in the F1. Clones of telomere proximal sequences were used to map restriction fragment length loci at the distal ends of eight of 20 chromosome arms.     

Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.     

Mitochondrial telomeres: surprising diversity of repeated telomeric DNA sequences among six species of Tetrahymena

The DNA sequences at the ends of the linear mtDNA of 6 species of Tetrahymena encompassing 13 strains were determined. All the strains have variable numbers of a tandemly repeated DNA sequence, 31 bp to 53 bp in size, at their mtDNA termini. Based upon the size and nucleotide sequence of the terminal repeats, the telomeres can be separated into four classes. T. pigmentosa, hyperangularis, and hegewischi have different telomeric repeats on the two ends of their mtDNAs. The only conserved feature of the mtDNA termini is the presence of tandem repeats. The function of the repeats might be to promote unequal crossing over during recombination, thereby overcoming the problem of telomere replication for these linear DNAs.     

Genomic organization of telomeric and subtelomeric sequences of Leishmania (Leishmania) amazonensis

Telomeres are DNA-protein complexes that protect linear chromosomes from degradation and fusions. Telomeric DNA is repetitive and G-rich, and protrudes towards the end of the chromosomes as 3'G-overhangs. In Leishmania spp., sequences adjacent to telomeres comprise the Leishmania conserved telomere associated sequences (LCTAS) that are around 100 bp long and contain two conserved sequence elements (CSB1 and CSB2), in addition to non-conserved sequences. The aim of this work was to study the genomic organization of Leishmania (Leishmania) amazonensis telomeric/subtelomeric sequences. Leishmania amazonensis chromosomes were separated in a single Pulsed Field Gel Electrophoresis (PFGE) gel as 25 ethidium bromide-stained bands. All of the bands hybridized with the telomeric probe (5'-TTAGGG-3')3 and with probes generated from the conserved subtelomeric elements (CSB1, CSB2). Terminal restriction fragments (TRF) of L. amazonensis chromosomes were analyzed by hybridizing restriction digested genomic DNA and chromosomal DNA separated in 2D-PFGE with the telomeric probe. The L. amazonensis TRF was estimated to be approximately 3.3 kb long and the telomeres were polymorphic and ranged in size from 0.2 to 1.0 kb. Afa I restriction sites within the conserved CSB1 elements released the telomeres from the rest of the chromosome. Bal 31-sensitive analysis confirmed the presence of terminal Afa I restriction sites and served to differentiate telomeric fragments from interstitial internal sequences. The size of the L. amazonensis 3' G-overhang was estimated by non-denaturing Southern blotting to be approximately 12 nt long. Using similar approaches, the subtelomeric domains CSB1 and CSB2 were found to be present in a low copy number compared to telomeres and were organized in blocks of 0.3-1.5 kb flanked by Hinf I and Hae III restriction sites. A model for the organization of L. amazonensis chromosomal ends is provided.