Evolution of the dispersed SUC gene family of Saccharomyces by rearrangements of chromosome telomeres.

The SUC gene family of Saccharomyces contains six structural genes for invertase (SUC1 through SUC5 and SUC7) which are located on different chromosomes. Most yeast strains do not carry all six SUC genes and instead carry natural negative (suc0) alleles at some or all SUC loci. We determined the physical structures of SUC and suc0 loci. Except for SUC2, which is an unusual member of the family, all of the SUC genes are located very close to telomeres and are flanked by homologous sequences. On the centromere-proximal side of the gene, the conserved region contains X sequences, which are sequences found adjacent to telomeres (C. S. M. Chan and B.-K. Tye, Cell 33:563-573, 1983). On the other side of the gene, the homology includes about 4 kilobases of flanking sequence and then extends into a Y' element, which is an element often found distal to the X sequence at telomeres (Chan and Tye, Cell 33:563-573, 1983). Thus, these SUC genes and flanking sequences are embedded in telomere-adjacent sequences. Chromosomes carrying suc0 alleles (except suc20) lack SUC structural genes and portions of the conserved flanking sequences. The results indicate that the dispersal of SUC genes to different chromosomes occurred by rearrangements of chromosome telomeres.     

Comparison of two yeast invertase genes: conservation of the upstream regulatory region.

The yeast genome contains a dispersed family of invertase structural genes (SUC1-SUC5, SUC7). Five of these genes are located very close to telomeres and are flanked by large regions of homologous sequence; recombination between telomeres could account for the dispersal of these SUC genes to different chromosomes. The SUC2 locus, in contrast, is not near a telomere and does not share large regions of flanking homology with the other loci. We examine here the relationship between SUC2 and one of the telomeric genes, SUC7. Sequence comparison revealed homology extending from about position -624 to +1791, which is close to the end of the mRNA. The 5' noncoding sequence includes two highly conserved regions: the region between -140 and +1, which contains the TATA box and presumably other promoter elements, and a second region extending from -508 to -400, which corresponds to the upstream regulatory region.     

Organization of subtelomeric repeats in Plasmodium berghei.

Several (but not all) Plasmodium berghei chromosomes bear in the subtelomeric position a cluster of 2.3-kilobase (kb) tandem repeats. The 2.3-kb unit contains 160 base pairs of telomeric sequence. The resulting subtelomeric structure is one in which stretches of telomeric sequences are periodically spaced by a 2.1-kb reiterated sequence. This periodic organization of internal telomeric sequences might be related to chromosome-size polymorphisms involving the loss or addition of subtelomeric 2.3-kb units.     

Cohabitation of insulators and silencing elements in yeast subtelomeric regions

In budding yeast, the telomeric DNA is flanked by a combination of two subtelomeric repetitive sequences, the X and Y' elements. We have investigated the influence of these sequences on telomeric silencing. The telomere-proximal portion of either X or Y' dampened silencing when located between the telomere and the reporter gene. These elements were named STARs, for subtelomeric anti-silencing regions. STARs can also counteract silencer-driven repression at the mating-type HML locus. When two STARs bracket a reporter gene, its expression is no longer influenced by surrounding silencing elements, although these are still active on a second reporter gene. In addition, an intervening STAR uncouples the silencing of neighboring genes. STARs thus display the hallmarks of insulators. Protection from silencing is recapitulated by multimerized oligonucleotides representing Tbf1p- and Reb1p-binding sites, as found in STARs. In contrast, sequences located more centromere proximal in X and Y' elements reinforce silencing. They can promote silencing downstream of an insulated expressed domain. Overall, our results suggest that the silencing emanating from telomeres can be propagated in a discontinuous manner via a series of subtelomeric relay elements.     

应用PCR进行水稻染色体末端区域作图

利用RAPD引物介寻的不对称复性温度PCR的方法（RM－PCR）,发展了一种基于端粒重复序列的新型分子标记。并在一个籼粳杂交来源（窄叶青8号×京系17）的水稻双单倍体群体中进行了端粒重复相关顺序的遗传定位。在23个定位位点中,有12个定位在水稻8个染色体臂的最远端,并将所在染色体分别向外延长了7．7～22．6cM。其中有些可能是定位在亚端粒区。有5个位点被定位在着丝粒区,另外6个位点定位于染色体内其他区域。     

Chromosomal polymorphism and adaptation to specific industrial environments of Saccharomyces strains

Several industrial Saccharomyces strains, including bakers', wine, brewers' and distillers' yeasts, have been characterized with regards to their DNA content, chromosomal polymorphism and homologies with the DNA of laboratory strains. Measurement of the DNA contents of cells suggested that most of the industrial yeasts were aneuploids. Polymorphisms in the electrophoretic chromosomal pattern were so large that each strain could be individually identified. However, no specific changes relating to a particular group were observed. Hybridization using different probes from laboratory strains was very strong in all cases, indicating that all industrial strains possess a high degree of DNA homology with laboratory yeasts. Probes URA3, CUP1, LEU2, TRP1, GAL4 or ADC1 demonstrated the presence of one or two bands, two especially in bakers' strains. Also, results indicate that all hybridized genes are located on the same chromosomes both in laboratory and industrial strains. Translocation from chromosome VIII to XVI seems to have occurred in a distillers' strain, judging by the location of the CUP1 probe. Finally, when the SUC2 probe is used, results indicate a very widespread presence of the SUC genes in only bakers' and molasses alcohol distillers' strains. This clearly suggests that amplification of SUC genes is an adaptive mechanism conferring better fitness upon the strains in their specific industrial conditions. The widespread presence of Ty1 and Ty2 elements as well as Y′ subtelomeric sequences could account for the inter- and intrachromosomal changes detected. Received: 21 July 1997 / Accepted: 25 August 1997     

Comparative genetics of yeasts: A novel β-fructosidase gene <Emphasis Type="Italic">SUC8</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Molecular and genetic analyses revealed that the distillers race XII, which is an ancestor of Saccharomyces cerevisiae Peterhof and Gatchina genetic lines, has three polymeric β-fructosidase genes: SUC2, SUC5, and SUC8. The latter gene located on the X chromosome was identitied in this work for the first time. The presence of the single SUC2 gene in yeasts used in the international project on sequencing of the S. cerevisiae genome is discussed.     

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.     

Characterisation of the subtelomeric regions of Giardia lamblia genome isolate WBC6

Giardia trophozoites are polyploid and have five chromosomes. The chromosome homologues demonstrate considerable size heterogeneity due to variation in the subtelomeric regions. We used clones from the genome project with telomeric sequence at one end to identify six subtelomeric regions in addition to previously identified subtelomeric regions, to study the telomeric arrangement of the chromosomes. The subtelomeric regions included two retroposons, one retroposon pseudogene, and two vsp genes, in addition to the previously identified subtelomeric regions that include ribosomal DNA repeats. The presence of vsp genes in a subtelomeric region suggests that telomeric rearrangements may contribute to the generation of vsp diversity. These studies of the subtelomeric regions of Giardia may contribute to our understanding of the factors that maintain stability, while allowing diversity in chromosome structure.     

Telomeric co-localization of the modified base J and contingency genes in the protozoan parasite Trypanosoma cruzi

Base J or beta-d-glucosylhydroxymethyluracil is a modification of thymine residues within the genome of kinetoplastid parasites. In organisms known to contain the modified base, J is located mainly within the telomeric repeats. However, in Trypanosoma brucei, a small fraction of J is also located within the silent subtelomeric variant surface glycoprotein (VSG) gene expression sites, but not in the active expression site, suggesting a role for J in regulating telomeric genes involved in pathogenesis. With the identification of surface glycoprotein genes adjacent to telomeres in the South American Trypanosome, Trypanosoma cruzi, we became interested in the telomeric distribution of base J. Analysis of J and telomeric repeat sequences by J immunoblots and Southern blots following DNA digestion, reveals approximately 25% of J outside the telomeric repeat sequences. Moreover, the analysis of DNA sequences immunoprecipitated with J antiserum, localized J within subtelomeric regions rich in life-stage-specific surface glycoprotein genes involved in pathogenesis. Interestingly, the pattern of J within these regions is developmentally regulated. These studies provide a framework to characterize the role of base J in the regulation of telomeric gene expression/diversity in T. cruzi.     

Organization of the SUC gene family in Saccharomyces.

The SUC gene family of yeast (Saccharomyces) includes six structural genes for invertase (SUC1 through SUC5 and SUC7) found at unlinked chromosomal loci. A given yeast strain does not usually carry SUC+ alleles at all six loci; the natural negative alleles are called suc0 alleles. Cloned SUC2 DNA probes were used to investigate the physical structure of the SUC gene family in laboratory strains, commercial wine strains, and different Saccharomyces species. The active SUC+ genes are homologous. The suc0 allele at the SUC2 locus (suc2(0) in some strains is a silent gene or pseudogene. Other SUC loci carrying suc0 alleles appear to lack SUC DNA sequences. These findings imply that SUC genes have transposed to different chromosomal locations in closely related Saccharomyces strains.     

The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var gene silencing

Plasmodium falciparum origin recognition complex 1 (ORC1) protein has been implicated in DNA replication and silencing var gene family. However, the mechanism and the domain structure of ORC1 related to the regulation of var gene family are unknown. Here we show that the unique N-terminus of PfORC1 (PfORC1N(1-238)) is targeted to the nuclear periphery in vivo and this region binds to the telomeric DNA in vitro due to the presence of a leucine heptad repeats. Like PfORC1N(1-238), endogenous full length ORC1, was found to be associated with sub telomeric repeat regions and promoters of various var genes. Additionally, binding and propagation of ORC1 to telomeric and subtelomeric regions was severely compromised in PfSir2 deficient parasites suggesting the dependence of endogenous ORC1 on Sir2 for var gene regulation. This feature is not previously described for Plasmodium ORC1 and contrary to yeast Saccharomyces cerevisiae where ORC function as a landing pad for Sir proteins. Interestingly, the overexpression of ORC1N(1-238) compromises the binding of Sir2 at the subtelomeric loci and var gene promoters consistent with de-repression of some var genes. These results establish role of the N-terminus of PfORC1 in heterochromatin formation and regulation of var gene expression in co-ordination with Sir2 in P. falciparum.     

A complex network of interactions governs DNA methylation at telomeric regions

DNA methylation modulates telomere function. In Arabidopsis thaliana, telomeric regions have a bimodal chromatin organization with unmethylated telomeres and methylated subtelomeres. To gain insight into this organization we have generated TAIR10-Tel, a modified version of the Arabidopsis reference genome with additional sequences at most chromosome ends. TAIR10-Tel has allowed us to analyse DNA methylation at nucleotide resolution level in telomeric regions. We have analysed the wild-type strain and mutants that encode inactive versions of all currently known relevant methyltransferases involved in cytosine methylation. These analyses have revealed that subtelomeric DNA methylation extends 1 to 2 kbp from Interstitial Telomeric Sequences (ITSs) that abut or are very near to telomeres. However, DNA methylation drops at the telomeric side of the telomere-subtelomere boundaries and disappears at the inner part of telomeres. We present a comprehensive and integrative model for subtelomeric DNA methylation that should help to decipher the mechanisms that govern the epigenetic regulation of telomeres. This model involves a complex network of interactions between methyltransferases and subtelomeric DNA sequences.     

Natural variation in a subtelomeric region of Arabidopsis: implications for the genomic dynamics of a chromosome end

We investigated genome dynamics at a chromosome end in the model plant Arabidopsis thaliana through a study of natural variation in 35 wild accessions. We focused on the single-copy subtelomeric region of chromosome 1 north (approximately 3.5 kb), which represents the relatively simple organization of subtelomeric regions in this species. PCR fragment-length variation across the subtelomeric region indicated that the 1.4-kb distal region showed elevated structural variation relative to the centromere-proximal region. Examination of nucleotide sequences from this 1.4-kb region revealed diverse DNA rearrangements, including an inversion, several deletions, and an insertion of a retrotransposon LTR. The structures at the deletion and inversion breakpoints are characteristic of simple deletion-associated nonhomologous end-joining (NHEJ) events. There was strong linkage disequilibrium between the distal subtelomeric region and the proximal telomere, which contains degenerate and variant telomeric repeats. Variation in the proximal telomere was characterized by the expansion and deletion of blocks of repeats. Our sample of accessions documented two independent chromosome-healing events associated with terminal deletions of the subtelomeric region as well as the capture of a scrambled mitochondrial DNA segment in the proximal telomeric array. This natural variation study highlights the variety of genomic events that drive the fluidity of chromosome termini.     

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.     

An interspersed repeated sequence specific for human subtelomeric regions.

A family of DNA loci (DNF28) from the pseudoautosomal region of the human sex chromosomes is characterized by a repeated element (STIR: subtelomeric interspersed repeat) which detects homologous sequences in the telomeric regions of human autosomes by in situ hybridization. Several STIR elements from both the pseudoautosomal region and terminal parts of autosomes were cloned and sequenced. A conserved 350 bp sequence and some characteristic structural differences between the autosomal and pseudoautosomal STIRs were observed. Screening of the DNA sequence databases with a consensus sequence revealed the presence of STIRs in several human loci localized in the terminal parts of different chromosomes. We mapped single copy probes flanking the cloned autosomal STIRs to the subtelomeric parts of six different chromosomes by in situ hybridization and genetic linkage analysis. The linkage data show a greatly increased recombination frequency in the subtelomeric regions of the chromosomes, especially in male meiosis. The STIR elements, specifically located in subtelomeric regions, could play a role in the peculiar recombination properties of these chromosomal regions, e.g. by promoting initiation of pairing at meiosis.     

Duplication of the bcr and gamma-glutamyl transpeptidase genes.

The Philadelphia (Ph') translocation involves rearrangement of the bcr gene located on chromosome 22. Hybridization experiments revealed the presence of multiple bcr gene-related loci within the human genome. Two of these were molecularly cloned and characterized. Both loci contain exons and introns corresponding to the 3' region of the bcr gene. Restriction enzyme and DNA sequence analysis indicate a very high degree of conservation between bcr and the two related genomic sequences. Both bcr-related loci are located on chromosome 22, one centromeric, the other telomeric, of the bcr gene. Within the two bcr related genomic sequences, fragments or the complete coding sequences of an unrelated gene were found to be present. This gene was identified; it encodes gamma-glutamyl transferase, an enzyme involved in the glutathione metabolism.     

Molecular polymorphism of β-fructosidase SUC genes in the Saccharomyces yeasts

The molecular polymorphism of SUC genes that encode β-fructosidase has been investigated in the yeast genus Saccharomyces. We have determined the nucleotide sequences of subtelomeric SUC3, SUC5, SUC7, SUC8, SUC9, and SUC10 genes of S. cerevisiae and the SUCa gene of S. arboricola. Comparisons of the nucleotide sequences of all known SUC genes revealed the predominance of C → T transitions in the third codon position, which were silent. The amino acid sequences of β-fructosidases studied have identity of 88–100%. SUCa (S. arboricola) and SUCb (S. bayanus) proteins, which had amino acid identity with other SUC proteins of less than 92%, were the most divergent. It was determined that accumulation of the polymeric SUC genes takes place in industrial populations of S. cerevisiae, while the other Saccharomyces species (S. arboricola, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, and S. paradoxus) each harbor only one SUC gene. Subtelomeric repeats of β-fructosidase SUC genes could appear in the genome of S. cerevisiae under the effect of selection in the course of their domestication.     

A comparative analysis of the composition and organization of two subtelomeric repeat families in <Emphasis Type="Italic">Aegilops speltoides</Emphasis>Tausch. and related species

The structural organization and evolution of two tandemly repeated families, Spelt1 and Spelt52, located in the subtelomeric regions of Aegilops speltoides chromosomes were studied. The Spelt1 family of sequences with a monomer length of 178 bp was characterized by cloning and sequence analysis of polymerase chain reaction (PCR) products. Members of the Spelt1 family revealed sequence similarities exceeding 95\%. This conservation has remained despite divergence of species in Aegilops section Sitopsis and after independent multiple amplification events in the genome of Ae. speltoides. Sequences representing the Spelt52 family were cloned, sequenced and compared with other sequences in databases. The Spelt52 repeat family contains monomers of two types, Spelt52.1 and Spelt52.2. The two monomers share a homologous stretch of 280 bp and have two regions without sequence similarity of 96 bp and 110 bp, respectively. PCR analysis was conducted to 15 lines in Ae. speltoidesTausch., Ae. longissimaSchw.&Mushc.,Ae. sharonensisEig.,Ae. bicornis(Forssk)Jaub.&Sp., andAe. searsii Feld.&Kis. using primers to the homologous and non- homologous regions of Spelt52 family. Intraspecies and interspecies differences in the occurrence and abundance of combinations of Spelt52.1 and Spelt52.2 monomers were detected. The use of primers to telomeric and subtelomeric repeats followed by Southern hybridization, cloning, and sequence analysis demonstrated that Spelt1 and Spelt52 are localized close to each other and to telomeric repeats. The efficiency of a PCR approach for the analysis of telomeric/subtelomeric junction regions of chromosomes is discussed.