Telomeric Sites Research Articles

Cytogenetics of donkey chromosomes: nomenclature proposal based on GTG-banded chromosomes and depiction of NORs and telomeric sites.

With the expansion of comparative genome analysis across different mammals, there is an increasing need to have well-defined banded karyotypes for the species chosen for investigation. In this context, the steadily growing gene mapping data in the donkey urgently require a framework whereby alignment/comparison of genetic information can be readily made with equids and other mammalian species. Hence a GTG-banded karyotype of the donkey (Equus asinus; EAS) is presented, along with schematic drawings and nomenclature of the banded chromosomes. In addition, the most characteristic features of individual chromosomes are described and their relative size estimated. Using the FISH approach, the location of nucleolous organizer regions (NORs) and telomeric repeat sequences (TTAGGG) were detected. Where possible, information on asine chromosomes is supplemented with known/likely equine and human homologues. The study thus primarily aims to provide an appropriate cytogenetic basis for the donkey chromosomes, so that research focused on gene mapping and comparative genomics in this species can be reported under a common format.

Cytogenetics and systematic approach on a newOryzomys species, of thenitidusgroup (Sigmodontinae, Rodentia) from Northeastern Brazil

Abstract The diploid number 2n=76 was found in five specimens (four males and one female) of a new species of Oryzomys belonging to the nitidus group (Sigmodontinae,Rodentia) from Pacoti, state of Ceara, Northeastern Brazil. Cytogenetic data included CBG and GTG-banding, Ag-NOR staining as well as fluorescence in situ hybridization (FISH) with (T2AG3)n as probe, which revealed exclusive signals in all telomeres of all chromosomes. The karyotype comprises two large pairs(1 and 3) and four small metacentric pairs (34 to 37); 31 acrocentric pairs from large to small (pairs 2, and pairs 4 to 33). The X chromosome is a large subtelocentric and the Y is a medium size chromosome with two different morphologies:acrocentric and submetacentric. Pairs 1 and 3 are the result of Robertsonian rearrangements when compared with the 2n=80 karyotypes observed in Oryzomys russatus, O. nitidus and O. emmomae. Interstitial telomeric sites (ITS) after FISH were not detected in those specific chromosomes.

Identification of wheat and tritordeum chromosomes by genomic in situ hybridization using total Hordeum chilense DNA as probe

Total genomic Hordeum chilense DNA probe was hybridized to somatic chromosome spreads of Triticum aestivum 'Chinese Spring' and to four advanced tritordeum lines, the latter being the fertile amphiploid between H. chilense and durum wheat (2n = 6x = 42, AABBH(ch)H(ch)). The probe hybridized strongly to the B-genome chromosomes and to one or two bands on the A-genome chromosomes present in both wheat and tritordeum alloploids. Bands on chromosomes 1D, 2D, and 7D from hexaploid wheat were also detected. Genomic H. chilense DNA probe identified 16 chromosome pairs of the chromosome complement of hexaploid wheat and all A- and B-genome chromosomes present in the tritordeum amphiploids. The in situ hybridization patterns observed correspond to those previously reported in wheat by both N-banding and in situ hybridization with the GAA-satellite sequence (Pedersen and Langridge 1997), allowing the identification of these chromosomes. Variation among the tritordeum amphiploids for hybridization sites on chromosomes 2A, 4A, 6A, 7A, 4B, 5B, and 7B was observed. Despite of this polymorphism, all lines shared the general banding pattern. When used as probe, total H. chilense genomic DNA labeled the H. chilense chromosomes over their lengths allowing the identification of 14 H. chilense chromosomes present in the tritordeum amphiploids. In addition, chromosome-specific telomeric, interstial, and centromeric hybridization sites were observed. These hybridization sites coincide with N-banded regions in H. chilense allowing the identification of the individual H. chilense chromosomes in one of the amphiploid. The N-banded karyotypes of H. chilense (accessions H1 and H7) are presented.

TRF1 binds a bipartite telomeric site with extreme spatial flexibility.

TRF1 is a key player in telomere length regulation. Because length control was proposed to depend on the architecture of telomeres, we studied how TRF1 binds telomeric TTAGGG repeat DNA and alters its conformation. Although the single Myb-type helix-turn-helix motif of a TRF1 monomer can interact with telomeric DNA, TRF1 predominantly binds as a homodimer. Systematic Evolution of Ligands by Exponential enrichment (SELEX) with dimeric TRF1 revealed a bipartite telomeric recognition site with extreme spatial variability. Optimal sites have two copies of a 5'-YTAGGGTTR-3' half-site positioned without constraint on distance or orientation. Analysis of binding affinities and DNase I footprinting showed that both half-sites are simultaneously contacted by the TRF1 dimer, and electron microscopy revealed looping of the intervening DNA. We propose that a flexible segment in TRF1 allows the two Myb domains of the homodimer to interact independently with variably positioned half-sites. This unusual DNA binding mode is directly relevant to the proposed architectural role of TRF1.

Physical mapping of wheat-Aegilops longissimabreakpoints in mildew-resistant recombinant lines using FISH with highly repeated and low-copy DNA probes

Fluorescence in situ hybridization (FISH) with multiple probes, consisting of highly repeated DNA sequences (pSc119.2 and pAs1) and of a low-copy, 3BS-specific RFLP sequence (PSR907), enabled determination of the physical position of the wheat-alien breakpoints (BPs) along the 3BS and 3DS arms of common wheat recombinant lines. These lines harbour 3SlS Aegilops longissima segments containing the powdery mildew resistance gene Pm13. In all 3B recombinants, the wheat-Aegilops longissima physical BPs lie within the interval separating the two most distal of the three pSc119.2 3BS sites. In all such recombinants a telomeric segment, containing the most distal of the pSc119.2 3BS sites, was in fact replaced by a homoeologous Ae. longissima segment, marked by characteristic pSc119.2 hybridization sites. Employment of the PSR907 RFLP probe as a FISH marker allowed to resolve further the critical region in the various 3B recombinant lines. Three of them, like the control common wheat, exhibited between the two most distal pSc119.2 sites a single PSR907 FISH site, which was missing in a fourth recombinant line. The amount of alien chromatin can thus be estimated to represent around 20% of the recombinant arm in the three former lines and a maximum of 27% in the latter. A similar physical length was calculated for the alien segment contained in three 3D recombinants, all characterized by the presence of the Ae. longissima pSc119.2 sites distal to the nearly telomeric pAs1 sites of normal 3DS. Comparison between the FISH-based maps and previously developed RFLP maps of the 3BS-3SlS and 3DS-3SlS arms revealed substantial differences between physical and genetic map positions of the wheat-alien BPs and of molecular markers associated with the critical chromosomal portions.Key words: wheat-alien recombinants, chromosome engineering, fluorescence in situ hybridization, highly repeated and low-copy DNA probes, physical versus genetic maps.

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

The yeast telomere length counting machinery is sensitive to sequences at the telomere-nontelomere junction.

Saccharomyces cerevisiae telomeres consist of a continuous 325 +/- 75-bp tract of the heterogeneous repeat TG1-3 which contains irregularly spaced, high-affinity sites for the protein Rap1p. Yeast cells monitor or count the number of telomeric Rap1p molecules in a negative feedback mechanism which modulates telomere length. To investigate the mechanism by which Rap1p molecules are counted, the continuous telomeric TG1-3 sequences were divided into internal TG1-3 sequences and a terminal tract separated by nontelomeric spacers of different lengths. While all of the internal sequences were counted as part of the terminal tract across a 38-bp spacer, a 138-bp disruption completely prevented the internal TG1-3 sequences from being considered part of the telomere and defined the terminal tract as a discrete entity separate from the subtelomeric sequences. We also used regularly spaced arrays of six Rap1p sites internal to the terminal TG1-3 repeats to show that each Rap1p molecule was counted as about 19 bp of TG1-3 in vivo and that cells could count Rap1p molecules with different spacings between tandem sites. As previous in vitro experiments had shown that telomeric Rap1p sites occur about once every 18 bp, all Rap1p molecules at the junction of telomeric and nontelomeric chromatin (the telomere-nontelomere junction) must participate in telomere length measurement. The conserved arrangement of these six Rap1p molecules at the telomere-nontelomere junction in independent transformants also caused the elongated TG1-3 tracts to be maintained at nearly identical lengths, showing that sequences at the telomere-nontelomere junction had an effect on length regulation. These results can be explained by a model in which telomeres beyond a threshold length form a folded structure that links the chromosome terminus to the telomere-nontelomere junction and prevents telomere elongation.

Telomeric (TTAGGG)n sequences are associated with nucleolus organizer regions (NORs) in the wood lemming.

The distribution of the (TTAGGG)n telomeric sequence was studied in chromosomes of the wood lemming, Myopus schisticolor, by fluorescence in-situ hybridization. As expected, the hybridization signals were observed at telomeres of all chromosomes. However, quite a number of interstitial telomeric sites were present in the pericentric heterochromatic regions. Consistent strong hybridization signals were also seen at one terminus of chromosomes 5, 7 and 12--15. By post-hybridization G-banding and silver-staining, the large blocks of the telomeric sequences on chromosomes 5 and 12 were localized to nucleolus organizer regions (NORs).

TRF1 binds a bipartite telomeric site with extreme spatial flexibility

TRF1 binds a bipartite telomeric site with extreme spatial flexibility

Structural and functional heterogeneity of rap1p complexes with telomeric and UASrpg-like DNA sequences

Rap1p binds to a variety of related DNA sequences. We studied complexes of Rap1p and its DNA-binding domain with two of these sequences, the UASrpg sequence (5′-ACACCCATACATTT-3′) and the Saccharomyces cerevisiae telomeric consensus (5′-ACACCCACACACCC-3′). When cloned in front of a minimal CYC1 promoter, the two sequences differed in their transcriptional potential. Whereas UASrpg or telomeric single binding sites activated transcription with approximately the same strength, adjacent UASrpg sequences showed higher synergistic activity and orientation-dependence than telomeric sequences. We also found different sequence requirements for Rap1p binding in vitro to both sequences, since a single base-pair that severely reduced binding of Rap1p to UASrpg sequences had very little effect on the telomeric sequence. The Rap1p binding domain distorted DNA molecules encompassing the UASrpg sequence or the telomeric-like sequence, as revealed by both KMnO 4 hypersensitivity and by hydroxyl radical foot-printing analysis. We propose that Rap1p is able to form structurally and functionally different complexes, depending on the type of DNA sequence the complex is assembled from. This functional and structural heterogeneity may be responsible for the multiple functions that Rap1p binding sites appear to have in vivo.

Human gamma-aminobutyric acid B receptor gene: complementary DNA cloning, expression, chromosomal location, and genomic organization

Background: The 6p21.3 region of human chromosome 6 is a genetic locus for schizophrenia, juvenile myoclonic epilepsy, and dyslexia. Methods: Due to our interest in these disorders we performed complementary DNA (cDNA) hybridization selection on genomic DNA clones spanning this region to identify potential positional-candidate genes. Results: We identified a full-length cDNA with an open reading frame of 2883 bp corresponding to a predicted protein of 961 amino acids that shares greater than 95% homology with the rat gamma-aminobutyric acid B (GABA B) receptor. Northern blot hybridization identified a 4.4-kb transcript in human brain. The human gene mapped to two sites on 6p21.3 separated by 2 Mb. Sequence analysis of both sites showed that the centromeric gene is transcribed, whereas the telomeric site is likely a pseudogene. The transcribed gene is distributed over 22 exons spanning 18 kb of genomic DNA. Conclusions: The genomic location, tissue expression, and function of the human GABA B receptor gene suggest that it is an important positional-candidate for the neurobehavioral disorders with a genetic locus on 6p21.3. In addition, delineation of the genomic organization will now permit it to be integrated as part of pharmacogenetic studies in trials of anxiolytic, narcotic, antiepileptic, and fluoxetine therapies.

Biosynthesis and function of the modified DNA base beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei.

beta-D-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. In Trypanosoma brucei, the majority of J is present in repetitive DNA but the partial replacement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG gene expression sites. To gain a better understanding of the function of J, we studied its biosynthesis in T. brucei and found that it is made in two steps. In the first step, thymine in DNA is converted into hydroxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosylated by an enzyme that shows no obvious sequence specificity. We identified analogs of thymidine that affect the J content of the T. brucei genome upon incorporation into DNA. These analogs were used to study the function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decrease in J content and caused a partial derepression of silent VSG gene expression site promoters, suggesting that J might strengthen transcriptional repression. Incorporation of hydroxymethyldeoxyuridine, resulting in a 15-fold increase in the J content, caused a reduction in the occurrence of chromosome breakage events sometimes associated with transcriptional switching between VSG gene expression sites in vitro. We speculate that these effects are mediated by the packaging of J-containing DNA into a condensed chromatin structure.

Selection for activation of a new variant surface glycoprotein gene expression site in Trypanosoma brucei can result in deletion of the old one

The African trypanosome Trypanosoma brucei expresses the active variant surface glycoprotein (VSG) gene in a telomeric VSG gene expression site. We have generated trypanosomes with a neomycin resistance gene inserted behind an active VSG gene expression site promoter, and a hygromycin resistance gene behind a silent one. By alternating drug selection, we could select for trypanosomes that had switched between the two marked VSG gene expression sites. Surprisingly, trypanosomes that had activated a new VSG gene expression site had often lost the old one. Using polymerase chain reaction (PCR), we screened large numbers of switched trypanosomes and found that sequences lost invariably included the drug marker near the promoter, as well as the telomeric VSG gene many tens of kilobases away. We postulate that stable activation of a new expression site requires silencing of the old one. If silencing does not occur at a sufficient rate by normal switch-off, stable activation of the new site can only occur if the old site is lost in random deletion events. The fact that we pick up these normally infrequent deletions, indicates that inactivation of the old VSG expression site could be rate limiting during switching in our strain of T. brucei.

In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei

In Trypanosoma brucei, the active variant surface glycoprotein genes ( vsg) are located at telomeric expression sites (ES), whose expression is highly regulated during the life cycle. In the procyclic form, all ESs are repressed. In the bloodstream form, where antigenic variation occurs, only one of approximately 20 ESs is active at a given time. We have investigated chromatin structure and DNA sequence around the ES promoter to identify cis-acting regulatory regions. A marker gene, inserted 1 kb downstream of the ES promoter, was used as a specific probe to map the position of nuclease hypersensitive sites. A prominent hypersensitive site was detected within the core promoter. This site was present in both active and inactive ES promoters, suggesting that a protein complex is bound to the promoter irrespective of its transcriptional state. However, none of the regions showed differential nuclease sensitivity between active and inactive transcriptional states. A systematic deletion analysis of the sequences surrounding the active ES promoter in situ confirmed the absence of cis-regulatory elements. We find that only 70 bp within the ES promoter are necessary to support ES regulation. Analysis of the reporter activities in an inactive bloodstream-form ES revealed the existence of an intermediate promoter activity in some clones, but we never observed full activation of more than one ES. The vsg mRNA from this intermediate ES was expressed less efficiently.

Analysis of a variant surface glycoprotein gene expression site promoter of Trypanosoma brucei by remodelling the promoter region

Trypanosoma brucei survives in the mammalian bloodstream by antigenic variation of its variant surface glycoprotein (VSG) coat. VSG genes are found in telomeric expression sites (ESs), and only one ES is fully transcribed at a time. The parasite changes its coat by either bringing another VSG gene into the active ES, or by switching on another ES and silencing the first. It has previously been shown that the promoter of an active ES can be replaced by a ribosomal promoter without affecting the function of the ES. This study has now analysed the conserved sequences flanking the ES promoter by deletion or replacement of these sequences in intact trypanosomes. The results show that the sequences 3′ of the promoter and extending down to the first protein-coding gene, ESAG 7, are not required in the bloodstream-form parasite either for high-level transcription or for switching of the ES. Transformants in which the sequences 5′ of the promoter extending up to simple-sequence 50-bp repeats had been removed were not obtained unless the 5' ES sequences were replaced with exogenous DNA, or unless the ES promoter was replaced by a ribosomal promoter, and even these transformants were rare. Transformants lacking the 5′ ES sequences displayed a less complete transcriptional repression of silent ESs. These results indicate that the area 5′ of an ES promoter is required for optimal functioning of an ES.

Ordered arrangement and rearrangement of chromosomes during spermatogenesis in two species of planarians (Plathelminthes).

The pattern of distribution of telomeric DNA (TTAGGG), 28S rDNA, and 5S rDNA has been studied using fluorescence in situ hybridization (FISH) and primed in situ labelling during spermatogenesis and sperm formation in the filiform spermatozoa of two species of planarians, Dendrocoelum lacteum and Polycelis tenuis (Turbellaria, Plathelminthes). In both species, the positions of FISH signals found with each probe sequence are constant from cell to cell in the nuclei of mature sperm. Chromosome regions containing 5S and 28S rDNA genes are gathered in distinct bundles of spiral form. In early spermatids with roundish nuclei, the sites of a given sequence on different chromosomes remain separate. Centromeres (marked by 5S rDNA) gather into a single cluster in the central region of the slightly elongated sperm nucleus. During spermatid maturation, this cluster migrates to the distal pole of the nucleus. In Polycelis, telomeric sites gather into three distinct clusters at both ends and in the middle of the moderately elongated nucleus. These clusters retain their relative positions as the spermatid matures. All the chromosome ends bearing 28S rDNA gather only into the proximal cluster. Our data suggest that structures in the nucleus selectively recognise chromosome regions containing specific DNA sequences, which helps these regions to find their regular places in the mature sperm nucleus and causes clustering of the sites of these sequences located on different chromosomes. This hypothesis is supported by observations on elongated sperm of other animals in which a correlation exists between ordered arrangement of chromosomes in the mature sperm nucleus and clustering of sites of the same sequence from different chromosomes during spermiogenesis.

Telomeres and mechanisms of Robertsonian fusion.

The Robertsonian (Rb) fusion, a chromosome rearrangement involving centric fusion of two acro-(telo)centric chromosomes to form a single metacentric, is one of the most frequent events in mammalian karyotype evolution. Since one of the functions of telomeres is to preserve chromosome integrity, a prerequisite for the formation of Rb fusions should be either telomere loss or telomere inactivation. Possible mechanisms underlying the formation of various types of Rb fusion are discussed here. For example, Rb fusion in wild mice involves complete loss of p-arm telomeres by chromosome breakage within minor satellite sequences. By contrast, interstitial telomeric sites are found in the pericentromeric regions of chromosomes originating from a number of vertebrate species, suggesting the occurrence of Rb-like fusion without loss of telomeres, a possibility consistent with some form of telomere inactivation. Finally, a recent study suggests that telomere shortening induced by the deletion of the telomerase RNA gene in the mouse germ-line leads to telomere loss and high frequencies of Rb fusion in mouse somatic cells. Thus, at least three mechanisms in mammalian cells lead to the formation of Rb fusions.

Accelerated Telomere Shortening in Fibroblasts After Extended Periods of Confluency

Telomere length in MRC-5 fibroblasts remains constant if the cells are proliferation-inhibited for up to 3 months by confluency. However, the apparent frequency of single-stranded sites in telomeres, measured as sensitivity to degradation by S1 nuclease, increases about fourfold during this extended inhibition of proliferation. After release of the cells, the frequency of telomeric single-stranded sites decreases to control values, and the telomere shortening rate increases about threefold as compared to controls proliferating without inhibition. This acceleration is transitory, the telomere shortening rate decreases to control values after about two population doublings after release. Finally, temporarily arrested fibroblast populations senesce at a lower cumulative population doubling level, but at about the same telomere length, as continuously proliferating controls. The data suggest that metabolic time-dependent single-strand degradation is a major cause of telomere shortening. They support the idea that telomere shortening plays an important role in triggering cellular senescence.

Origin of human chromosome 2 revisited

Similarities in chromosome banding patterns and hornologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chrornosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low-stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all three great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.

The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA.

TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA.