Telomeric Sites Research Articles

Gene amplification mechanisms: the role of fragile sites.

We studied the early stages of gene amplification in a Chinese hamster cell line and identified two distinct amplification mechanisms, both relying on an unequal segregation of gene copies at mitosis. In some cases, a sequence containing the selected gene is looped out, generating an acentric circular molecule, and amplification proceeds through unequal segregation of such extrachromosomal elements in successive cell cycles. In other cases, the accumulation of intrachromosomally amplified copies is driven by cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere, which leads to bridge formation and further break in mitosis (BFB cycles). We showed that some clastogenic drugs specifically trigger the intrachromosomal amplification pathway and strictly correlated this induction of BFB cycles to the ability of these drugs to activate fragile sites. In three model systems, we also established, that the location of centromeric and telomeric fragile sites relative to the selected genes determines the size and sequence content of the early amplicons.

Frequent loss of the active site during variant surface glycoprotein expression site switching in vitro in Trypanosoma brucei.

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid being killed by their mammalian hosts. The active VSG gene is located in one of many telomeric expression sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Trypanosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thymidine kinase (TK) gene. Following integration of the TK gene downstream of the 221a VSG expression site promoter, transformant cell lines became sensitive to the nucleoside analog 1-(2-deoxy-2-fluoro-8-D-arabinofuranosyl)-5-iodouracil. These TK trypanosomes were able to revert to resistance at a rate approaching 10(-5) per cell per generation. The majority of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched variants showed that some had shut down TK expression via an in situ expression site switch. However, most variants had the complete 221 expression site deleted and another VSG expression site activated. We speculate that a new VSG expression site cannot switch on without inactivation of the old site.

The role of transferrin-receptor variation in the host range of Trypanosoma brucei.

Trypanosoma brucei is a unicellular parasite transmitted between African mammals by tsetse flies. T. brucei multiplies freely in the bloodstream of many different mammals, and survives by antigenic variation of the main component of its surface coat, variant surface glycoprotein (VSG). Trypanosomes take up transferrin through a heterodimeric transferrin receptor, the genes for which are expressed in telomeric expression sites along with the VSG gene. There are up to 20 of these expression sites per trypanosome nucleus, but usually only one is active at a time. Different expression sites encode transferrin receptors that are similar but not identical. Here we show that these small differences between transferrin receptors can have profound effects on the binding affinity for transferrins from different mammals, and on the ability of trypanosomes to grow in the sera of these mammals. Our results suggest that the ability to switch between different transferrin-receptor genes allows T. brucei to cope with the large sequence diversity in the transferrins of its hosts.

Telomeres and radiation-induced chromosome breakage.

Molecular characterization of some cytologically apparent terminal deletions in human tumours revealed that these were subtelomeric cryptic translocations undetectable by classical cytogenetic methods. To determine whether subtelomeric cryptic translocations occur following exposure of mammalian cells to ionizing radiation we used four cell lines exhibiting variable telomere lengths. Our G1 and G2 radiation experiments revealed that a small percentage of broken chromosomes exhibited telomeric signals. This occurred exclusively in cell lines exhibiting FISH-detectable telomeres, suggesting that telomeric signals at radiation-induced chromosome breaks were the result of subtelomeric cryptic translocations. In addition, telomeric signals at G2 chromatid breaks were usually paired with telomeres of intact sister chromatids, further supporting the notion that subtelomeric cryptic translocations are responsible for the presence of telomeric sequences at radiation-induced chromosome breaks. In one of the cell lines we identified what looked like de novo telomeric signals at derived chromatid breaks observed 20 h following irradiation. Our previous study suggested that these signals may be the result of amplification of interstitial telomeric sites in the first cell cycle and spontaneous breakage of interstitial telomeric sites in subsequent cell cycles. Taken together our results suggest that a small percentage of radiation-induced chromosome/chromatid breaks may be modified by subtelomeric cryptic translocations and that interstitial telomeric sites may be involved in radiation-induced chromosome instability.

Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein.

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

Localization of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei.

African trypanosomes such as Trypanosoma brucei undergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing the variant surface glycoprotein (VSG) gene expressed. The transcribed VSG gene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change the VSG gene expressed is by activating a new site and inactivating the previously active one. The mechanisms that control expression site switching are unknown, but have been suggested to involve epigenetic regulation. We have found previously that VSG genes in silent (but not active) expression sites contain modified restriction endonuclease cleavage sites, and we have presented circumstantial evidence indicating that this is attributable to the presence of a novel modified base beta-D-glucosyl-hydroxymethyluracil, or J. To directly test this, we have generated antisera that specifically recognize J-containing DNA and have used these to determine the precise location of this modified thymine in the telomeric VSG expression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomeric VSG genes in silenced expression sites and not in actively transcribed telomeric VSG genes. J was absent from inactive chromosome-internal VSG genes. DNA modification was also found at the boundaries of expression sites. In the long 50-bp repeat arrays upstream of the promoter and in the telomeric repeat arrays downstream of the VSG gene, J was found both in silent and active expression sites. This suggests that silencing results in a gradient of modification spreading from repetitive DNA flanks into the neighboring expression site sequences. In this paper, we discuss the possible role of J in silencing of expression sites.

β- d-glucosyl-hydroxymethyluracil, a novel base in African trypanosomes and other Kinetoplastida

A novel base, β- d-glucosyl-hydroxymethyluracil or J for short, was recently discovered in DNA of bloodstream form Trypanosoma brucei. The base is predominantly found in the hexameric repeat arrays of chromosome telomeres and in adjacent repetitive sub-telomeric DNA, and it is made by modification of specific thymines in DNA. J is present in inactive telomeric variant surface glycoprotein (VSG) genes, but not in active ones, suggesting a link between the presence of J and repression of the telomeric expression sites for VSG genes. The presence of J in DNA is specific for bloodstream form trypanosomes, as J is absent in insect form (procyclic) T. brucei. In addition to African trypanosomes, J has been found in DNA from other Kinetoplastida that do not undergo antigenic variation, such as Leishmania and Crithidia. The biological function of J remains to be deciphered.

TRF1 is a dimer and bends telomeric DNA.

TRF1 is a mammalian telomeric protein that binds to the duplex array of TTAGGG repeats at chromosome ends. TRF1 has homology to the DNA-binding domain of the Myb family of transcription factors but, unlike most Myb-related proteins, TRF1 carries one rather than multiple Myb-type DNA-binding motifs. Here we show that TRF1 binds DNA as a dimer using a large conserved domain near the N-terminus of the protein for TRF1-TRF1 interactions. Dimerization was observed both in a complex with DNA and in the yeast two-hybrid assay. TRF1 dimers were found to require both Myb repeats for the formation of a stable complex with DNA, indicating a parallel between the DNA-binding mode of TRF1 and other Myb-related proteins. TRF1 was found to have a number of biochemical similarities to Rap1p, a distantly related DNA-binding protein that functions at telomeres in yeast. Rap1p and TRF1 both require two Myb motifs for DNA binding and both factors bind along their cognate telomeric sequences without showing strong cooperative interactions between adjacent proteins. Furthermore, TRF1 was found to bend its telomeric site to an angle of -120 degrees. Since Rap1p similarly distorts telomeric DNA, we propose that DNA bending is important for the function of telomeres in yeast and mammals.

Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences.

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid immune system-mediated killing by their mammalian host. An important mechanism for switching the expressed VSG gene is the duplicative transposition of a silent VSG gene into one of the telomeric VSG expression sites of the trypanosome, resulting in the replacement of the previously expressed VSG gene. This process appears to be a gene conversion reaction, and it has been postulated that sequences within the expression site may act to initiate and direct the reaction. All bloodstream form expression sites contain huge arrays (many kilobase pairs) of 70-bp repeat sequences that act as the 5' boundary of gene conversion reactions involving most silent VSG genes. For this reason, the 70-bp repeats seemed a likely candidate to be involved in the initiation of switching. Here, we show that deletion of the 70-bp repeats from the active expression site does not affect duplicative transposition of VSG genes from silent expression sites. We conclude that the 70-bp repeats do not appear to function as indispensable initiation sites for duplicative transposition and are unlikely to be the recognition sequence for a sequence-specific enzyme which initiates recombination-based VSG switching.

Physical mapping of human 7q and 14q subtelomeric DNA sequences in the great apes.

Phylogenetic divergence of the members of the Pongidae family has been based on genetic evidence. The terminal repeat array (T2AG3) has lately been considered as an additional basis to analyze genomes of highly related species. The recent isolation of subtelomeric DNA probes specific for human (HSA) chromosomes 7q and 14q has prompted us to cross-hybridize them to the chromosomes of the chimpanzee (PTR), gorilla (GGO) and orangutan (PPY) to search for its equivalent locations in the great ape species. Both probes hybridized to the equivalent telomeric sites of the long (q) arms of all three great ape species. Hybridization signals to the 7q subtelomeric DNA sequence probe were observed at the telomeres of HSA 7q, PTR 6q, GGO 6q and PPY 10q, while hybridization signals to the 14q subtelomeric DNA sequence probe were observed at the telomeres of HSA 14q, PTR 15q, GGO 18q and PPY 15q. No hybridization signals to the chromosome 7-specific alpha satellite DNA probe on the centromeric regions of the ape chromosomes were observed. Our observations demonstrate sequence homology of the subtelomeric repeat families D7S427 and D14S308 in the ape chromosomes. An analogous number of subtelomeric repeat units exists in these chromosomes and has been preserved through the course of differentiation of the hominoid species. Our investigation also suggests a difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes, possibly derived from interchromosomal transfers and subsequent amplification of ancestral alpha satellite sequences.

Telomere exchange can be an important mechanism of Variant Surface Glycoprotein gene switching in Trypanosoma brucei

Trypanosoma brucei undergoes antigenic variation by changing its Variant Surface Glycoprotein (VSG) coat. Although there are up to a thousand VSG genes, only one is transcribed at a time from a telomeric VSG expression site. Switching can involve DNA rearrangements exchanging the active VSG gene, or transcriptional activation of a new expression site and transcriptional silencing of the old one. Determining the mechanism mediating a switch has not always been easy, as the many virtually identical copies of VSG gene expression sites complicate transcriptional analysis. To overcome this problem, we have used bloodstream form T. brucei with a single copy VSG gene in an active expression site marked with a hygromycin resistance gene. We allowed these transformants to undergo switching of the active VSG gene, via three different experimental methods. We were able to select large numbers of switched trypanosomes from a single infected mouse using a new microtitre-dish based procedure developed for this purpose. The drug sensitivity of the switched trypanosomes allowed us to determine the transcriptional state of the marked expression site, and polymerase chain reaction (PCR) amplification was used to determine whether the single copy drug resistance gene and VSG gene present in the marked expression site had been retained. These studies showed that telomere exchange, which has been considered rare, can in some cases be an important mechanism of VSG gene switching. We describe 4 telomere exchange events between the active VSG 221 expression site and 4 different chromosomes.

Spontaneous and radiation-induced chromosomal breakage at interstitial telomeric sites

Spontaneous and radiation-induced chromosomal breakage at interstitial telomeric sites

Spontaneous and radiation-induced chromosomal breakage at interstitial telomeric sites

The Chinese hamster genome contains a total of 18 cytologically detectable arrays of interstitial telomeric sequences. A combination of G-banding and two-colour fluorescence in situ hybridization revealed that 25 out of 27 (93%) breakpoints of spontaneously occurring terminal deletions in four immortalized Chinese hamster cell lines were located in chromosomal regions containing interstitial telomeric sequences. Each of the four immortalized Chinese hamster cell lines expressed telomerase. Radiation experiments revealed the sensitivity of interstitial telomeric sequences to radiation-induced chromosomal breakage in all telomerase-positive cell lines. However, radiation-induced chromosomal breakage at interstitial telomeric sites in non-transformed, primary Chinese hamster cells was almost non-existent. Telomerase activity in primary Chinese hamster cells was not detected. These results indirectly suggest that interstitial telomeric sites represent a favourable substrate for chromosomal healing.

The Crystal Structure of the DNA-Binding Domain of Yeast RAP1 in Complex with Telomeric DNA

Telomeres, the nucleoprotein complexes at the ends of eukaryotic chromosomes, are essential for chromosome stability. In the yeast S. cerevisiae, telomeric DNA is bound in a sequence-specific manner by RAP1, a multifunctional protein also involved in transcriptional regulation. Here we report the crystal structure of the DNA-binding domain of RAP1 in complex with a telomeric DNA site at 2.25 Å resolution. The protein contains two similar domains that bind DNA in a tandem orientation, recognizing a tandemly repeated DNA sequence. The domains are structurally related to the homeodomain and the proto-oncogene Myb, but show novel features in their DNA-binding mode. A structured linker between the domains and a long C-terminal tail contribute to the binding specificity. This structure provides insight into the recognition of the conserved telomeric DNA sequences by a protein.

Molecular characterization and cytogenetic analysis of highly repeated DNAs of lake trout, Salvelinus namaycush

The chromosomes of lake trout (Salvelinus namaycush) contain a considerable amount of heterochromatin located at the centromeres and/or telomeres of several chromosomes, including a sex-specific block located distally on the X chromosome. In order to investigate further the repetitive DNAs of lake trout, genomic DNA from a female was size fractionated (<600 bp) with the restriction endonuclease AluI and fragments were cloned into the bacteriophage M13. A total of 42 clones were isolated. Relative copy number of individual inserts within the lake trout genome was estimated by Southern analysis. Twelve clones were determined to be highly repetitive and were chosen for further investigation. Inserts of these clones contained sequences similar to the AluI/RsaI, EcoRI/DraI, DraI/BstEII, and MboI/BglII families reported from Arctic char (Salvelinus alpinus). The chromosomal location of several of these fragments was determined in lake trout by fluorescence in situ hybridization (FISH). Two related AluI/RsaI sequences (Type A, approximately 140 bp, and Type B,approximately 120 bp) showed differential hybridization. Type A hybridized to the centromeres of all metacentric as well as several acrocentric chromosomes. Type B hybridized to the centromeres of most acrocentric chromosomes. A sequence with homology to the EcoRI/DraI family hybridized to the centromeres of several acrocentric chromosomes. Sequences with partial similarity to the DraI/BstEII family hybridized to the major rDNA sites (nucleolar organizer regions, NORs) and several minor telomeric sites. The interstitial and telomeric heterochromatin of lake trout, including that of the X chromosome, appears to comprise sequences belonging to the MboI/BglII family.

A ribosomal DNA promoter replacing the promoter of a telomeric VSG gene expression site can be efficiently switched on and off in T. brucei

Trypanosoma brucei survives in the mammalian blood-stream by regularly changing its variant surface glycoprotein (VSG) coat. The active VSG gene is located in a telomeric expression site, and coat switching occurs either by replacing the transcribed VSG gene or by changing the expression site that is active. To determine whether VSG expression site control requires promoter-specific sequences, we replaced the active VSG expression site promoter in bloodstream-form T. brucei with a ribosomal DNA (rDNA) promoter. These transformants were fully infective in laboratory animals, and the rDNA promoter, which is normally constitutively active, was efficiently inactivated and reactivated in the context of the VSG gene expression site. As there is no sequence similarity between the VSG expression site promoter and the rDNA promoter, VSG expression site control does not involve sequences specific to the VSG expression site promoter. We conclude that an epigenetic mechanism, such as telomeric silencing, is involved in VSG expression site control in bloodstream-form T. brucei.

Loss of telomeric sites in the chromosomes of Mus musculus domesticus (Rodentia: Muridae) during Robertsonian rearrangements.

Mouse chromosomes possessing multiple Robertsonian rearrangements (Rb chromosomes) have been examined using fluorescence in situ hybridization with the telomeric consensus sequence (TTAGGG)n. No hybridization signals were detected at the primary constriction of Rb chromosomes. This observation leads us to conclude that the formation of Rb chromosomes in the mouse is invariably associated with the loss of telomeric regions. More significantly, a further alteration in regions flanking the primary constrictions was observed after hybridizing with a minor satellite DNA probe to Rb chromosomes. It seems likely that the breakpoints required for a Robertsonian process do not include telomeric sites exclusively but extend to the adjacent pericentromeric regions of the original acrocentric chromosomes. In contrast to previous reports, these observations demonstrate the elimination of substantial amounts of chromosomal DNA during the formation of mouse Rb chromosomes.

Control of gene expression in trypanosomes.

Trypanosomes are protozoan agents of major parasitic diseases such as Chagas' disease in South America and sleeping sickness of humans and nagana disease of cattle in Africa. They are transmitted to mammalian hosts by specific insect vectors. Their life cycle consists of a succession of differentiation and growth phases requiring regulated gene expression to adapt to the changing extracellular environment. Typical of such stage-specific expression is that of the major surface antigens of Trypanosoma brucei, procyclin in the procyclic (insect) form and the variant surface glycoprotein (VSG) in the bloodstream (mammalian) form. In trypanosomes, the regulation of gene expression is effected mainly at posttranscriptional levels, since primary transcription of most of the genes occurs in long polycistronic units and is constitutive. The transcripts are processed by transsplicing and polyadenylation under the influence of intergenic polypyrimidine tracts. These events show some developmental regulation. Untranslated sequences of the mRNAs seem to play a prominent role in the stage-specific control of individual gene expression, through a modulation of mRNA abundance. The VSG and procyclin transcription units exhibit particular features that are probably related to the need for a high level of expression. The promoters and RNA polymerase driving the expression of these units resemble those of the ribosomal genes. Their mutually exclusive expression is ensured by controls operating at several levels, including RNA elongation. Antigenic variation in the bloodstream is achieved through DNA rearrangements or alternative activation of the telomeric VSG gene expression sites. Recent discoveries, such as the existence of a novel nucleotide in telomeric DNA and the generation of point mutations in VSG genes, have shed new light on the mechanisms and consequences of antigenic variation.

Control of gene expression in trypanosomes.

Trypanosomes are protozoan agents of major parasitic diseases such as Chagas' disease in South America and sleeping sickness of humans and nagana disease of cattle in Africa. They are transmitted to mammalian hosts by specific insect vectors. Their life cycle consists of a succession of differentiation and growth phases requiring regulated gene expression to adapt to the changing extracellular environment. Typical of such stage-specific expression is that of the major surface antigens of Trypanosoma brucei, procyclin in the procyclic (insect) form and the variant surface glycoprotein (VSG) in the bloodstream (mammalian) form. In trypanosomes, the regulation of gene expression is effected mainly at posttranscriptional levels, since primary transcription of most of the genes occurs in long polycistronic units and is constitutive. The transcripts are processed by transsplicing and polyadenylation under the influence of intergenic polypyrimidine tracts. These events show some developmental regulation. Untranslated sequences of the mRNAs seem to play a prominent role in the stage-specific control of individual gene expression, through a modulation of mRNA abundance. The VSG and procyclin transcription units exhibit particular features that are probably related to the need for a high level of expression. The promoters and RNA polymerase driving the expression of these units resemble those of the ribosomal genes. Their mutually exclusive expression is ensured by controls operating at several levels, including RNA elongation. Antigenic variation in the bloodstream is achieved through DNA rearrangements or alternative activation of the telomeric VSG gene expression sites. Recent discoveries, such as the existence of a novel nucleotide in telomeric DNA and the generation of point mutations in VSG genes, have shed new light on the mechanisms and consequences of antigenic variation.

Site-Specific Chromosome Damage and Myeloid Leukaemogenesis in the CBA Mouse

Abstract Deletion and/or rearrangement of chromosome 2 is a very early and possibly initiating event for acute myeloid leukaemia in the CBA mouse. Here an outline is given of a series of cytogenetic and molecular studies which imply that the expression of radiation damage to this chromosome is highly non-random, being dependent upon the presence of specific telomere-like DNA repeat sequences at certain interstitial chromosomal locations. The secondary four-stranded DNA structures that may be formed at such telomeric chromosome sites and the potential role of these recombinogenic structures as preferential targets for radiation induced cytogenetic change are discussed.