Subtelomeric Loci Research Articles

Generation of locus-specific probes for interphase fluorescence in situ hybridisation—application in Barrett's esophagus

Despite the wide range of probes commercially available for interphase fluorescence in situ hybridisation (FISH), the supply of locus-specific probes is limited to genes or chromosomal regions commonly altered in genetic diseases or during carcinogenesis. Generation of these probes is therefore desirable to accommodate individual research requirements. Hence, we detail the methodology required to design and produce custom locus-specific interphase FISH probes for any human genomic region of interest and their application was illustrated in cytogenetic investigations of Barrett's tumourigenesis. Previously utilising FISH, we observed that Barrett's tissues demonstrated chromosome 4 hyperploidy [Gut 52 (2003) 623], but as centromeric probes were used in this analysis, it was not known if the whole chromosome was amplified. We consequently generated single-copy sequence probes for the 4p16.3 and 4q35.1 subtelomeric loci. Multicolour FISH was subsequently performed on interphase preparations originating from patients with Barrett's esophagus at varying histological grades, thus demonstrating the whole region of chromosome 4 was amplified within the tissues. Additionally, probes for the DNA methyltransferase genes were produced to determine if gene dosage alterations were responsible for increasing methylation activity during Barrett's neoplastic progression. No significant alterations at the DNMT1 and DNMT3a loci were detected. An increased copy number of these genes is therefore not the basis for the hypermethylation commonly observed in this premalignant lesion.

Transcription of subtelomerically located var gene variant in Plasmodium falciparum appears to require the truncation of an adjacent var gene.

The Plasmodium falciparum R29 clone preferentially transcribes the R29var gene variant on rosette selection, unlike other isogenic clones from the same parasite lineage. Characterisation of the R29var gene locus revealed that this gene lies internal to, and is in a tail-to-tail orientation with, a second var gene variant (A4var) at one end of chromosome 13. In the R29 clone, a spontaneous deletion event between these two var variants deletes all of the A4var gene and the subtelomeric repetitive sequence arrays. We have previously shown that a simple disruption of the A4var gene is not sufficient to preferentially activate the R29var gene in rosette-selected parasites. We therefore hypothesised that the truncation of the chromosome end may be a key factor in predisposing the R29var variant to transcription under rosette selection conditions. Here, we have generated a panel of isogenic parasite clones with both intact and truncated A4var-R29var loci, and show that R29var transcription is only detected in rosette-selected clones with a truncated locus. Furthermore, we present provisional data describing the relative frequency with which this spontaneous deletion event occurs. These data have implications in our understanding of how spontaneous deletion events within subtelomeric var loci may affect transcription of these var gene variants.

The mitotic stability of the minichromosomes of Trypanosoma brucei.

The principal immune-evasion strategy of the extracellular protozoan parasite Trypanosoma brucei is one of antigenic variation (reviewed in [1–4]). In the mammalian host bloodstream, the parasite adopts a dense cell surface coat composed predominantly of a single immunogenic variable surface glycoprotein (VSG). Periodic switching of the expressed VSG gene results in successive changes in the antigenic identity of cells, prolonging parasitaemia and increasing probability of transmission. To this end, the organism maintains a population of around 100 small linear chromosomes which carry a library of divergent VSG genes at subtelomeric locations (see [5,6]). These minichromosomes (MCs) are between 30 and 150 kb in size and consist of a large palindromic core region made up of 177 bp tandem repeats (Wickstead et al., submitted for publication), subtelomeric VSG genes [7,8], canonical eukaryotic telomeres [9], and smaller portions of other repetitive DNAs (for example, 70 bp repeats [10]). They are also transcriptionally silent—minichromosomal VSG genes only become active upon duplicative transposition to active VSG expression sites (VSG-ESs) occurring on larger chromosomes. Central to the role of MCs in T. brucei is the diversity of VSG genes that they carry. A large diversity within the population of MCs can be maintained only if individual MCs are segregated with fidelity at mitosis—without mitotic fidelity, stochastic impetus will inevitably reduce the

Evidence for the importance of genetic structuring to the structural and functional specialization of the Plasmodium falciparum var gene family.

The var gene family encodes Plasmodium falciparum erythrocyte membrane 1 (PfEMP1) proteins that act as virulence factors responsible for both antigenic variation and cytoadherence of infected erythrocytes. These proteins orchestrate infected erythrocyte sequestration from blood circulation and contribute to adhesion-based complications of P. falciparum malaria infections. For this study, we analysed the genetic organization and strain structure of var genes and present evidence for three separately evolving groups that have, in part, functionally diverged and differ between subtelomeric and central chromosomal locations. Our analyses suggest that a recombination hierarchy limits reassortment between groups and may explain why some var genes are unusually conserved between parasite strains. This recombination hierarchy, coupled with binding and immune selection, shapes the variant antigen repertoire and has structural, functional and evolutionary consequences for the PfEMP1 protein family that are directly relevant to malaria pathogenesis.

Mapping the Wolf-Hirschhorn Syndrome Phenotype Outside the Currently Accepted WHS Critical Region and Defining a New Critical Region, WHSCR-2

In an attempt to define the distinctive Wolf-Hirschhorn syndrome (WHS) phenotype, and to map its specific clinical manifestations, a total of eight patients carrying a 4p16.3 microdeletion were analyzed for their clinical phenotype and their respective genotypes. The extent of each individual deletion was established by fluorescence in situ hybridization, with a cosmid contig spanning the genomic region from MSX1 (distal half of 4p16.1) to the subtelomeric locus D4S3359. The deletions were 1.9-3.5 Mb, and all were terminal. All the patients presented with a mild phenotype, in which major malformations were usually absent. It is worth noting that head circumference was normal for height in two patients (those with the smallest deletions [1.9 and 2.2 Mb]). The currently accepted WHS critical region (WHSCR) was fully preserved in the patient with the 1.9-Mb deletion, in spite of a typical WHS phenotype. The deletion in this patient spanned the chromosome region from D4S3327 (190 b4 cosmid clone included) to the telomere. From a clinical point of view, the distinctive WHS phenotype is defined by the presence of typical facial appearance, mental retardation, growth delay, congenital hypotonia, and seizures. These signs represent the minimal diagnostic criteria for WHS. This basic phenotype maps distal to the currently accepted WHSCR. Here, we propose a new critical region for WHS, and we refer to this region as "WHSCR-2." It falls within a 300-600-kb interval in 4p16.3, between the loci D4S3327 and D4S98-D4S168. Among the candidate genes already described for WHS, LETM1 (leucine zipper/EF-hand-containing transmembrane) is likely to be pathogenetically involved in seizures. On the basis of genotype-phenotype correlation analysis, dividing the WHS phenotype into two distinct clinical entities, a "classical" and a "mild" form, is recommended for the purpose of proper genetic counseling.

Virulence-related surface glycoproteins in the yeast pathogen Candida glabrata are encoded in subtelomeric clusters and subject to RAP1- and SIR-dependent transcriptional silencing.

Candida glabrata is an important opportunistic pathogen causing both mucosal and bloodstream infections. C. glabrata is able to adhere avidly to mammalian cells, an interaction that depends on the Epa1p lectin. EPA1 is shown here to be a member of a larger family of highly related genes encoded in subtelomeric clusters. Subtelomeric clustering of large families of surface glycoprotein-encoding genes is a hallmark of several pathogens, including Plasmodium, Trypanosoma, and Pneumocystis. In these other pathogens, a single surface glycoprotein is expressed, whereas other genes in the family are transcriptionally silent. Similarly, whereas EPA1 is expressed in vitro, EPA2-5 are transcriptionally repressed. This repression is shown to be due to regional silencing of the subtelomeric loci. In Saccharomyces cerevisiae, subtelomeric silencing is initiated by Rap1p binding to the telomeric repeats and subsequent recruitment of the Sir complex by protein-protein interaction. We demonstrate here that silencing of the subtelomeric EPA loci also depends on functional Sir3p and Rap1p. This identification and analysis of the EPA gene family provides a compelling example in an ascomycete of chromatin-based silencing of natural subtelomeric genes and provides for the first time in a pathogen, molecular insight into the transcriptional silencing of large subtelomeric gene families.

Ex vivo and in vitro identification of a consensus promoter for VSG genes expressed by metacyclic-stage trypanosomes in the tsetse fly.

The trypanosome variant surface glycoprotein (VSG) is first expressed during differentiation to the infective, metacyclic population in tsetse fly salivary glands. Unlike the VSG genes expressed by bloodstream form trypanosomes, metacyclic VSGs (MVSGs) have their own promoters. The scarcity of metacyclic cells has meant that only indirect approaches have been used to study these promoters, and not even their identities have been agreed on. Here, we isolated trypanosomes by dissection from salivary glands and used an approach involving 5' rapid amplification of cDNA ends to identify the transcription start site of three MVSGs. This shows that the authentic start site is that proposed for the MVAT series of MVSGs (K. S. Kim and J. E. Donelson, J. Biol. Chem. 272:24637-24645, 1997). In the more readily accessible procyclic trypanosome stage, where MVSGs are normally silent, we used reporter gene assays and linker scanning analysis to confirm that the 67 bp upstream of the start site is a promoter. This is confirmed further by accurate initiation in a homologous in vitro transcription system. We show also that MVSG promoters become derepressed when tested outwith their endogenous, subtelomeric loci. The MVSG promoters are only loosely conserved with bloodstream VSG promoters, and our detailed analysis of the 1.63 MVSG promoter reveals that its activity depends on the start site itself and sequences 26 to 49 bp and 56 to 60 bp upstream. These are longer than those necessary for the bloodstream promoter.

Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity.

We investigated the influence of telomere proximity and composition on the expression of an EGFP reporter gene in human cells. In transient transfection assays, telomeric DNA does not repress EGFP but rather slightly increases its expression. In contrast, in stable cell lines, the same reporter construct is repressed when inserted at a subtelomeric location. The telomeric repression is transiently alleviated by increasing the dosage of the TTAGGG repeat factor 1 (TRF1). Upon a prolongated treatment with trichostatin A, the derepression of the subtelomeric reporter gene correlates with the delocalization of HP1alpha and HP1beta. In contrast, treating the cells with 5 azacytidin, a demethylating agent, or with sirtinol, an inhibitor of the Sir2 family of deacetylase, has no apparent effect on telomeric repression. Overall, position effects at human chromosome ends are dependent on a specific higher-order organization of the telomeric chromatin. The possible involvement of HP1 isoforms is discussed.

Karyotype and C‐Banding Patterns of Mitotic Chromosomes in Diploid Bromegrass (Bromus riparius Rehm)

Previous cytogenetic studies of the genus Bromus L. were limited to chromosome counts and construction of karyotypes on the basis of Feulgen staining. Since the chromosomes of Bromus are similar in morphology, these karyotypes are of limited use for chromosome identification and genome analysis. The objectives of this study were to develop and evaluate a Giemsa C‐banding procedure to use in identification of individual bromegrass chromosomes and to develop a karyotype for diploid Bromus riparius Rehm. (2n = 14; PI 440215). All chromosomes had one or more C‐bands which were located mainly at telomeric regions. A group (I) of four pairs of chromosomes had telomeric bands on only one arm and could be differentiated. In this group, one pair had an interstitial C‐band along with a telomeric band, one pair had a nucleolus organizer region (NOR) at a subtelomeric location on the short arm, and the other two pair could be distinguished by centromere location. The other group (II) of three pairs of chromosomes had telomeric bands on both arms. The unequivocal identification of specific chromosomes of Group II was not possible in all cells because of their similarity and differential condensation of chromosomes. Chromosomes of both groups were either metacentric or submetacentric. The total length of individual chromosomes ranged from 5.58 to 6.87 μm and the arm ratios ranged from 1.02 to 1.5. The homologous chromosomes were paired and assigned numbers I to VII in decreasing length. A karyotype was constructed by means of the C‐bands, mean chromosome lengths, and arm ratios. The C‐banding procedure used in this study could be used to developed karyotypes for the other species of the genus Bromus and these C‐banded karyotypes could be used to compare genomes within the genus.

Transition between two forms of heterochromatin at plant subtelomeres.

The manner of packing of the terminal DNA loci into nucleosomes and higher order structures may strongly influence their functional interactions. Besides the structural flexibility of telomeric DNA sequences, conserved features of their chromatin including short nucleosome phasing (157 bp) and nucleosome sliding have been described previously. To gain a complementary knowledge of subtelomeres, we have analysed the chromatin structure of two subtelomeric tandem repeats from the plant Silene latifolia: X43.1 and 15Ssp. X43.1 shows two distinct nucleosome periodicities--157 and 188 bp. Preferred positions of its two nucleosomes have been mapped at both low and high resolution and the experimental results correspond to computer-predicted positions. 15Ssp is a newly-discovered sequence showing a telomere-associated position by PCR and a subtelomeric location by pulsed-field gel electrophoresis and fluorescence in situ hybridisation. Its 159 bp sequence unit shows a tandem arrangement and the presence of micrococcal nuclease-hypersensitive sites when either naked DNA or chromatin is digested. Use of a chemical nuclease results in a regular nucleosome ladder of 157 bp periodicity. Moreover, 15Ssp mononucleosomes show instability and absence of specific positioning, features typical for telomeric chromatin.

Polymorphic open reading frames encoding secretory proteins are located less than 3 kilobases from Theileria parva telomeres

Polymorphic, multicopy gene families are frequently located in subtelomeric regions of the genomes of parasitic protozoa. Theileria parva telomere-associated (TA) DNA from two chromosomes contained long open reading frames (ORFs) 54% identical at the N-termini, whose 3′ ends were 2670 and 2680 bp from the telomeric repeats. Probes derived from these ORFs revealed related sequences close to additional telomeres. The 3′ end of an unrelated ORF was approximately 2720 bp from a third telomere. These are among the closest ORFs to telomeres in any organism. Reverse transcription PCR detected transcripts originating within the telomeric multicopy gene family. Additional ORFs, with complex sequence similarities, were located centromeric to the telomere-adjacent ORFs. Transcripts from the schizont stage of T. parva, containing domains with significant amino acid similarity to a 3529 codon ORF located 6900 bp upstream of the telomeric repeats, were mapped to a subtelomeric locus at a fourth telomere. Five telomeric ORFs contained predicted N-terminal signal peptides and one of these signal peptides was functional in a heterologous system. Hybridisation data suggested extensive strain polymorphism between ORFs. Two of the telomere-adjacent ORFs were absent from the genome of a cloned T. parva parasite which can, nonetheless, be passaged through ticks and cattle. T. parva is unusual, among organisms so far studied, in the high density of potential coding sequences located directly adjacent to telomeres and the apparent absence of extensive tracts of repeated sequences within the TA DNA.

Subsenescent Telomere Lengths in Fibroblasts Immortalized by Limiting Amounts of Telomerase

Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250-400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres.

Analysis of Sir2p domains required for rDNA and telomeric silencing in Saccharomyces cerevisiae.

Silent information regulator (Sir) 2 is a limiting component of the Sir2/3/4 complex, which represses transcription at subtelomeric and HM loci. Sir2p also acts independently of Sir3p and Sir4p to influence chromatin organization in the rDNA locus. Deleted and mutated forms of Sir2p have been tested for their ability to complement and/or to disrupt silencing. The highly conserved C-terminal domain of Sir2p (aa 199-562) is insufficient to restore repression at either telomeric or rDNA reporters in a sir2Delta background and fails to nucleate silencing when targeted to an appropriate reporter gene. However, its expression in an otherwise wild-type strain disrupts telomeric repression. Similarly, a point mutation (P394L) within this conserved core inactivates the full-length protein but renders it dominant negative for all types of silencing. Deletion of aa 1-198 from Sir2(394L) eliminates its dominant negative effect. Thus we define two distinct functional domains in Sir2p, both essential for telomeric and rDNA repression: the conserved core domain found within aa 199-562 and a second domain that encompasses aa 94-198. Immunolocalization and two-hybrid studies show that aa 94-198 are required for the binding of Sir2p to Sir4p and for the targeting of Sir2p to the nucleolus through another ligand. The globular core domain provides an essential silencing function distinct from that of targeting or Sir complex formation that may reflect its reported mono-ADP-ribosyl transferase activity.

Modulation of life-span by histone deacetylase genes in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has a limited life-span, which is measured by the number of divisions that individual cells complete. Among the many changes that occur as yeasts age are alterations in chromatin-dependent transcriptional silencing. We have genetically manipulated histone deacetylases to modify chromatin, and we have examined the effect on yeast longevity. Deletion of the histone deacetylase gene RPD3 extended life-span. Its effects on chromatin functional state were evidenced by enhanced silencing at the three known heterochromatic regions of the genome, the silent mating type (HM), subtelomeric, and rDNA loci, which occurred even in the absence of SIR3. Similarly, the effect of the rpd3Delta on life-span did not depend on an intact Sir silencing complex. In fact, deletion of SIR3 itself had little effect on life-span, although it markedly accelerated the increase in cell generation time that is observed during yeast aging. Deletion of HDA1, another histone deacetylase gene, did not result in life-span extension, unless it was combined with deletion of SIR3. The hda1Delta sir3Delta resulted in an increase in silencing, but only at the rDNA locus. Deletion of RPD3 suppressed the loss of silencing in rDNA in a sir2 mutant; however, the silencing did not reach the level found in the rpd3Delta single mutant, and RPD3 deletion did not overcome the life-span shortening seen in the sir2 mutant. Deletion of both RPD3 and HDA1 caused a decrease in life-span, which resulted from a substantial increase in initial mortality of the population. The expression of both of these genes declines with age, providing one possible explanation for the increase in mortality during the life-span. Our results are consistent with the loss of rDNA silencing leading to aging in yeast. The functions of RPD3 and HDA1 do not overlap entirely. RPD3 exerts its effect on chromatin at additional sites in the genome, raising the possibility that events at loci other than rDNA play a role in the aging process.

The FSHD Region on Human Chromosome 4q35 Contains Potential Coding Regions among Pseudogenes and a High Density of Repeat Elements

The distal end of chromosome 4q contains the locus involved in facioscapulohumeral muscular dystrophy (FSHD1). Specific genomic deletions within a tandem DNA repeat (D4Z4) are associated with the disease status, but no causal genes have yet been discovered. In a systematic search for genes, a 161-kb stretch of genomic DNA proximal to D4Z4 was sequenced, analyzed for homologies, and subjected to gene prediction programs. A major fraction (45%) of the subtelomeric region is composed of repeat sequences attributable mainly to LINE-1 elements. Apart from the previously identified FRG1 and TUB4q sequences, several additional potential coding regions were identified by analyzing the sequence with exon prediction programs. So far, we have been unable to demonstrate transcripts by RT-PCR or cDNA library hybridization. However, several retrotransposed pseudogenes were identified. The high density of pseudogenes and repeat elements is consistent with the subtelomeric location of this region and explains why previous transcript identification studies have been problematic.

Neuroblastoma in Two Siblings Supports the Role of 1p36 Deletion in Tumor Development

Familial neuroblastoma occurs rarely. We studied a family with three children; one of them has a disseminated (stage 4) and another has a localized (stage 2) neuroblastoma. We observed subtelomeric locus D1Z2 (1p36) deletion in both tumors by using double-color fluorescence in situ hybridization. The MYNC gene was found in single copy in both tumors. Loss of heterozygosity (LOH) and restriction fragment length polymorphism analyses were performed by using DNA from frozen tumor cells and from microdissected tumor areas excised from paraffin-embedded sections. We detected somatic LOH at locus D1S468 (1p36) in a tumor-cell population with a trisomy 1 of the stage-2 patient. Neuroblastoma cells of the stage-4 patient were diploid and showed allelic loss at the following loci: D1S172, D1S80, D1S94, D1S243, D1S468, D1S214, D1S241, and D1S164. Haplotype study showed that the siblings inherited the same paternal 1p36→pter chromosome region by homologous recombination and that, in the two tumors, arm 1p of different chromosomes of maternal origin was damaged. Our results suggest that the siblings inherited the predisposition to neuroblastoma associated with paternal 1p36 region and that tumors developed as a consequence of somatic loss of the maternal 1p36 allele.

Genomic organization, sequence interrelationship, and physical localization using in situ hybridization of two tandemly repeated DNA sequences in the genus Olea

Two tandemly repeated DNA sequences, the 81-bp family and pOS218, have been isolated from a Sau3AI Olea europaea ssp. sativa partial genomic library. Sequencing of the 81-bp element showed the monomer to be between 78 and 84 bases long and to contain 51-58% adenine and thymidine residues. Comparison between the monomers revealed heterogeneity of the sequence primary structure. The clone pOS218 is 218 bases long, and sequence comparison between the two elements revealed that an internal region of the pOS218 repeated DNA sequence had 79% homology to the 81 bp repeat sequence. A breakage-reunion mechanism, involving the CAAAA sequence, could be responsible for the derivation of pOS218 from the 81 bp family element. By using double target in situ hybridization, co-localization of the two sequences on Olea chromosomes was observed. The sequences were present at DAPI stained heterochromatic regions, as major or minor sites having a subtelomeric or interstitial location. Methylation studies using two sets of isoschizomers, Sau3AI-MboI and MspI-HpaII, demonstrated that most cytosine residues in the GATC sites and the internal cytosine in the CCGG sites of both elements were methylated in O. europaea ssp. sativa. No major difference in methylation was apparent between DNA extracted from young leaves or from callus of O. europaea ssp. sativa. Both elements are also present in Olea chrysophylla, Olea oleaster, and Olea africana, but are absent from other Oleaceae genera, including Phillyrea, Forsythia, Ligustrum, Parasyringa, and Jasminum.

A 169-base pair tandem repeat DNA marker for subtelomeric heterochromatin and chromosomal rearrangements in aphids of the Myzus persicae group.

Numerous copies of a 169-base pair DNA sequence (Myzus persicae group repeat; MpR) occur at subtelomeric locations on all chromosomes of three members of the Myzus persicae species group (Myzus persicae, M. antirrhinii, M. certus). MpR occurs in large tandem arrays at both ends of all autosomes of the standard 2n = 12 karyotype, and near one end of the X chromosome (the end opposite to the nucleolar organizer) and is estimated to make up about 5% of the genome (a total of about 200000 copies). Locations of MpR were compared in various karyotypes to determine the likely nature of the rearrangements (fusions, dissociations, translocations) that are found in this species group which, like other Hemiptera, has holocentric chromosomes that are devoid of morphological markers. Aphid clones heterozygous for autosome dissociations do not have any detectable MpR at 'new' chromosome ends, indicating that this sequence is not involved in 'capping' of chromosomes. However, a clone with a de novo autosome fusion had an interstitial block of MpR marking the point of fusion, and clones heterozygous for an autosomal 1,3 translocation had MpR from autosome 1 translocated to a new site on autosome 3. The isolation from M. antirrhinii of the telomeric repeat TTAGG, which is found in several insect groups, is also reported.

Members of the olfactory receptor gene family are contained in large blocks of DNA duplicated polymorphically near the ends of human chromosomes.

We have identified three new members of the olfactory receptor (OR) gene family within a large segment of DNA that is duplicated with high similarity near many human telomeres. This segment is present at 3q, 15q, and 19p in each of 45 unrelated humans sampled from various populations. Additional copies are present polymorphically at 11 other subtelomeric locations. The frequency with which the block is present at some locations varies among populations. While humans carry seven to 11 copies of the OR-containing block, it is located in chimpanzee and gorilla predominantly at a single site, which is not orthologous to any of the locations in the human genome. The observation that sequences flanking the OR-containing segment are duplicated on larger and different sets of chromosomes than the OR block itself demonstrates that the segment is part of a much larger, complex patchwork of subtelomeric duplications. The population analyses and structural results suggest the types of processes that have shaped these regions during evolution. From its sequence, one of the OR genes in this duplicated block appears to be potentially functional. Our findings raise the possibility that functional diversity in the OR family is generated in part through duplications and inter-chromosomal rearrangements of the DNA near human telomeres.

Expression of var genes located within polymorphic subtelomeric domains of Plasmodium falciparum chromosomes.

Plasmodium falciparum var genes encode a diverse family of proteins, located on the surfaces of infected erythrocytes, which are implicated in the pathology of human malaria through antigenic variation and adhesion of infected erythrocytes to the microvasculature. We have constructed a complete representative telomere-to-telomere yeast artificial chromosome (YAC) contig map of the P. falciparum chromosome 8 for studies on the chromosomal organization, distribution, and expression of var genes. Three var gene loci were identified on chromosome 8, two of which map close to the telomeres at either end of the chromosome. Analysis of the previously described chromosome 2 contig map and random P. falciparum telomeric YAC clones revealed that most, if not all, 14 P. falciparum chromosomes contain var genes in a subtelomeric location. Mapping the chromosomal location of var genes expressed in a long-term culture of the P. falciparum isolate Dd2 revealed that four of the five different expressed var genes identified map within subtelomeric locations. Expression of var genes from a chromosomal domain known for frequent rearrangements has important implications for the mechanism of var gene switching and the generation of novel antigenic and adhesive phenotypes.