t-loops at trypanosome telomeres

Mammalian telomeres form large duplex loops (t-loops) that may sequester chromosome ends by invasion of the 3' TTAGGG overhang into the duplex TTAGGG repeat array. Here we document t-loops in Trypanosoma brucei, a kinetoplastid protozoan with abundant telomeres due to the presence of many minichromosomes. These telomeres contained 10-20 kb duplex TTAGGG repeats and a 3' TTAGGG overhang. Electron microscopy of psoralen/UV cross-linked DNA revealed t-loops in enriched telomeric restriction fragments and at the ends of isolated minichromosomes. In mammals, t-loops are large (up to 25 kb), often comprising most of the telomere. Despite similar telomere lengths, trypanosome t-loops were much smaller (approximately 1 kb), indicating that t-loop sizes are regulated. Coating of non-cross-linked minichromosomes with Escherichia coli single-strand binding protein (SSB) often revealed 3' overhangs at both telomeres and several cross-linked minichromosomes had t-loops at both ends. These results suggest that t-loops and their prerequisite 3' tails can be formed on the products of both leading and lagging strand synthesis. We conclude that t-loops are a conserved feature of eukaryotic telomeres.     

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.     

Isolation and characterization of a human telomere.

A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human. One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide. Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres. This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions. However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions. Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA. Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells.     

Rearrangements of highly polymorphic regions near telomeres of Saccharomyces cerevisiae.

We have examined the mitotic and meiotic properties of telomeric regions in various laboratory strains of yeast. Using a sequence (Y probe) derived from a cloned yeast telomere (J. Szostak and E. Blackburn, Cell 29:245-255, 1982), we found that various strains of Saccharomyces cerevisiae show extensive polymorphisms of restriction endonuclease fragment length. Some of the variation in the lengths of telomeric fragments appears to be under the control of a small number of genes. When DNA from various strains was digested with endonuclease KpnI, nearly all of the fragments homologous to the Y probe were found to be of different size. The pattern of fragments in different strains was extremely variable, with a greater degree of polymorphism than that observed for fragments containing the mobile TY1 element. Tetrad analysis of haploid meiotic segregants from diploids heterozygous for many different Y-homologous KpnI fragments revealed that most of them exhibited Mendelian (2:0) segregation. However, only a small proportion of these fragments displayed the obligate 2:2 parental segregation expected of simple allelic variants at the same chromosome end. From the segregations of these fragments, we concluded that some yeast telomeres lack a Y-homologous sequence and that the chromosome arms containing a Y-homologous sequence are different among various yeast strains. Regions near yeast telomeres frequently undergo rearrangement. Among eight tetrads from three different diploids, we have found three novel Y-homologous restriction fragments that appear to have arisen during meiosis. In all three cases, the appearance of a new fragment was accompanied by the loss of another band. In one of these cases, the rearrangement leading to a novel fragment arose in an isogenic diploid, in which both homologous chromosomes should have been identical. Among these same tetrads we also found examples of apparent mitotic gene conversions and mitotic recombination involving telemetric regions.     

Cloned embryonic DNA sequences flanking the mouse immunoglobulin C gamma 3 and C gamma 1 genes

To investigate the DNA surrounding genes for immunoglobulin heavy chain constant (CH) regions, we have isolated two clones bearing a C gamma 3 gene and two bearing a C gamma 1 gene from a library of mouse embryo DNA fragments. The C gamma 3 clones span 8.6 kilobase pairs (kb) on the 5' side of the gene and 6.7 kb on its 3' side, while the C gamma 1 clones together span 13 kb of 5' flanking sequence and 2.5 kb of 3' flanking sequence. Restriction mapping of the C gamma 3 gene indicates that intervening sequences divide the gene into segments of domain size, as in other CH genes. Hybridization of clone fragments to restriction digests of mouse DNA indicates that both the C gamma 1 and C gamma 3 genes probably occur as single copies in the genome. Moreover, the entire cloned sequences on the 5' side of both genes appear to be unique in the genome, indicating that no large common sequences flank CH genes. Restriction data suggest that the C gamma 3 gene is 37-40 kb 5' to the C gamma 1 gene.     

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.     

Changes of telomere lengths in human intracranial tumours

The termini of human chromosomes comprise stretches of G-rich repeats that are about 5–20 kilobase (kb) in length. The size of the telomeres can be determined by hybridization with probes specific for these (ttaggg)_n sequences after digestion of chromosomal DNA with appropriate restriction enzymes and electrophoretic separation of the fragments. Here, probing with the ³²P-labelled synthetic (TTAGGG)₃ oligonucleotide revealed length changes of the telomeres occurring in intracranial tumours. Among 60 samples analysed, 41.7% showed telomere elongation, and 21.7% telomere reduction, whereas 36.7% of the tumours exhibited equal lengths compared with the patients' peripheral blood leukocytes. Most of the elongated glioma telomeres exceeded in length those of untransformed astrocytes derived from human fetal tissue.     

Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580.

To determine the physical length of the chromosome of Campylobacter jejuni, the genome was subjected to digestion by a series of restriction endonucleases to produce a small number of large restriction fragments. These fragments were then separated by pulsed-field gel electrophoresis with the contour-clamped homogeneous electric field system. The DNA of C. jejuni, with its low G+C content, was found to have no restriction sites for enzymes NotI and SfiI, which cut a high-G+C regions. Most of the restriction enzymes that were used resulted in DNA fragments that were either too numerous or too small for genome size determination, with the exception of the enzymes SalI (5' ... G decreases TCGAG ... 3'), SmaI (5' .... CCC decreases GGG .... 3'), and KpnI (5' ... GGTAC decreases C .... 3'). With SalI, six restriction fragments with average values of 48.5, 80, 110, 220, 280, and 980 kilobases (kb) were obtained when calibrated with both a lambda DNA ladder and yeast Saccharomyces cerevisiae chromosome markers. The sum of these fragments yielded an average genome size of 1.718 megabases (Mb). With SmaI, nine restriction fragments with average values ranging from 39 to 371 kb, which yielded an average genome size of 1.726 Mb were obtained. With KpnI, 11 restriction fragments with sizes ranging from 35 to 387.5 kb, which yielded an average genome size of 1.717 Mb were obtained. A SalI restriction map was derived by partial digestion of the C. jejuni DNA. The genome sizes of C. laridis, C. coli, and C. fetus were also determined with the contour-clamped homogeneous electric field system by SalI, SmaI, and KpnI digestion. Average genome sizes were found to be 1.714 Mb for C. coli, 1.267 Mb for C. fetus subsp. fetus, and 1.451 Mb for C. laridis.     

Preliminary study on the molecular structure of 3'''' region of Duchenne muscular dystrophy (DMD) gene in Chinese

Number and order of HindⅢ exon-containing fragments (Hd) at 3' region of DMD gene were studied systematically using 16 partly-overlapping cDNA subprobes which were produced from dystrophin cDNA 9- 14 with each of 9 restriction endonudeases. There are 25 Hd fragments corresponding to cDNA 9 -14 in DMD gene. Since then, the exact length and the new order of Hd fragments are established. A new 2.1 kb fragment (Hd 55) is revealed, a 5.2 kb fragment (formely designated as Hd 59) is excluded and the existence of a controversial 3.2 kb fragment (Hd 64) is confirmed. Besides, three new exons were revealed by comparing the PvuⅡ and the XbaⅠ hybridization patterns with the Hindlll hybridization patterns for these cDNA subprobes. It is concluded that there are at least 66 Hd fragments, or 79 exons in DMD gene basing on the discovery of three additional exons. The corresponding relationship between the 66 Hd fragments and the SfiⅠ large scale physical map has been studied, and at least 17 Hd fragments or 19 exo     

DNA length polymorphism located 5'' to the human myelin basic protein gene.

A region of DNA 5' to the human myelin basic protein (MBP) gene, located on the long arm of chromosome 18, is a site of restriction-fragment-length polymorphism (RFLP) showing 37% heterozygosity in 40 subjects studied. Southern transfer analysis using a 0.9-kb genomic fragment encompassing the first exon of the human MBP gene reveals this polymorphism with at least nine restriction enzymes, indicating that insertion, deletion, or both is the basis for the DNA length variation. Double restriction-enzyme digest analysis suggests that this polymorphism is within the region 0.5-2.0 kb upstream of the coding region of the first exon of the human MBP gene. Eleven different allelic RFLPs were identified, differing in size by as many as 450 bp. The distribution of insertion/deletion-size variants from this region is bimodal, with most restriction fragments varying in size over a 0.1-kb range. Pedigree analysis of polymorphism at this site in one three-generation family shows Mendelian assortment of parental haplotypes. The form and frequency of polymorphism generated by this site is similar to that reported for human DNA regions comprised of homologous short tandem repeats.     

Unusual chromatin in human telomeres.

We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere.     

Tetrahymena micronuclear sequences that function as telomeres in yeast.

We explored the ability of S. cerevisiae to utilize heterologous DNA sequences as telomeres by cloning germline (micronuclear) DNA from Tetrahymena thermophila on a linear yeast plasmid that selects for telomere function. The only Tetrahymena sequences that functioned in this assay were (C4A2)n repeats. Moreover, these repeats did not have to be derived from Tetrahymena telomeres, although we show that micronuclear telomeres (like macronuclear telomeres) of Tetrahymena terminate in (C4A2)n repeats. Chromosome-internal restriction fragments carrying (C4A2)n repeats also stabilized linear plasmids and were elongated by yeast telomeric repeats. In one case, the C4A2 repeat tract was approximately 1.5 kb from the end of the genomic Tetrahymena DNA fragment that was cloned, but this 1.5 kb of DNA was missing from the linear plasmid. Thus, yeast can utilize internally located tracts of telomere-like sequences, after the distal DNA is removed. The data provide an example of broken chromo-some healing, and underscore the importance of the telomeric repeat structure for recognition of functional telomeric DNA in vivo.     

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.     

Telomere looping in P. sativum (common garden pea)

Telomeres vary greatly in size among plants and, in most higher plants, consist of a long array of 5'-TTTAGGG-3'/3'-AAATCCC-5' (TTTAGGG) repeats. Recently, telomeric DNA in human, mouse, oxytricha, and trypanosome chromosomes have been found arranged into loops (t-loops), proposed to sequester the telomere from unwanted repair events and prevent activation of DNA damage checkpoints. We have asked whether t-loops exist in the higher order plant Pisum sativum (garden pea). DNA was isolated from the shoots and root tips of germinating seeds. Analysis of the telomeric restriction fragments showed that DNA hybridizing to a (TTTAGGG)n probe migrated as a smear centering around 25 kb, and direct sequencing verified the repeat to be (TTTAGGG)n. Total DNA in isolated nuclei was photo-cross-linked, and the telomeric restriction fragments were purified by gel filtration. Electron microscopic (EM) analysis revealed DNA molecules arranged as t-loops with a size distribution consistent with that seen by gel electrophoresis. Some molecules had loops as large as 75 kb. These results show that the arrangement of telomeric DNA into loops occurs in higher plants.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

The size heterogeneity of rat insulin-like growth factor-I mRNAs is due primarily to differences in the length of 3'-untranslated sequence

Previous studies have revealed multiple size classes of rat insulin-like growth factor-I (IGF-I) of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases (kb). Available sequence information accounts for only 2.1 kb of the 7.5-7.0 kb IGF-I mRNAs. We used oligomer directed ribonuclease H (RNase H) mapping to define the extent to which the unknown sequence in the large molecular weight mRNAs lies 5' or 3' to known sequence. Rat liver polyadenylated RNAs were incubated with oligomer probes complementary to internal rat IGF-I precursor (E domain) coding sequences. RNase H was used to hydrolyze IGF-I mRNAs at the point of annealment with the oligomers. Resultant 5' and 3'-IGF-I mRNA fragments were analyzed on Northern blots. A probe specific for type 1 (class C) 5'-sequences (the most predominant of multiple 5'-sequence types found on rat IGF-I mRNAs) identifies intact IGF-I mRNAs of 7.5-7.0, 1.9-1.5 and 1.2-0.9 kb but, after oligomer directed RNase cleavage of these mRNAs, identified only a single IGF-I mRNA 5'-fragment. Major differences in the length of sequence 5' to the IGF-I coding sequence therefore, do not account for the multiple size classes of type 1 (class C) IGF-I mRNAs. The size of the 5'-fragment suggests that the extent of sequence 5' to the IGF-I coding sequence is 0.4-0.7 kb in type 1 (class C) IGF-I mRNAs. Identification of multiple 3'-fragments of IGF-I mRNAs demonstrated heterogeneity in the 3'-ends of rat IGF-I mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes.

The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin.     

Vectorettes for long chromosome walking in genomic DNA of the human p53 gene

Chromosome walking in mammalian DNA by vectorette PCR is not always very specific, and the walks have been limited to distances <1 kb. To improve the method, we have designed new vectorettes, which unlike the currently used ones have very little repetitive sequences or homology with known DNA sequences of various origins in the data banks. We have tested these new vectorettes for chromosome walking in human p53 tumor suppressor gene, human tissue-type plasminogen activator gene, and mouse stanniocalcin gene with good success. In chromosome walking of the human p53 gene, we isolated gene-specific fragments of 2.4. kb, and by walking in a bacterial artificial chromosome (BAC) clone carrying the mouse stanniocalcin gene, we isolated fragments up to about 7 kb in size. We further sequenced the 5' region of the p53 gene and found that the nucleotides upstream of -1009 are transcribed in antisense orientation into a messenger RNA (mRNA) (flj10385) encoding a putative serine/threonine kinase.     

Intracisternal A-particle genes in Mus musculus: a conserved family of retrovirus-like elements.

The structural organization of intracisternal A-particle genes has been studied, using isolates from a mouse gene library in lambda phage Charon 4A. The predominant gene form among the isolates was 7.3 kilobases (kb) in length. R-loops between the 7-kb (35S) A-particle genomic ribonucleic acid and several of these genes were colinear, with no visible evidence of intervening deoxyribonucleic acid sequences. One recombinant was found with an A-particle gene that contained a 1.7-kb deletion. Using the deletion as a reference, the deoxyribonucleic acid and ribonucleic acid homology regions were localized with respect to one another and to the restriction map: the 5' terminus of the ribonucleic acid was several hundred base pairs within the 5' end of the deoxyribonucleic acid homology region. Restriction endonuclease fragments encompassing the 5' and 3' regions of one 7.3-kb gene were separately subcloned into pBR322. Heteroduplexes between the two subclones revealed an approximately 300-base pair segment of terminally redundant sequences. The cloned 3' fragment hybridized with restriction fragments from the 5' end of several other A-particle genes, demonstrating the presence of common (though not necessarily identical) terminally repeated sequences. A-particle genes varied in the occurrence of specific restriction sites at characteristic internal loci. However, heteroduplexes between several variant 7.3-kb genes showed continuous homology regions even when spread under stringent hybridization conditions. The relative abundance of restriction site variants was highly conserved in 12 laboratory strains of Mus musculus, in embryonic and adult tissues of a single inbred strain, and in the SC-1 cell line of feral mouse origin, but appeared to differ in a feral Japanese substrain, Mus musculus molossinus. Some evidence suggests that subsets of A-particle genes may have similar flanking sequences. The results are discussed in terms of the evolution of this multigene family.