Identification of a human chromosome-specific interstitial telomere-like sequence (ITS) at 22q11.2 using double-strand PRINS

Interstitial telomeric sequences (ITSs), telomere-like repeats at intrachromosomal sites, are common in mammals and consist of tandem repeats of the canonical telomeric repeat, TTAGGG, or a repeat similar to this. We report that the ITS in human chromosome region 22q11.2 is, in the sequenced genome database, 101 tandem repeats of the sequence TTAGGGAGG. Using the primed in situ labeling (PRINS) technique and primers against the canonical telomeric repeat (TTAGGG), we illuminated telomeric sites for all chromosomes and an ITS locus at 22q11.2. Using the TTAGGGAGG sequence, we designed PRINS primers that efficiently and specifically illuminate the 22q11.2 ITS locus without illuminating telomeric and other ITS loci. The 22q11.2 locus has more repeat units than other ITSs loci enabling an unprecedented high detection frequency for this interstitial telomere locus. The 22q11.2 is associated with hot spots for disease-related chromosome breaks for multiple disorders, such as DiGeorge syndrome and chronic myeloid leukemia. We describe our findings that the ITS at 22q11.2 is in the same area of, and proximal to the common rearrangement region of multiple disorders. We suggest that the ITS might be involved in DNA repair processes in this area to protect the chromosome from more serious damage.     

The influence of interstitial telomeric sequences on chromosome instability in human cells

Although most telomere repeat sequences are found at the ends of chromosomes, some telomeric repeat sequences are also found at intrachromosomal locations in mammalian cells. Several studies have found that these interstitial telomeric repeat sequences can promote chromosome instability in rodent cells, either spontaneously or following ionizing radiation. In the present study we describe the extensive cytogenetic analysis of three different human cell lines with plasmids containing telomeric repeat sequences integrated at interstitial sites. In two of these cell lines, Q18 and P8SX, instability has been detected in the chromosome containing the integrated plasmid, involving breakage/fusion/bridge cycles or amplification of the plasmid DNA, respectively. However, the data suggest that the instability observed is characteristic of the general instability in these cell lines and that the telomeric repeat sequences themselves are not responsible. Consistent with this interpretation, the chromosome containing an integrated plasmid with 500 bp of telomeric repeat sequences is highly stable in the third cell line, SNG28, which has a relatively stable genome. The stability of the chromosome containing the integrated plasmid sequences in SNG28 makes this an excellent cell line to study the effect of ionizing radiation on the stability of interstitial telomeric sequences in human cells.     

Autosomal localization of interstitial telomeric sites (ITS) in brook trout, Salvelinus fontinalis (Pisces, Salmonidae)

Cytogenetic analysis of brook trout performed with molecular and conventional methods led to identification of interstitial telomeric sites on one or two subtelocentric chromosomes within the same pair. Morphology and specific patterns of these chromosomes using fluorochromes associated with A/T- or G/C-rich DNA proved that these chromosomes are not sex related. The chromomycin-positive region was located on the short arms of the ITS bearing chromosome pair and flanked by telomeric sequences, suggesting that this part of the chromosome had been translocated from another one. Our observations confirm that GC-rich regions are highly mobile genetic structures, and led to ITS formation on brook trout chromosomes.     

Distribution of spontaneous chromosome breaks in human chromosomes

Summary Localization of chromosome breaks in human chromosomes was analyzed in 264 peripheral lymphocyte cultures. Three hundred and sixty-nine chromosome breaks could be exactly localized to a chromosome band or region of the Paris Conference nomenclature. The distribution of breaks in the chromosome regions was found to be nonrandom. Chromosome 3 alone had 23% of the breaks and region 3p2 had 13% of the total breaks. Some other chromosome regions, such as 5p1, 9q1, 14q2, and 16q2 also displayed clustering of breaks. Sex chromosomes had less breaks than expected. Spontaneous chromosome breaks were almost exclusively located in the lightly stained G bands.Supported by grants from the Foundation for Pediatric Research and Research Foundation of Orion Corporation Ltd.     

A sequence-ready map of the human chromosome 1q telomere

A 260-kb half-YAC clone derived from human chromosome 1q was mapped at high resolution using cosmid subclone fingerprint analysis and was integrated with overlapping clones from the telomeric end of a separately derived 1q44 BAC contig to create a sequence-ready map extending to the molecular telomere of 1q. Analysis of 100 kb of sample sequences from across the 260-kb region encompassed by the half-YAC revealed the presence of EST sequence matches corresponding to 12 separate Unigene clusters and to 12 separate unclustered EST sequences. Low-copy subtelomeric repeats typical of many human telomere regions are present within the distal-most 30 kb of 1q. The previously isolated and radiation hybrid-mapped markers Bda84F03, 1QTEL019, and WI11861 localized at distances approximately 32, 88, and 99 kb, respectively, from the 1q terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 1q and will enable telomeric closure of the human chromosome 1q DNA reference sequence by connecting the molecular 1q telomere to an internal BAC contig.     

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.     

Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene

A radiation-induced hybrid cell line containing 10-20 million base pairs of DNA derived from the terminal part of human 4p16 in a background of hamster chromosomes has been used to construct a genomic library highly enriched for human sequences located close to the Huntington disease (HD) gene. Recombinant phage containing human inserts were isolated from this library and used as hybridization probes against two other radiation hybrids containing human fragments with chromosomal breaks in 4p16 and against a human-hamster somatic cell hybrid that retains only the 4p15-4pter part of chromosome 4. Of 121 human phage tested, 6 were mapped distal to the HD-linked D4S10 locus. Since the HD gene is located between D4S10 and the 4p telomere, all of these sequences are likely to be closer to HD than D4S10, and any one of them may be a distal flanking marker for the disease locus. Long-range restriction map analysis performed with a field-inversion gel system shows that the six new loci are distributed in different places within 4p16. Although it is not possible to establish an order for the six sequences with the FIGE data, the results demonstrate that the region detected by these probes must span at least 2000 kb of DNA.     

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

DNA replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict and often tissue-specific temporal program. However, the genetic program that controls activation of replication origins has still not been fully elucidated in mammalian cells. Previously, we measured replication timing at the sequence level along human chromosomes 11q and 21q. In the present study, we sought to obtain a greater understanding of the relationship between replication timing programs and human chromosomes by analysis of the timing of replication of a single human chromosome 11 that had been transferred into the Chinese hamster ovary (CHO) cell line by chromosome engineering. Timing of replication was compared for three 11q chromosomal regions in the transformed CHO cell line (CHO(h11)) and the original human fibroblast cell line, namely, the R/G-band boundary at 11q13.5/q14.1, the centromere and the distal telomere. We found that the pattern of replication timing in and around the R/G band boundary at 11q13.5/q14.1 was similar in CHO(h11) cells and fibroblasts. The 11q centromeric region, which replicates late in human fibroblasts, replicated in the second half of S phase in CHO(h11) cells. By contrast, however, the telomeric region at 11q25, which is late replicating in fibroblasts (and in several other human cell lines), replicated in the first half of S phase or in very early S phase in CHO(h11) cells. Our observations suggest that the replication timing programs of the R/G-band boundary and the centromeric region of human chromosome 11q are maintained in CHO(h11) cells, whereas that for the telomeric region is altered. The replication timing program of telomeric regions on human chromosomes might be regulated by specific mechanisms that differ from those for other chromosomal regions.     

Generation of a complete set of human telomeric band painting probes by chromosome microdissection

Chromosomal rearrangements involving telomeric bands have been frequently detected in many malignancies and congenital diseases. To develop a useful tool to study chromosomal rearrangements within the telomeric band effectively and accurately, a whole set of telomeric band painting probes (TBP) has been generated by chromosome microdissection. The intensity and specificity of these TBPs have been tested by fluorescence in situ hybridization and all TBPs showed strong and specific signals to target regions. TBPs of 6q and 17p were successfully used to detect the loss of the terminal band of 6q in a hepatocellular carcinoma cell line and a complex translocation involving the 17p terminal band in a melanoma cell line. Meanwhile, the TBP of 21q was used to detect a de novo translocation, t(12;21), and the breakpoint at 21q was located at 21q22.2. Further application of these TBPs should greatly facilitate the cytogenetic analysis of complex chromosome rearrangements involving telomeric bands.     

Functional complementation of the radiation-sensitive mutant M10 cell line by human chromosome 5

The mouse lymphoma (L5178Y) cell mutant M10 is defective in rejoining DNA double-strand breaks and is hypersensitive to ionizing radiation. The introduction of human chromosome 5 into M10 cells by microcell mediated chromosome transfer complemented the ionizing-radiation hypersensitivity defect of this cell line. The presence of chromosome 5 in the microcell hybrids was shown using PCR with chromosome-specific primers and fluorescence in situ hybridization. From this data we conclude that the gene that corrects the radiation hypersensitivity of M10 cells is located on chromosome 5 and tentatively assigned to the 5q14 to 5pter region. We designate this gene XRCC4L.     

Isolation of 1001 new markers from human chromosome 11, excluding the region of 11p13-p15.5, and their sublocalization by a new series of radiation-reduced somatic cell hybrids.

The determination of the physical map of human chromosome 11 will require more clones than are currently available. We have isolated an additional 1001 new markers in a bacteriophage vector from a somatic cell hybrid cell line that contains most of chromosome 11, except the middle of the short arm. These markers were localized to five different regions, 11p15-pter, 11p12-cen, 11q11-q14, 11q14-q23, and 11q23-qter, by a panel of previously characterized somatic cell hybrids. The region 11q11-14 harbors genes that have been shown to be important in breast cancer, B-cell lymphomas, centrocytic lymphomas, asthma, and multiple endocrine neoplasia, type 1 (MEN1). To determine the positions of the recombinant clones located there, we developed a new series of radiation-reduced somatic cell hybrids. These hybrids, together with those previously characterized, allowed us to map the 11q11-q14 markers into 11 separate segregation groups.     

Effect of telomere length on telomeric gene expression.

Telomeres gradually shorten as human somatic cells divide and a correlation has been observed between the average telomere length and cell senescence. It has been proposed that the genes responsible for cell senescence are located near the telomere and are activated when telomere length reaches a critical point. This is consistent with evidence from Saccharomyces cerevisiae, in which genes are regulated differently depending on their distance from the telomere. We investigated the possibility that differential gene expression is conferred by telomere length in human cells. A plasmid containing the neomycin phosphotransferase (neo) gene was transfected into the SV40-transformed human fibroblast cell line LM217. In one transfectant the plasmid was integrated at the telomere of chromosome 13. Subclones of this cell line that had various lengths of telomeric repeat sequences on the end of this chromosome were isolated. No effect on neo gene expression was found when the length of the telomere varied between 25 and 0.5 kb, as demonstrated by colony forming ability, growth rates and RNA blot analysis. These results therefore suggest that putative chromatin structural differences conferred by telomere length do not affect the expression of genes located near telomeres.     

Spontaneous and radiation-induced chromosomal breakage at interstitial telomeric sites

The Chinese hamster genome contains a total of 18 cytologically detectable arrays of interstitial telometic sequences. A combination of G-banding and twocolour 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.     

Regional assignment of the human thymidylate synthase (TS) gene to chromosome band 18p11.32 by nonisotopic in situ hybridization

Summary The human thymidylate synthase (TS) gene was regionally assigned to chromosome band 18p11.32 by nonisotopic in situ hybridization using biotinylated cDNA (1.1kb insert) and genomic DNA (6.8kb insert) probes of the human gene. There have been two provisional assignments for the TS gene to 18pter-q12 and 18q21-qter. The present result confirmed the first of these and further localized the TS gene to the telomeric region of the short arm of chromosome 18. The TS gene appears to be a novel telomeric anchor point for the construction of both physical and genetic linkage maps of human chromosome 18.     

The sites of radiation induced-breakage in human lymphocyte chromosomes, determined by quinacrine fluorescence

We have examined the distribution of 298 radiation-induced breakpoints in human chromosomes by quinacrine fluorescence. Very little damage was discovered which would not have been detectable with simple staining methods. The data suggest an excessive number of breaks in chromosomes 3 and a deficit in No. 16. The participation of chromosomes in rearrangements appears to be proportional to chromosome length. Within chromosome arms, breakpoints are selectively placed in telomeric regions.     

Genomic organization and chromosomal localization of the mouse protein kinase C alpha gene

In Chinese hamster extended blocks of telomeric-like repeats were previously detected by in situ hybridization at the pericentromeric region of most chromosomes and short arrays were localized at several interstitial sites. In this work, we analyzed the molecular organization of internal telomeric sequences (ITs) in the Chinese hamster genome. In genomic transfers hybridized with a telomeric probe, multiple Bal31 insensitive fragments were detected. Most of the fragments ranged in size between less than 1 kb and more than 100 kb and some were polymorphic. Fluorescence in situ hybridization experiments on DNA fibers and on elongated chromosomes showed that the pericentromeric ITs are composed of extensive and essentially continuous arrays of telomeric-like sequences. We then isolated three genomic regions which contain short ITs. These ITs are localized at interstitial sites (3q13-15, 3q21-26, 1p26) and are composed of 29-126 bp of (TTAGGG)(n) repeats. A peculiar feature of all the three ITs is the AT richness of the flanking sequences. Since AT-rich DNA is known to be unstable and characteristic of several mammalian fragile sites, we propose that the three ITs were inserted at these sites during the repair of double strand breaks.     

Misrejoining of DNA double-strand breaks in primary and transformed human and rodent cells: a comparison between the HPRT region and other genomic locations.

Many studies of radiation response and mutagenesis have been carried out with transformed human or rodent cell lines. To study whether the transfer of results between different cellular systems is justified with regard to the repair of radiation-induced DNA double-strand breaks (DSBs), two assays that measure the joining of correct DSB ends and total rejoining in specific regions of the genome were applied to primary and cancer-derived human cells and a Chinese hamster cell line. The experimental procedure involves Southern hybridization of pulsed-field gel electrophoresis blots and quantitative analysis of specific restriction fragments detected by a single-copy probe. The yield of X-ray-induced DSBs was comparable in all cell lines analyzed, amounting to about 1 x 10(-2) breaks/Mbp/Gy. For joining correct DSB ends following an 80 Gy X-ray exposure all cell lines showed similar kinetics and the same final level of correctly rejoined breaks of about 50%. Analysis of all rejoining events revealed a considerable fraction of unrejoined DSBs (15-20%) after 24 h repair incubation in the tumor cell line, 5-10% unrejoined breaks in CHO cells and complete DSB rejoining in primary human fibroblasts. To study intragenomic heterogeneity of DSB repair, we analyzed the joining of correct and incorrect break ends in regions of different gene density and activity in human cells. A comparison of the region Xq26 spanning the hypoxanthine guanine phosphoribosyl transferase locus with the region 21q21 revealed identical characteristics for the induction and repair of DSBs, suggesting that there are no large variations between Giemsa-light and Giemsa-dark chromosomal bands.     

Chromosomal distribution, localization and expression of the human endogenous retrovirus ERV9

ERV9 is a class I family of human endogenous retroviral sequences. Somatic cell hybrid genomic hybridization experiments using a mono-chromosomal panel indicate the presence of approximately 120 ERV9 loci in the human genome distributed on most chromosomes. Fluorescence in situ hybridization (FISH) using an ERV9 cDNA probe containing gag, pol and env sequences, verified this observation and a consistent signal was found at the chromosome region 11q13.3-->q13.5. By analysis of a panel of radiation hybrids, an ERV9 locus was mapped to within a 300-kbp region at the chromosome site 11q13. The marker cCLGW567 and the locus MAP3K11/D11S546 centromeric and telomeric flanked it, respectively. Northern blot analysis, using an ERV9 LTR probe, indicated that most normal tissues examined expressed low abundant ERV9 LTR driven mRNAs of various sizes. The most prominent expression was found in adrenal glands and testis. However, the level of expression varied in the same tissues among different individuals indicating that ERV9 mRNA expression probably is inducible in certain tissues or at various cell stages.     

Determination of the chromosomal site for the human radiosensitive ataxia telangiectasia gene by chromosome transfer

The chromosomal localization of the gene which complements radiation hypersensitivity of AT cells was studied by microcell-mediated chromosome transfer. A 6-thioguanine-resistant derivative of an immortalized AT cell line, AT2KYSVTG, was used as a recipient for microcell-mediated chromosome transfer from 4 strains of mouse A9 cells, 3 of which carried a human X/11 recombinant chromosome containing various regions of chromosome 11, while the other carried an intact X chromosome. HAT-resistant microcell hybrids were isolated and examined for their radiosensitivity and chromosome constitution. The microcell hybrid clones obtained from the transfer of an intact X chromosome or an X/11 chromosome bearing the pter → q13 region of chromosome 11 did not show a difference in radiosensitivity from parental AT cells, while those obtained from the transfer of X/11 chromosomes bearing either the p11 → qter or the pter → q23 region of chromosome 11 exhibited a marked radioresistance which was comparable to normal human fibroblasts. A HAT-resistant but radiosensitive variant was further obtained from the microcell fusion with an A9 cell strain carrying an X/11 chromosome bearing the 11p11 → qter region, in which a deletion at the 11q23 region was found. The results indicate that the gene which complements a radiosensitive phenotype of AT is located at the q23 region of chromosome 11.     

Regional asssignments of the loci AK3, ACONS, and ASS on human chromosome 9.

Experiments are described in which human cells carrying balanced reciprocal translocations involving four different regions of chromosome 9 were fused with a Chinese hamster cell line and the resulting hybrids used to obtain subchromosomal assignments of the loci ASS, AK3, and ACONS. ASS was localized on the distal portion of the long arm of chromosome 9, in the region 9q34 leads to 9qter, and AK3 and ACONS on the short arm, in the region 9pter leads to 9p13.