Relative DNA contents of somatic nuclei of ox,sheep and goat

Diploid ox nuclei contain about 14% more DNA than nuclei from sheep of the same sex. Goat nuclei have a similar DNA content to those of sheep. In view of the similar chromosome banding patterns in these species, it appears that chromosome evolution must have involved numerous minute interstitial deletions or additions of DNA. Although chromosomes which have similar banding patterns in these three species may be regarded as homologous in this respect, and can be regarded as having a common evolutionary origin, they are not homologous for the quantity of their DNA.     

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.     

Huntington disease-linked restriction fragment length polymorphism localized within band p16.1 of chromosome 4 by in situ hybridization.

A 5.5-kilobase (kb) single sequence DNA fragment (G8) reveals the DNA polymorphic locus D4S10 on Southern blot analysis. This locus is closely linked to Huntington disease and has been mapped to chromosome 4 short arm using human-mouse somatic cell hybrids, and specifically to chromosome 4 band p16 using DNA from individuals with deletions of chromosome 4 short arm who exhibit Wolf-Hirschhorn syndrome. With in situ hybridization techniques, we have confirmed the location of D4S10 on chromosome 4 and further localized it within band p16 utilizing five patients, four with overlapping chromosome 4 short-arm aberrations. The DNA segment G8 was hybridized to the mataphase chromosomes of the five patients. Two of them have different interstitial deletions of one of the chromosome 4 short arms (TA and BA), two have different chromosome 4 short-arm terminal deletions (RG and DQ), and one has a normal male karyotype. By noting the presence or absence of hybridization to the partially deleted chromosomes with known precise breakpoints, we were able to more accurately localize probe G8 to the distal half of band p16.1 of chromosome 4.     

Increased sensitivity of subtelomeric regions to DNA double-strand breaks in a human cancer cell line

We previously reported that a single DNA double-strand break (DSB) near a telomere in mouse embryonic stem cells can result in chromosome instability. We have observed this same type of instability as a result of spontaneous telomere loss in human tumor cell lines, suggesting that a deficiency in the repair of DSBs near telomeres has a role in chromosome instability in human cancer. We have now investigated the frequency of the chromosome instability resulting from DSBs near telomeres in the EJ-30 human bladder carcinoma cell line to determine whether subtelomeric regions are sensitive to DSBs, as previously reported in yeast. These studies involved determining the frequency of large deletions, chromosome rearrangements, and chromosome instability resulting from I-SceI endonuclease-induced DSBs at interstitial and telomeric sites. As an internal control, we also analyzed the frequency of small deletions, which have been shown to be the most common type of mutation resulting from I-SceI-induced DSBs at interstitial sites. The results demonstrate that although the frequency of small deletions is similar at interstitial and telomeric DSBs, the frequency of large deletions and chromosome rearrangements is much greater at telomeric DSBs. DSB-induced chromosome rearrangements at telomeric sites also resulted in prolonged periods of chromosome instability. Telomeric regions in mammalian cells are therefore highly sensitive to DSBs, suggesting that spontaneous or ionizing radiation-induced DSBs at these locations may be responsible for many of the chromosome rearrangements that are associated with human cancer.     

Initiation of DNA Replication from Non-Canonical Sites on an Origin-Depleted Chromosome

Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins). In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism.     

Chromosome 7 long-arm deletions in myeloid disorders: terminal DNA sequences are commonly conserved and breakpoints vary

Deletions in the long arm of chromosome 7 are common recurrent abnormalities in secondary leukemias and myelodysplastic syndromes. To learn more about the basic mechanisms involved, we used Southern blot analysis to study four patients with different 7q--deletions to determine the exact break-points and to define the extent of the deletions. Several genes and DNA sequences from 16 different loci were found to be deleted, as judged by the absence or considerable weakening of an allelic band in granulocytic DNA in patients with constitutional heterozygosity. A terminal segment was present in each of the partially deleted chromosomes, as shown by heterozygosity for probes from the region 7q35----qter in granulocyte DNA. This indicated that the chromosome 7q deletions were interstitial, rather than terminal, in each of these patients. The length of the preserved terminal segment varied among the patients. Our results support gene loss as a mechanism contributing to leukemogenesis. Since the deletions are interstitial, hybrid genes may be formed at the junction, but the variation in breakpoints argues against the existence of a common hybrid gene of importance to the malignant process.     

Effect of large targeted deletions on the mitotic stability of an extra chromosome mediating drug resistance in Leishmania

A mitotically stable linear extra chromosome obtained in a Leishmania donovani strain rendered mycophenolic acid-resistant has been physically mapped. This 290-kb chromosome has an inverted duplicated structure around a central inversion region, and is derived from a conservative amplification event of a ~140-kb subtelomeric end of chromosome 19. Large-sized targeted deletions of the central region were performed through homologous recombination using three specific transfection vectors. The size of the extra chromosome was thus successfully reduced from 290 to 260, 200 and 120 kb respectively. The mitotic stability of these chromosomes was then analysed in drug-free cultures over >140 days. Results differed according to the deletion created. By contrast with the smallest deletion the two largest deletions altered mitotic stability, leading to progressive loss of the size-reduced chromosomes with similar kinetics in both mutants. The 30-kb region common to both deletions may therefore be considered as involved in mitotic stability. A 44-kb contig covering this region could be assembled and sequenced. The analysis of this sequence did not reveal any sequence elements typical of centromeric DNA. By contrast, its enrichment in homopolymer tracts suggests that this region might contain an origin of replication.     

Viable deletions of a telomere from a Drosophila chromosome

Destabilization of a P element transposon inserted in the subtelomeric region induced a set of similar chromosomal rearrangements. These rearrangements appear to be terminal deletions with endpoints clustered at the centromere-distal end of the transposon. The terminally deleted chromosome progressively loses sequences from the broken end at a rate of approximately 50-100 bp per fly generation, suggesting that the replication of this end may be incomplete. In most cases, capping of the broken end by readdition of new sequences was not observed. Past failures to recover terminal deletions of Drosophila chromosomes following X-ray mutagenesis may have been due to a cell cycle arrest in response to unrepaired DNA damage rather than to an absolute requirement for the telomere.     

Common fragile sites as targets for chromosome rearrangements

Common fragile sites are large chromosomal regions that preferentially exhibit gaps or breaks after DNA synthesis is partially perturbed. Fragile site instability in cultured cells is well documented and includes gaps and breaks on metaphase chromosomes, translocation and deletions breakpoints, and sister chromosome exchanges. In recent years, much has been learned about the genomic structure at fragile sites and the cellular mechanisms that monitor their stability. The study of fragile sites has merged with that of cell cycle checkpoints and DNA repair, with multiple proteins from these pathways implicated in fragile site stability, including ATR, BRCA1, CHK1, and RAD51. Since their discovery, fragile sites have been implicated in constitutional and cancer chromosome rearrangements in vivo and recent studies suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early tumorigenesis. Here we review the relationship of fragile sites to chromosome rearrangements, particularly in tumor cells, and discuss the mechanisms that may be involved.     

Chromosome breakage and recombination at fragile sites.

Chromosomal fragile sites are points on chromosomes that usually appear as nonstaining chromosome or chromatid gaps. It has frequently been suggested that fragile sites may be involved in chromosome breakage and recombination events. We and others have previously shown that fragile sites predispose to intrachromosomal recombination as measured by sister-chromatid exchanges. These findings suggested that fragile site expression often, if not always, is accompanied by DNA strand breakage. In the present report, fragile sites are shown to predispose to deletions and interchromosomal recombination. By use of somatic cell hybrids containing either human chromosome 3 or the fragile X chromosome, deletions and translocations were induced by FUdR or aphidicolin with breakpoints at the fragile sites Xq27 or 3p14.2 (FRA3B) or at points so close to the fragile sites as to be cytogenetically indistinguishable. Southern blot analysis of DNA from a panel of chromosome 3 deletion and translocation hybrids was then utilized to detect loss or retention of markers flanking FRA3B and to corroborate the cytogenetic evidence that the breakpoints were at this fragile site. One cell line with a reciprocal translocation between human chromosome 3 (with breakpoint at 3p14.2) and a hamster chromosome showed cytogenetically that the fragile site was expressed on both derivative chromosomes, supporting the hypothesis that the fragile site represents a repeated sequence. The approach described provides a means of generating specific rearrangements in somatic cell hybrids with a breakpoint at a fragile site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence Heterogeneity in Closed Simian Virus 40 Deoxyribonucleic Acid

The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved. The undenatured DNA from viruses passaged twice at multiplicities of infection much less than 1 plaque-forming unit (PFU) per cell appeared to be homogeneous in size. The heteroduplexes formed by this DNA indicated that approximately 2% of the molecules carried deletions, but that substitutions were below the level of detection. In contrast, undenatured DNA from viruses grown by passaging undiluted lysates seven times or by infection with stock virus at a multiplicity of infection of 5 PFU per cell contained a large frequency of molecules shorter than the full length. The heteroduplex samples indicated that 12 and 7% of the undenatured material contained base substitutions, and 13 and 11% contained deletions. The deletions and substitutions appear to occur in separate molecules. Length measurements on heteroduplexes displaying the loop characteristic of substitutions have established that these molecules are from true sequence substitutions, and not from adjacent or overlapping deletions. More than 80% of the molecules carrying substitutions are shorter than the native SV40 length. On the average, the substituted sequence is about 20% of the length of SV40, but it replaces a sequence about 30% of the native length. The substituted sequences may be host cell nuclear DNA, possibly arising from integration of SV40 into the chromosome followed by excision of the SV40 DNA together with chromosomal DNA.     

A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.     

The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells.

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.     

The chromosomal DNA of Streptomyces lividans 66 is linear

Two copies of a DNA sequence similar or identical to one end of the linear plasmid SLP2 were found on the Streptomyces lividans chromosome. Restriction mapping showed that these sequences represented free ends. Electrophoretic retardation and glass-binding studies indicated that the telomeres carry covalently bound proteins. Moreover, the chromosome migrated as an 8Mb linear DNA in pulsed-field gel electrophoresis. A similar finding with the chromosomes of six other Streptomyces species suggested that a linear chromosome may be characteristic of the genus. The S. lividans chromosome can be circularized by joining the two ends by artificial targeted recombination or by spontaneous deletions spanning both telomeres. Thus the chromosome appears to be able to exist, in viable bacteria, as a linear or a circular molecule.     

Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus

Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.     

Molecular evidence that chromosome breakage by Ds elements is caused by aberrant transposition.

The transposable Dissociation (Ds) element of maize was first discovered as a site of high-frequency chromosome breakage. Because both Ds-mediated breakage and transposition require the presence of the Activator (Ac) element, it has been suggested that chromosome breakage may be the outcome of an aberrant transposition event. This idea is consistent with the finding that only complex structures containing multiple Ds or Ac and Ds elements have been correlated with chromosome breakage. In this report, we describe two chromosome-breaking maize alleles that contain pairs of closely linked but separate Ds elements inserted at the Waxy locus. A polymerase chain reaction assay was utilized to isolate intermediates in the breakage process. The DNA sequence of these intermediates reveals deletions and base pair changes consistent with transposon footprints that may represent the junctions between fused sister chromatids. These results provide direct molecular evidence that chromosome breakage is the result of aberrant transposition events.