Genetic effects of specific DNA lesions in mammalian cells

Accurate dosimetry for chemical mutagens is extremely difficult, and precise manipulation of the frequency of a particular lesion is ordinarily impossible. With 8-MOP plus UVA, however, both are possible because 8-MOP, when photoactivated by one photon of UVA, forms monoadducts whilst crosslinks are formed only if a second photon of light photoactivates the monoadducts. If 8-MOP molecules that are unreacted after a UVA exposure are removed from cells by washing, the effect of a subsequent UVA irradiation can be attributed only to the conversion of monoadducts to DNA interstrand crosslinks. Using this experimental procedure and L5178Y mouse lymphoma cells, we have shown that DNA interstrand crosslinks are at least 10-fold more effective at causing both sister-chromatid exchanges and chromosomal aberrations than are monoadducts. In contrast, crosslinks are no more effective than monoadducts in mutation induction. These experiments identify directly for the first time that a particular chemically induced lesion, DNA interstrand crosslinks, can, like thymine dimers, cause chromosomal aberrations and sister-chromatid exchanges. The results also show that sister-chromatid exchanges can be induced independently of mutations.     

Mapping of sister-chromatid exchanges in human chromosomes using G-banding and autoradiography.

Lumphocytes were pulse-labelled with [3H] thymidine. Following G-banding, the cells were autoradiographed and 46 in their third post-labelling division selected. The locations of 611 sister-chromatid exchanges (SCE's) which had occurred in the previous two cell cycles were recorded as label discontinuities along identified chromosomes. Between particular chromosomes, SCE frequency was proportional to chromosome length. SCE frequency distributions within particular chromosomes fitted Poisson expectations. There was no over-representation of exchanges in centromeric regions, or in the C-banded regions of chromosomes 1, 9 and 16. A trend of increased frequency of SCE in darkly G-banded regions and in relatively darkly banded chromosomes was evident. The apparent excess of SCE in dark G-bands could be considered to be a consequence of the more condensed state of the DNA in these regions in the interphase nucleus relative to the DNA in pale G-band regions. Such compaction could result in an enhanced probability of SCE and a reduced probability of gross inter- or intra-change involving these regions. In contrast, the more extended interphase state of the DNA in pale G-banded regions would allow non-homologous exchange and account for the preferred location of X-ray-induced exchange events to pale G-bands.     

A possible new type of chromosome rearrangement: telomere-centromere translocation (tct) followed by double duplication

Summary The reassessment of a case of complex interchromosomal rearrangement after breakage at centromeric and telomeric regions, and the comparison with four other independently published cases suggested the existence of a new type of rearrangement. It would consist of: formation of an isochromosome after breakage at a centromeric region, duplication of the telomeric region of another chromosome, and reassociation of the nonduplicated arm of the first chromosome with the duplicated telomeric region of the second chromosome.     

Repair of 8-methoxypsoralen monoadducts in mouse lymphoma cells

Studies of the repair of DNA lesions at biologically important doses is extremely difficult for most mutagens. With 8-methoxypsoralen (8-MOP) plus longwave ultraviolet light (UVA) as the lesion-inducing agent, however, it is easy to manipulate the relative frequency of different DNA adducts by means of a special experimental protocol (the tap-and-test protocol) and this can be used to measure repair of DNA adducts. Three classes of photoadducts are produced by 8-MOP plus UVA treatment: 3,4-cyclobutane monoadducts, 4',5'-cyclobutane monoadducts, and 8-MOP-DNA interstrand crosslinks. A monoadduct is formed when a photoactivated 8-MOP molecule reacts with a pyrimidine base. An 8-MOP-DNA interstrand crosslink is formed when an existing monoadduct is photoactivated to react with another pyrimidine base on the opposite DNA strand. Thus monoadducts are formed by absorption of one photon of light and crosslinks by absorption of two. In the tap-and-test experiments, cells were exposed to UVA in the presence of 8-MOP and then re-exposed to UVA in the absence of free 8-MOP so that only crosslinks can be produced by the second UVA treatment. By means of this technique we have previously shown that DNA crosslinks are much more effective than monoadducts at producing chromosomal damage (sister-chromatid exchanges and micronuclei) but not mutations (Liu-Lee et al., 1984). If L5178Y mouse lymphoma cells were able to remove monoadducts, incubation prior to the second UVA treatment should lead to decreases in the effect of re-irradiation, because fewer monoadducts would be available for crosslink formation. In this way, we have found that psoralen monoadducts are repaired in these cells and that about 70% of those capable of crosslink formation are removed or otherwise made unavailable for crosslink formation in 6 h.     

Structural analysis and complete physical map of Arabidopsis thaliana chromosome 5 including centromeric and telomeric regions.

Previously, we have reported a fine physical map of Arabidopsis thaliana chromosome 5, except for the centromeric and telomeric regions, by ordering clones from YAC, P1, TAC, and BAC libraries of the genome consisting of the two contigs of upper arm and lower arm, 11.6 M bases and 14.2 M bases, respectively. Here, the remaining centromeric and telomeric regions of chromosome 5 are completely characterized by the ordering of clones and PCR amplifications. Chromosome 5 of Arabidopsis thaliana ecotype Columbia is about 28.4 M bases long. The centromeric region is estimated at about 2 M bases long between two 5S-rDNA clusters. The 180-bp repeat region mainly consists of blocks of 180-bp tandem family and various type retroelements dispersed over a 500-kb region. The telomeric regions of chromosome 5 are characterized by PCR cloning, sequencing and hybridization. The telomere repeats at both ends are about 2.5-kb long and interestingly, telomere-associated repeats (approximately 700 bp) are found near both ends of chromosome 5.     

Analysis of the frequency and distribution of sister chromatid exchanges in cultured human lymphocytes

Summary Lymphocytes from 20 normal subjects (11 male and 9 female) were examined for the frequency and location of sister chromatid exchanges (SCE) by the BrdU—Giemsa method. The mean frequency of SCE was 6.37 with little significant variation. One subject had a high number of exchanges in chromosome 1 while the remainder showed a random distribution of exchanges between chromosomes. The frequency of exchanges generally increased with chromosome length. However, chromosome 1, 2 and the B group had more exchanges than expected while the E, F and G groups had less than expected. The distribution of exchanges in chromosomes 1, 2 and the B group was non-random with a concentration of exchanges below the centromere and to a lesser extent on the distal portion of the long arm. The majority of exchanges appeared to occur at the junction between the dark and light G bands. It is suggested that the concentration of exchanges may reflect differences in BrdU incorporation along the length of the chromosome.     

Induction of sister-chromatid exchange in human blood lymphocytes by aqueous extract of palmyrah (Borassus flabellifer) flour

pPalmyrah palm (Borassus flabellifer) is widely consumed by people in certain tropical countries. The incidence of human malignant lymphomas, mutagenicity and toxicity in rats and bacteria encouraged us to study the potency of palmyrah crude aqueous extracts in inducing sister-chromatid exchanges (SCEs) in human blood lymphocytes in vitro. The extracts induced SCEs in a dose-related manner in both females and males. These effects apparently showed no consistency between batches. This result may be due to the intrinsic variation of different donors in their response to the induction of SCEs by palmyrah extracts. SCE frequency was proportional to chromosome length and SCEs at the centromeric region showed no difficulty in being scored. Concerning methods of short-term cytogenetic testing for detecting mutagenic and carcinogenic chemicals, we found that the SCE test was not more sensitive than the classic chromosome-breakage test.     

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.     

Detection of sister chromatid exchanges by 4′-6-diamidino-2-phenylindole fluorescence

This paper describes a 4-6-diamidino-2-phenylindole (DAPI) fluorescent technique for differentiation of sister chromatids and for the study of sister chromatid exchanges (SCE) in mouse chromosomes. The advantages of the DAPI fluorescent technique are also described. Differences in the occurrence of SCE between the centromeric heterochromatin (C-banded) and the chromosomal arm chromatin were studied in mouse cells (RAG) with or without mitomycin C treatment. Single strand exchanges between the DNA double helices in the sister chromatids were not detected. SCE and chromosome breakage appeared to occur more frequently in the centromeric region than in the chromosomal arm. This might play an important role in chromosome evolution in mice.     

Identification,characterisation and clinical applications of cosmids from the telomeric and centromeric regions of the long arm of chromosome 22

Using human telomeric repeats and centromeric alpha repeats, we have identified adjacent single copy cosmid clones from human chromosome 22 cosmid libraries. These single copy cosmids were mapped to chromosome 22 by fluorescence in situ hybridisation (FISH). Based on these cosmids, we established contigs that included part of the telomeric and subtelomeric regions, and part of the centromeric and pericentromeric regions of the long arm of human chromosome 22. Each of the two cosmid contigs consisted of five consecutive steps and spanned approximately 100–150 kb at both extreme ends of 22q. Moreover, highly informative polymorphic markers were identified in the telomeric region. Our results suggest that the telomere specific repeat (TTAGGG) _n encompasses a region that is larger than 40 kb. The cosmid contigs and restriction fragment length polymorphism markers described here are useful tools for physical and genetic mapping of chromosome 22, and constitute the basis of further studies of the structure of the subtelomeric and pericentromeric regions of 22q. We also demonstrate the use of these clones in clinical diagnosis of different chromosome 22 aberrations by FISH.     

Cytogenetic analysis of some Brazilian marsupials (Didelphidae: Marsupialia)

Three species of marsupials from the Amazon region (Marmosa cinerea, Caluromys lanatus, and Didelphis marsupialis) and two from the region of S?o Paulo (Didelphis marsupialis and Didelphis albiventris) were studied. The G-banding pattern of the species with 2n = 14 (M. cinerea and C. lanatus) was very similar, as well as the pattern of G-bands in the species with 22 chromosomes (Didelphis). All of the autosomes of M. cinerea and D. albiventris have centromeric C-bands and the Y chromosome is totally C-band positive. The long arm of the M. cinerea X chromosome is completely C-band positive except for a negative band close to the centromeric region. In D. albiventris the long arm of the X chromosome is C-band positive except for a negative band close to the telomeric region. In M. cinerea the silver-stained nucleolar organizer regions (Ag-NORs) are found in the acrocentric chromosomes, being located in the telomeric region of one pair and in the centromeric region of the other pair. Caluromys lanatus has centromeric Ag-NORs in one acrocentric and in one submetacentric chromosome pairs. Didelphis marsupialis has three chromosome pairs with telomeric Ag-NORs. In D. albiventris the Ag-NORs are terminal and located in both arms of one pair and in the long arm of two pairs of chromosomes.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

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.     

Linkage disequilibrium of three polymorphic RFLP markers in the apolipoprotein AI-CIII gene cluster on chromosome 11

A panel of glial tumors consisting of 11 low grade gliomas, 9 anaplastic gliomas, and 29 glioblastomas were analyzed for loss of heterozygosity by examining at least one locus for each chromosome. The frequency of allele loss was highest among the glioblastomas, suggesting that genetic alterations accumulate during glial tumor development. The most common genetic alteration detected involved allele losses of chromosome 10 loci; these losses were observed in all glioblastomas and in three of the anaplastic gliomas. In order to delineate which chromosome 10 region or regions were deleted in association with glial tumor development, a deletion mapping analysis was performed, and this revealed the partial loss of chromosome 10 in eight glioblastomas and two of the anaplastic gliomas. Among these cases, three distinct regions of chromosome 10 were indicated as being targeted for deletion: one telomeric region on 10p and both telomeric and centromeric locations on 10q. These data suggest the existence of multiple chromosome 10 tumor suppressor gene loci whose inactivation is involved in the malignant progression of glioma.     

姐妹染色单体互换在鹌鹑胚胎细胞染色体间及染色体上的分布

SCE在鹌鹑胚胎细胞间的分布为Poisson分布;在染色体间的分布是非随机的,与染色体的相对长度成正相关（P<0.05）,但也不完全按各染色体的相对长度分布。着丝粒区的SCE相对很高,按染色体的相对长度分布。胚胎的不同性别对每个细胞的SCE平均值无显著影响,但对性染色体Z上的SCE是否有影响还不能做出肯定的结论。     

Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

Effect of mitomycin C on the structure of human chromosomes observed in metaphase after labelling by moderate thermal denaturation]

Mitomycin C induced chromosome rearrangements were analysed in cultured human leukocytes by reverse banding technique. Breaks and chromosomal exchanges involved preferentialy the entromeric region of some chromosomes (1, 5, 9, 16, and 20). Associations between acrocentric chromosomes was not found to be increased. But acrocentric associations with centromeric regions were frequently present. The differences between the mechanism of exchanges and breaks are discussed. The part of heterochromatin in post replication DNA repair is considered.     

Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac

The frequency of sister chromatid exchanges (SCEs) was determined for the chromosomes (except Y2) of the Indian muntjac stained by the fluorescence plus Giemsa (FPG) or harlequin chromosome technique. The relative DNA content of each of the chromosomes was also measured by scanning cytophotometry. After growth in bromodeoxyuridine (BrdU) for two DNA replication cycles. SCEs were distributed according to the Poisson formula in each of the chromosomes. The frequency of SCE in each of the chromosomes was directly proportional to DNA content. A more detailed analysis of SCEs was performed for the three morphologically distinguishable regions of the X-autosome composite chromosome. The SCE frequency in the euchromatic long arm and short arm were proportional to the amount of DNA. In contrast, the constitutive heterochromatin in the neck of this chromosome contained far fewer SCEs than expected on the basis of the amount of DNA in this region. A high frequency of SCE, however, was observed at the point junctions between the euchromatin and heterochromatin.     

Frequency of sister chromatid exchanges in a balanced reciprocal whole-arm translocation

Summary The frequency of sister chromatid exchanges (SCEs) in the centromere of chromosomes involved in a whole-arm translocation t(1;19) was evaluated in altogether 911 metaphases of translocation carriers (n=5) and of normal controls (n=6). Comparison of the two groups reveals no significant differences in the SCE rate (x ²=3.06, n _f =1). The question as to whether the possible increase of the SCE rate at the translocation point could be detected by light microscopy is discussed. Parameters included in the discussion are the ratio of the SCE frequency at the translocation point to the SCE frequency at any of the possible breakage points in the centromeric region and the number of possible breakage points in the centromeric region.     

Diversification of a ZZ/ZW sex chromosome system in Characidium fish (Crenuchidae, Characiformes)

Karyotype and other chromosomal markers of Characidium cf. gomesi were analyzed using conventional (Giemsa-staining, Ag-NOR and C-banding) and molecular (Fluorescent in situ hybridization (FISH) with 18S and 5S rDNA biotinylated probes) techniques. Both sexes had invariably diploid chromosome number 2n = 50 while karyotypes of males and females differed. That of male consisted of 32 metacentric + 18 submetacentric chromosomes and that of female consisted 31 metacentric + 18 submetacentric + 1 subtelocentric chromosomes. The Z chromosome was medium-sized metacentric, while W was highly heterochromatinized subtelocentric element. NORs as revealed by Ag-staining were situated at 2–7 telomeric regions while FISH with 18S probes showed consistently 10 signals at telomeric regions. FISH with 5S rDNA probe showed constantly signals at one metacentric pair. Distribution of centromeric heterochromatin was mostly in all chromosome pairs, besides some telomeric sites. The common origin of the sex chromosome system of ZZ/ZW type in the karyotypes of other representatives of the genus analyzed so far might be hypothesized based on biogeography and partial phylogeny of the group.