Hatao Kato

Spontaneous and induced sister chromatid exchanges as revealed by the BUdR-labeling method.

Publisher Summary This chapter discusses the spontaneous and induced sister chromatid exchanges (SCE) as revealed by the 5-bromo-2’-deoxyuridine (BUdR)-labeling method. The elaboration of techniques for labeling chromosomes efficiently in vivo and a comparison of the incidence of SCEs in cells from various tissues or organs of animals, especially immunocompetent cells, germ line cells, and embryonic cells, would provide important clues in determining the existence of spontaneous SCEs and their biological significance which has long remained an enigma. Recently an attempt has been made to explain the evolution of multiple gene families in eukaryotic genomes such as rRNA and immunoglobulin V-gene families in terms of the consecutive occurrence of unequal SCEs. It has been shown that many eukaryotic genomes contain a large (over 70%) DNA fraction in which middle repetitive sequences of about 300 nucleotides are interspersed between single-copy DNA sequences averaging 3000 nucleotides or less in length. A correspondence between the middle repetitive sequences and the sites of meiotic crossing-over has been suggested in liliaceous plants. Strand exchange involving the pairing of these sequences may well have a chance to undergo unequal recombination without affecting the array of structural genes in the chromosome. The remarkable susceptibility of SCEs to ultraviolet light or chemicals having similar modes of action, and the simplicity of scoring this event with the aid of the BUdR-labeling method, make SCE a new assay system for hazardous effects of various environmental mutagens and carcinogens.

Mechanisms for sister chromatid exchanges and their relation to the production of chromosomal aberrations

By taking advantage of the fact that fluorescent light (FL) induces strand breaks only in bromodeoxyuridine(BrdU)-substituted DNA, and that those breaks eventually lead to the formation of sister chromatid exchanges (SCEs), the response of SCEs to FL was studied carefully in Chinese hamster chromosomes in which, out of four DNA strands, BrdU-substitution had occurred either in one or three strands. The FL-induced SCE frequency did not differ greatly between these two types of chromosomes. However, when they were submitted to caffeine treatment, a drastic increase in the frequency was detected in the trifilarly-substituted chromosomes while a significant decrease occurred in the unifilarly-substituted chromosomes. Based on these results, a working hypothesis was developed that the SCE can arise by at least two different mechanisms, one operating at replicating points probably utilizing the machinery of DNA replication, and the other acting only in the post-replicational DNA portion, probably in a similar fashion as assumed in a general model of crossing over in the eukaryote. These dual mechanisms may account for the discrepancy encountered in the explanations of the induction of SCEs by various exogenous agents as well as spontaneous SCEs. The present study also showed that some, but clearly not all, of chromatid deletions are the outcome of the failure to complete SCEs arising through these mechanisms.

Banding patterns of Chinese hamster chromosomes revealed by new techniques

Two kinds of techniques were newly developed to reveal banding patterns of the Chinese hamster chromosomes. Both techniques were essentially the same as those used for the extraction of proteins, and one of them could induce bands in chromosome arms in only a few seconds. Banding patterns produced by these techniques appeared to be identical to those induced by the methods reported by previous workers, requiring post-fixation incubation of slides in a warm saline. —The banding pattern was typical for each chromosome pair, permitting unequivocal identification of several pairs which were hardly distinguished by the conventional staining procedures. It was confirmed that these patterns had been well preserved in the chromosomes of a cultured cell line.

Factors involved in the production of banded structures in mammalian chromosomes

Dozens of reagents were tested for their ability to produce bands in Chinese hamster chromosomes by incubating air-dried preparations in aqueous solutions of these reagents for a definite period prior to the Giemsa staining. Acids were found to be without effect on the band production. Many of salts were able to induce bands if their pH was alkaline. Strong bases were also found to be potent band inducing reagents. They produced bands only in a few seconds. Protein denaturants such as urea, guanidine-HCl and several surface active compounds were also effective in the band production. — In the light of these results, it seems very likely that the solubilization or extraction of some chromosomal proteins, probably of acid nature, would be the primary cause of the appearance of the banded structure in chromosome arms.

Analysis of crossing over in mouse meiotic cells by BrdU labelling technique.

Differential staining of sister chromatids in male mouse meiotic cells was achieved by continuous infusion with BrdU from the tail vein followed by Giemsa staining (the FPG technique of Perry and Wolff, 1974). Analysis of 341 bivalents including XY, and 21 univalents reveals that: (1) visible crossing over coincided exactly with the site of chiasmata; (2) no evidence was obtained in support of chiasma terminalisation; (3) an anomalous type of crossing over was found in the monochiasmatic bivalents, which could not be explained by the conventional hypothesis for crossing over; (4) some of the terminally associated bivalents might be achiasmatic, and univalents might have originated from such bivalents; (5) in XY bivalents, sister chromatid association was between lightly and darkly stained chromatids, suggesting a lack of crossing over and the existence of other genetic controls for this association; (6) during meiosis sister chromatid exchanges (SCEs) might be less frequent than crossovers.

DNA repair enzymes in ataxia telangiectasia and Bloom's syndrome fibroblasts

Ataxia telangiectasia, Blooms syndrome and normal fibroblasts were compared as to the capacity of their cellular extracts to enhance the priming activity of gamma-irradiated colicin E1 DNA for purified DNA polymerase. It was found that an ataxia strain had substantially lower, and a Blooms syndrome strain had slightly lower capacity than a normal strain; while the activities of apurinic site specific endonuclease in these extracts were comparable.

Cytogenetical survey of black rats, Rattus rattus, in southwest and Central Asia, with special regard to the evolutional relationship between three geographical types

A chromosome survey of the black rat, Rattus rattus, was made from animals collected at different localities in Southwest and Central Asia. Asian type black rats (2 n=42) were distributed in northern India, northern Pakistan, while the Oceanian type rats (2 n=38) were found in southern India, southern Pakistan and Central Asia. A border line of distribution of rats with Asian and Oceanian types can be drawn dividing India and Pakistan into northern and southern parts. A hybrid type between Asian and Oceanian types was found in Karachi, Pakistan. Rats with 40 chromosomes, probably a transient type from Asian to Oceanian type, were found in Sri Lanka (Ceylon). It is suggested that these three geographic variants have developed via sequential events of Robertsonian fusion of acrocentric chromosomes in Asian type black rats. This fusion probably took place somewhere in southern India. The Oceanian type black rats that thus developed in southern India migrated widely to the rest of the world through Central Asia and Europe accompanying the movement of mankind.

Stable telocentric chromosomes produced by centric fission in chinese hamster cells in vitro

Metaphase examination of pseudodiploid Chinese hamster cells revealed that spontaneous breaks or fission occurred rather frequently (2.9%) at the centromeric regions of subtelo- or metacentric chromosomes, resulting in the production of telocentric chromosomes. The centromeric fission appeared to occur in every member of the chromosome complement. An attempt was made to isolate cells possessing thus derived telocentrics from the cell population and gave two clonal lines which were retaining one and two telocentric chromosomes, respectively. Both banding and labeling patterns of these chromosomes indicated unequivocally their X chromosome origin. They were transmitted successively to the daughter cells during a 3-month culture period, showing no tendency to fuse to produce a metacentric chromosome.

In vivo sister chromatid exchange in cells of various organs of the mouse.

In vivo sister chromatid exchange (SCE) in mouse cells derived from various organs was studied by infusing BrdU from the tail vein. It was found that at BrdU concentrations ranging from 2.2–13.5 μg/g/h, the SCE frequency in bone marrow cells seemed to stay at a constant level (1.5–2/cell/two cell cycles) whereas it started to rise as the BrdU dose exceeded this dose range. When BrdU within this dose range was infused continuously from the tail vein for appropriate hours to label chromosomes in various organs, the average SCE frequencies per cell were found to be 1.64 in bone marrow cells, 1.82 in spermatogonia, 1.99 in splenic cells, 2.89 in intestinal cells and 3.69 in cells from adjuvant stimulated lymph nodes. It is suggested that the spontaneous level of the in vivo SCE frequency might be about 1.5–2/cell/two cell cycles in the mouse. In cells derived from intestine and adjuvant stimulated lymph node, some unknown factors might work as a inducer of SCEs resulting in a significant increase in the SCE frequency in these organs.

Preferential occurrence of sister chromatid exchanges at heterochromatin-euchromatin junctions in the wallaby and hamster chromosomes

Chromosomes of two mammalian species, the white-throated wallaby and the rat-like hamster, possessed large amounts of constitutive heterochromatin which is detectable as C bands. By making use of this character, the frequency of sister chromatid exchanges (SCEs) was determined for the C band and the euchromatic regions of the chromosome. In both species, the distribution of SCEs in the euchromatin of chromosomes was found to be proportional to its metaphase length, while the number of SCEs localized in the C band regions was clearly fewer than expected on the basis of the relative length of those regions at metaphase. Many SCEs were, however, detected at the junctions between the euchromatin and the C band heterochromatin. All of these findings were consistent with previous observations on the Indian muntjac and the kangaroo rat chromosomes.