Dermatoglyphic development on the volar pads of rats with chromosome abnormalities.

Dermatoglyphic abnormalities are often observed in patients with chromosome aberrations, but no similar observations have been made in animals. In the present study, palmar dermatoglyphics were examined in 4 rats with chromosome anomalies. Reciprocal translocations were induced by gamma-irradiation; the animals used were obtained from among offspring with abnormal karyotypes that were derived from the original mutant rats. As the epidermal surface of the volar pad of the rat is flat, dermatoglyphic characteristics were observed on the dermal surface following staining with toluidine blue. Unusual ridge configurations were found in some of them, suggesting that dermatoglyphic development in the rat reflects, to some extent, an abnormal chromosome constitution.

Preferential clustering of viral DNA sequences at or near the site of chromosomal rearrangement in fowl adenovirus type 1 DNA-transformed cell lines.

All six transformants obtained by inoculating fowl adenovirus type 1 (CELO virus) DNA or its fragments into a rat cell line of normal karyotype had more than 50 copy-equivalents of viral DNA sequences. Each of the transformants had almost all if not all of these viral DNA sequences clustered on a marker chromosome(s). Although the marker chromosome(s) differed from one cell line to another, viral DNA sequences preferentially clustered in or near the achromatic (or light-stained) region of the G-banded marker chromosomes where chromosomal rearrangement or translocation occurred. These results indicate that no particular chromosome is required to act as the integration site of viral DNA for the transformation of cells, but chromosomal rearrangement at or near the cluster of viral DNA sequences might contribute to the transformation.

Inheritance of dermatoglyphic configurations in the rat.

Inheritance of dermatoglyphic configurations was studied on the palmar III interdigital pad of the rat (Rattus norvegicus). Four rats from each of the WKS and ACI/N inbred strains were mated with each other, and 54 F1 and 88 F2 hybrids were obtained. In addition, 52 offspring were produced by backcross mating between F1 hybrids and WKS and ACI/N parents. Whorls were the predominant pattern in the WKS strain and triradial patterns characterized the ACI/N strains. The F1 hybrids showed a wide range of pattern types, consisting of whorls, loops, cusps, arches, and triradial patterns. These patterns were intermediate in size between the two inbred strains. The F2 hybrids presented a distribution of patterns with a similar range as the F1, but the frequencies of some types were different. Patterns in the offspring of each backcross demonstrated a slight shift towards the characteristic pattern of the parental inbred strain. No sex difference was observed. Generally, the bilateral differences were striking, with a radial direction predominating on the left palm, and an ulnar one on the right palm, respectively. This study suggests that the dermal patterns are genetically determined, but also are influenced by environmental factors, especially in the hybrids.

Malignant transformation of Bloom syndrome B-lymphoblastoid cell lines by carcinogens.

Three types of Bloom syndrome B-lymphoblastoid cell lines, as well as one derived from a normal person, treated with 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine (0.3 micrograms/ml for 24 hr), were studied for tumorigenicity in nude mice, colony formation in soft agar, cytogenetic changes, and immunoglobulin markers. When normal and Bloom syndrome cells with normal sister chromatid exchange (SCE) levels and karyotypes (type I) were treated with carcinogens, no significant changes occurred in the immunoglobulin profile and karyotype, only rare colony formation was seen, and no tumors were produced. In contrast, when Bloom syndrome cells with high SCE levels (type II with normal karyotype and type III with an abnormal karyotype) were treated with carcinogens, tumors were produced in 22 of 53 nude mice injected; a high rate of colony formation in soft agar was seen; the cells exhibited virtual loss of immunoglobulin markers; and structural changes in chromosomes 1, 2, 3, 4, 5, 7, 11, 14, and 15 were found in the tumors in addition to the original chromosome abnormalities present in the injected cells. It appears that Bloom syndrome B-lymphoblastoid cell lines with high levels of SCE are highly susceptible to the action of carcinogens, as evidenced by tumor formation in nude mice and colony formation in agar. Apparently, the carcinogens were capable of transforming only those cells that had a critical level of SCE (approximately 140 per cell) and not those with only mildly increased levels (approximately 13 per cell).

[Development of technics for observation of chromosomes by the differential staining of sister chromatids, high resolution banding and the in situ molecular hybridization].

Cytogenetical technique newly developed for the observation of chromosomes, such as differential staining of sister-chromatids the high-resolution banding and in situ molecular hybridization are introduced in this chapter. In the differential sister-chromatid staining, acricine-orange, BUdR and FPG techniques are described. In the high resolution banding, amethopterin, BUdR and ethidium methods are introduced. RNA/DNA and DNA/DNA in situ hybridization techniques are also included.

[Technique for the karyotype analysis].

Techniques for the karyotype analysis in the man and some laboratory animals are described in the present chapter. The international systems for the representation of the standard karyotypes in these materials due to the conventional and the several banding stainings are also introduced.

Inhibition of bromodeoxyuridine-associated sister chromatid exchanges in Bloom's syndrome cells with cycloheximide.

Effects of cycloheximide (CH) and deoxycytidine (dC) on the frequency of sister chromatid exchanges (SCEs) in normal and Bloom's syndrome (BS) cells labeled with bromodeoxyuridine (BrdU) during first, second, and third cell cycles were evaluated using endomitotic and three-way differentiation analyses. When CH at 0.2 and 2.0 ng/ml was added to normal and BS cultures of BrdU-labeled endomitoses, the rate of single SCEs was significantly decreased in BS cells, though the rate of reduction in single SCEs was slight in normal cells. No significant change was detected in the twin SCE rate. In BS cells, treatment with CH at 0.2 and 2.0 ng/ml produced significant reductions in SCEs in both the second (SCE2) and third (SCE3) cell cycles, sometimes reaching the normal level. Treatment with dC at 13 and 26 micrograms/ml resulted in almost no significant changes in rates of SCE during first, second, and third cell cycles. When CH was added to BrdU-labeled normal and BS cell cultures, the cell growth rates improved from 35% to 70% over the control level in the BS cells, though in normal cells, the addition of CH resulted in a close-dependent lower cell growth rate. Deoxycytidine did not noticeably affect the cell growth rates in BrdU-labeled normal and BS cultures. The finding that the reduction of BrdU-induced SCEs in BS is paralleled by cell growth improvement is of special interest.

[Cytogenetic techniques for the observation of chromosomes from the cultured cells of the blood and amniotic fluid].

Cytogenetical techniques of the observation of chromosomes in mammals (human) by the cultivation of the peripheral blood cells and also cells suspending in the amniotic fluid are described in this paper. In the blood cell cultivations, the common technique by use of the leukocytes and also a small amount of the whole blood are introduced. The culture methods of the cells including in the amniotic fluid are also described with the technique of the chromosome preparations.

Studies of Angiostrongylus cantonensis in two experimental hosts.

Lung infection with Angiostrongylus cantonensis was established in experimentally infected Indian soft-furred rats (Millardia meltada) and Indian spiny mice (Mus platythrix). Pulmonary arterial migration occurred earlier (at day 20 after infection) in M. meltada than in Wistar rats. M. meltada and M. platythrix could serve as useful animal models for comparative studies of mechanisms determining host specificity of A. cantonensis.

Genetic Differentiation among Karyotypic Forms of the Black Rat, RATTUS RATTUS.

The black rat, Rattus rattus, consists of five karyotypic forms-2n = 42 (high C-banding); 2n = 42 (low C-banding); 2n = 40; 2n = 38; 2n = 42 Mauritius. Here, we use isozyme electrophoresis and microcomplement fixation to elucidate the genetic distance and phylogenetic relationship among each of the various karyotypic forms of R. rattus and R. norvegicus . The results show that (1) the 2n = 42 Mauritius black rat (2n = 42Mau) is genetically very similar to the 2n = 38 form, suggesting that this island population has undergone very rapid chromosomal evolution; (2) the 2n = 40 form from the highlands of Sri Lanka is genetically distinct from the 2n = 38 form from the lowlands; the genetic difference is probably insufficient, however, to prevent future introgression; (3) the level of genetic differentiation occurring between the 2n = 42 forms on the one hand and the 2n = 38, 2n = 40 and 2n = 42 Mau forms on the other support the hybrid incompatability data in suggesting that the two groups are either full species or incipient species; (4) in contrast to data from amino acid composition of transferrin and from restriction endonuclease digests of mtDNA, the present data suggest that the various karyotypic forms of R. rattus are phylogenetically more closely related to each other than any is to R. norvegicus, and that they are related by a series 2n = 42 --> 2n = 40 --> 2n = 38; (5) the R. rattus/R. norvegicus divergence occurred 2-8 million years ago, whereas the various chromosomal forms of R. rattus diverged over the last 4 million years.

Frequency of sister-chromatid exchanges depending on the amount of 5-bromodeoxyuridine incorporated into parental DNA.

Chinese hamster D-6 cells were grown for two cell cycles. The effect of 5-bromodeoxyuridine (BrdU) on the frequencies of sister-chromatid exchanges (SCEs) in these cells was investigated by the BrdU-labeling method. A low concentration (5 microM) of BrdU was inoculated in the first cell cycle for SCE counting. When excess concentrations (100-1000 microM) of BrdU were added subsequently in the second cell cycle, a 1-2-fold increase of SCE frequencies was observed. When excess thymidine (dT) (100-1000 microM) was supplied instead of BrdU, the incidence of SCE also increased. When cells were exposed to high concentrations (50-200 microM) of BrdU in the first cell cycle, a 1-4-fold increase in SCE frequencies was observed. This incidence of SCE was largely dependent on the concentration of BrdU and dT used in the second cell cycle. These results suggest that efficient SCE induction by BrdU is related to the BrdU residue incorporated into parental DNA strands.

Analyses of bromodeoxyuridine-associated sister chromatid exchanges (SCEs) in Bloom syndrome based on cell fusion: single and twin SCEs in endoreduplication.

When Bloom syndrome (BS) cells labeled with bromodeoxyuridine (BrdUrd) for one round of DNA replication were fused with nonlabeled normal cells, the hybrid cells had a normal level of sister chromatid exchange (SCE) at the first mitosis after fusion. However, when normal cells treated with mitomycin C (MC) were fused with nontreated normal cells, the MC-induced SCE was not affected by fusion with normal cells. Single and twin SCEs were analyzed in the Colcemid-induced endoreduplicated normal and BS lymphoid B cells from diplochromosomes. In normal cells, the same number of SCEs occurs in each of the two cell cycles; the SCE ratio of single (6.30 SCEs per cell) to twin (2.92 SCEs per cell) was 2:1 on the endoreduplicated-cell basis, showing 1:1 on the diploid-cell basis. In BS cells, the SCE ratio of single (144.8 SCEs per cell) to twin (5.9 SCEs per cell) was 25:1 on the endoreduplicated-cell basis and was 12:1 on the diploid-cell basis. These studies strongly suggest that most of the BS SCEs occur during the second cell cycle when BrdUrd-containing DNA is used as template for replication and that the normal level of BS SCE observed at the first mitosis of the hybrid cells is the result of SCE inhibition resulting from the fusion with normal cells.

Analysis of single and twin sister chromatid exchanges in endoreduplicated normal and Bloom syndrome B-lymphoid cells.

Single and twin sister chromatid exchanges (SCEs) were analysed in the colcemid-induced endoreduplicated normal and Bloom syndrome (BS) B-lymphoid cells with diplochromosomes. In normal cells, an equal number of SCEs occur in each of the two cell cycles; the ratio of single (= 5.51 SCEs/cell) to twins (= 2.64 SCEs/cell) was 2:1 on the endoreduplicated-cell basis, and it was 1:1 on the diploid-cell basis. In contrast, in 29 endomitoses from one BS B-lymphoid line, a manyfold increase of single SCEs was detected and 139.4 single SCEs on the average were counted, whereas twin SCEs were rare and only 4.9 twin SCEs were countable. In BS cells, the ratio of single (= 139.4 SCEs/cell) to twins (4.9 SCEs/cell) was 28:1 on the endoreduplicated-cell basis, and it was 14:1 on the diploid cell-basis; the rates of S1 and S2 exchanges were 4.9 and 69.7 SCEs/cell, respectively. The present study strongly indicates that most of BS SCEs occur during the second cell cycle when BrdU-containing DNA is used as template for replication and that BrdU enhances BS SCEs.

Chromosome alteration and the development of tumors. XXIII. Banding karyotype analyses of methylcholanthrene-induced tumors in the Indian spiny mouse, Mus platythrix, with special regard to the anomalies of chromosomes with nucleolar organizer regions.

In the Indian spiny mouse, Mus platythrix (2n = 26), six tumors were induced by 3-methylcholanthrene, and their karyotypes were analyzed in the primary state by G-banding. The chromosome numbers of these tumors were widely distributed ranging from diploid to tetraploid, but the frequency of cells exhibiting diploidy was the highest. Among these cells, the frequency of the cells with a normal diploid karyotype was only 27%, but the remaining cells (73%) showed pseudo- or near-diploid karyotypes. Although several numerical and structural anomalies of the chromosomes were observed in these tumor cells, centric fusion and translocation was most commonly seen, and that of trisomy and monosomy ranked second. Among 13 chromosome pairs, higher frequencies of chromosome anomalies were observed in the chromosomes No. 5, 8, and 12. Anomalies of the other autosomes were related primarily to centric fusion with chromosomes No, 5, 8, or 12, those of the X chromosome were mainly numerical changes. Taking into account the nucleolar organizer regions (NORs), which always occurred in chromosome pairs No. 5, 8 and 12 in this species, a possible relationship between the anomalies of those chromosomes containing NORs and the malignant transformation of cells is proposed.

Polymorphisms of mitochondrial DNAs in Norway rats (Rattus norvegicus): cleavage site variations and length polymorphism of restriction fragments.

Extensive polymorphism was found in mitochondrial DNAs (mtDNAs) of Norway rats (Rattus norvegicus). The restriction endonuclease cleavage patterns of mtDNAs of laboratory rats, wild rats, tumor cells, and culture cells were compared. The polymorphism is defined by two criteria; one is cleavage site variation and the other is length polymorphism of restriction fragments. The cleavage site variation may be caused by point mutation, and the length polymorphism by sequence deletions or insertions. At least five types, types A-E, were identified by cleavage site variations, and two groups, a and b, were identified by length polymorphism of one HpaII fragment, Hpa5. All types except type C belonged to either group-a or group-b, whereas both groups were found in type C. Differentiation of polymorphic Norway rat mtDNA types and the experimental use of the polymorphism are discussed.

Mauritius type black rats with peculiar karyotypes derived from Robertsonian fission of small metacentrics.

All seventeen black rats collected from Mauritius Island were characterized by having many extra small acrocentric autosomes. Their basic karyotype was of Oceanian type, because of the presence of the large metacentric M1 and M2 pairs, but chromosome numbers in 13 specimens among them were 42, those of 3 specimens 43, and those of the remaining one specimen 44. Although the Oceanian type rat had 2 small acrocentric autosomes (pair no. 13), 16 Mauritius rats had 10 small acrocentrics, and the remaining one had 8 small acrocentrics. Comparative karyotype analysis between Oceanian and Mauritius type rats showed that the extra small acrocentrics found in Mauritius rats were due to Robertsonian fission of small metacentric pairs no. 14 and 18 of the original Oceanian type rat. Only one rat with 8 small acrocentrics showed the heteromorphic pair no. 18 consisting of one metacentric and two acrocentrics. The large metacentric M1 chromosome in 13 of 17 rats examined showed homologous pair, but two of them were heteromorphic by involving one metacentric M1 and two acrocentrics. In the remaining two rats M1 chromosome was not observed, but acrocentric pairs no. 4 and 7 were included. These acrocentrics were also suggested to be originated from Robertsonian fission of the large metacentric M1 chromosome. Robertsonian fission seemed to be one of the important mechanism found in karyotype evolution.

Evolutionary aspects of variant types of rat mitochondrial DNA'S.

Mitochondrial DNA's (mtDNAs) were prepared from various kinds of individual Norway rats, Rattus norvegicus, and from three types of individual black rats, Rattus rattus, (Asian type, Ceylon type, and Oceanian type). Intra- and interspecies divergence of their mtDNA sequences were calculated based on changes in restriction endonuclease cleavage sites. The extent of intraspecies divergence of black rats (about 8%) is much larger than that of Norway rats (1%) and the mtDNA of Asian-type black rats resembles the mtDNA of Norway rats more closely than it resembles the mtDNA of other types of black rats. These results strongly suggest that during the course of intraspecies differentiation of black rats, probably long after the separation of the three types of black rats, some Asian-type black rats were isolated sexually and formed a new species, Norway rats. On the basis of our observations we propose a hypothetical process to explain the evolution of animal mtDNA.