Masaki Kuro-o

Germ line-restricted, highly repeated DNA sequences and their chromosomal localization in a Japanese hagfish (Eptatretus okinoseanus)

The various species of Japanese hagfish, namely, Eptatretus okinoseanus (types A and B), Eptatretus burgeri and Myxine garmani, are known to eliminate a fraction of their chromosomes during early embryogenesis. High molecular weight DNA from germ line cells and somatic cells of these hagfish species was isolated and digested with different restriction enzymes. The DNA fragments were separated by agarose gel electrophoresis. Digestion with BamHI and DraI generated two weak bands and one weak band, respectively, that were estimated to be about 90, and 180 bp and about 90 bp long and were limited to the germ line DNA in both types of E. okinoseanus. DNA filter hybridization experiments showed that the two BamHI fragments and the one DraI fragment were present almost exclusively in the germ line DNA of E. okinoseanus. Thus, these DNA fragments appear to be eliminated during embryogenesis. Moreover, evidence was obtained that these fragments are highly and tandemly repeated. Molecular cloning and sequence analysis revealed that the BamHI fragments are mainly composed of a family of closely related sequences that are 95 bp long (EEEo1, for Eliminated Element of E. okinoseanus 1), and the DraI fragment is composed of another family of closely related sequences that are 85 bp long (EEEo2). The two DNA families account for about 19% of the total eliminated DNA in E. okinoseanus type A. Fluorescence in situ hybridization experiments demonstrated that the two families of DNA are located on several C-band-positive, small chromosomes that are limited to germ cells in both types of E. okinoseanus.

Germ line-restricted supernumerary (B) chromosomes in Eptatretus okinoseanus

Cytogenetic studies were performed on two types of Japanese hagfish (Eptatretus okinoseanus) that eliminate about 45% (type A) and 55% (type B) of their DNA from presumptive somatic cells during the differentiation of somatic cells. The observations revealed inter- and intraindividual variations in the number of chromosomes in germ cells of both types of hagfishes. Although the modal number of chromosomes in the germ cells was 54 in both types, the percentage of cells with the modal number was rather low (38.6% [51/132] in five specimens of type A and 22.7% [25/110] in eight specimens of type B). In addition, one of seven type B specimens clearly had a modal number of 62 chromosomes. Another specimen of type B had a bimodal distribution of chromosome numbers, with peaks of 54 and 59 chromosomes. The observation of interindividual variations was supported by data on the amount of DNA in germ cells of type B specimens. However, these variations were rarely observed in somatic cells. These results suggest that supernumerary (B) chromosomes are maintained in germ cells and are eliminated together with some other chromosomes and/or chromatin from somatic cells.

Cytogenetic studies of Hynobiidae (Urodela). X Morphological variation of chromosome 10 in ten pond‐type Hynobius from Korea and Japan, with comments on phylogenetic relationships

We have studied karyotypes of ten pond‐type Hynobius collected from 51 localities: H. leechii from Korea, and H. tsuensis, H. dunni, H. nebulosus, H. abei, H. tokyoensis, H. takedai, H. lichenatus, H. nigrescens and H. retardatus from Japan. C‐banding analyses have been performed on the specimens from 38 out of the 51 localities. Among the karyological variations observed, chromosome 10 showed intra‐ and interspecific variations in many species, which we considered suitable for phylogenetic analysis. Our phylogenetic inferences concerning the morphological variation of chromosome 10 in ten pond‐type Hynobius are summarized as follows: 1) similarity in the morphology and the amount of heterochromatin of chromosome 10 in species found in close geographic proximity to each other indicates a close phylogenetic relationship; 2) species distributed in widely separated geographic areas and yet with similar amounts of heterochromatin on chromosome 10 have conserved this chromosomal pattern from a common ancestor; 3) the main evolutionary changes of chromosome 10 are decreases in the C‐positive region and transformation of the biarmed type of this chromosome to the uniarmed type by a pericentric inversion involving the C‐positive region.

Cytogenetic studies of Hynobiidae (Urodela). IX. Karyological characteristics ofHynobius abei Sato by means of R-and C-banding

Chromosomal characteristics of the salamander speciesHynobius abei, from Ohimya (Kyoto) were revealed by the techniques of R-and C-banding. The karyotype ofH. abei was characterized by the shortness of an R-negative (C-positive) band in the terminal region of the long arm of chromosome 2 and a band encompassing the whole short arm of chromosome 10. These two bands inH. abei were the shortest among those of the variousHynobius species that have been examined. Otherwise no differences could be detected betweenH. abei and seven other pond-type species ofHynobius (2n=56) in terms of the banding patterns of 18 specifically identifiable pairs of their chromosomes.

Cytogenetic studies of Hynobiidae (Urodela) XVI. Comparative C-banded karyotype analysis of Pseudohynobius flavomaculatus (Fei et ye), Ranodon shihi (Liu) and Batrachuperus pinchonii (David)

Initial analysis of Pseudohynobius flavomaculatus chromosomes determined the chromosome number of this species to be 2n = 52. A re-examination of Ranodon shihi chromosomes detected 2n = 66 chromosomes, in contrast with a previous finding of 2n = 64. The C-banding patterns of these two species and that ofBatrachuperus pinchonii were compared with each other. Regions of homoeology in the C-banding pattern among these three species represented 33.51–48.30% of the total length of their chromosomes. We also detected two types of chromosome rearrangement in hynobiid species based on the results of the present and previous cytogenetic studies.

Cytogenetic studies of Hynobiidae (Urodela). XIV. Analysis of the chromosome of a Chinese salamander, Batrachuperus pinchonii (David)

The chromosome number of a Chinese salamander, Batrachuperus pinchonii, was re-examined. Adults and embryonic specimens had a diploid number of 66, with 33 bivalents during meiosis, in contrast to previous reported results. Furthermore, when C-banding analysis was performed with embryos, chromosomes with banding patterns homoeologous to those of Salamandrella keyserlingii and Hynobius species were found. It appears, therefore, that Batrachuperus, Salamandrella and Hynobius might be derived from a common ancestral species in eastern Asia.

Establishment and Characterization of BALB/c Retroperitoneal Sarcoma with Spontaneous Liver Metastases

The objective of this study was to examine the identity and characteristics of a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin that selectively metastasized to the liver. From the primary tumor, a permanent cell line, termed LMFS (liver metastasis from sarcoma) was established in vivo and in vitro. After a subcutaneous injection of more than 1 × 105 cells in the side back of mice, the LMFS cells proliferated at the inoculation site (100% take) and induced metastatic nodules spontaneously in the liver, but not in the lung. By the limiting dilution technique, a cloned cell line, LMFS‐1, was established in vitro. The LMFS‐1 cell line had similar morphological characteristics to the LMFS cells both in vitro and in vivo. The doubling time of the LMFS‐1 cell line was 10 h in passage 60. The number of chromosomes ranged from 71 to 108 and 93% of metaphases showed near‐tetraploidy. In microscopic examination, no specific arrangement of the LMFS tumor cells was seen; the LMFS cell had medium‐ to large‐sized atypical nuclei and clear and large cytoplasm. Electronmicroscopy showed that the cytoplasm of the LMFS cell had a moderate amount of rough‐surfaced endoplasmic reticulum but no desmosomes or microvilli. Immunohistochemically, the LMFS cells were positive for vimentin, but showed no reaction for keratin or cytokeratin. Therefore, the LMFS tumor was considered to be an undifferentiated sarcoma. The LMFS cell line should be a useful tool not only for studies of metastasis, but also for experiments on the therapy of hepatic tumors.