Evidence for heteromorphic sex chromosomes in males of Rana tagoi and Rana sakuraii in Nishitama district of Tokyo (Anura: Ranidae).

Karyotypes of the Tago brown frog Rana tagoi and stream Tago brown frog Rana sakuraii from a mountain region in the Nishitama district in Tokyo were examined by conventional Giemsa staining, C-banding and late replication (LR)-banding. Chromosome number was 2n = 26 in all cases. The 26 chromosomes consisted of five (1-5) pairs of large chromosomes and eight (6-13) pairs of small chromosomes. Chromosome 10 had a secondary constriction on the long arm. In all frogs, on chromosome pair 8, the XX/XY type sex chromosome was present. C-banding analysis indicated that, in R. sakuraii, neither the X nor Y chromosome possessed interstitial C-bands but each had centromere staining, while in R. tagoi, an interstitial C-band was present on the long arm of the X chromosome. The Y chromosome had no interstitial C-band. LR-banding analysis demonstrated the X and Y chromosomes to have a LR-band on the short arm and two LR-bands, each on the long arm, and the bands on both species to be essentially the same. Heteromorphic sex chromosomes in males of R. sakuraii and R. tagoi were identified for the first time in this study.

The site and time of expression of MIF in frog development.

A ventrally localized melanization-inhibiting factor (MIF) has been suggested to play a role in the expression of dorsal-ventral pigment patterns in amphibia. Here we investigate the onset and localization of MIF appearance in frog development. The expression of MIF was analyzed in the wild-type and gray-eyed mutant (g/g) of Rana japonica by immunoblotting and immunohistochemistry using an anti-MIF neutralizing monoclonal antibody. Western blot analysis revealed that the anti-MIF antibody recognized approximately 51 kDa and approximately 58 kDa bands. The 51 kDa band first appeared at the external gill stage, while 58 kDa band was additionally detected at the hindlimb bud stage. With the use of immunohistochemistry, it was found that the anti-MIF antibody stained the whole epidermis of the embryos at the external gill stage; however, the staining was stronger in lateral and ventral epidermis than in dorsal. Staining with the anti-MIF antibody was observed only in the outer epidermis of the ventral skin, but not in the dorsal skin during and after metamorphosis. The spatial expression of MIF in the wild-type was the same as that in the gray-eyed mutant. The same immunohistochemical result was obtained in the adults of R. nigromaculata. These results suggest that MIF is involved in the formation of the dorsal-ventral pigment pattern.

Melanosome formation in cultured amelanotic melanophores of Rana brevipoda by a frog tyrosinase cDNA Transfection.

A cDNA encoding tyrosinase of Rana nigromaculata was introduced into cultured, tyrosinase-negative amelanotic melanophores of R. brevipoda by a calcium phosphate precipitation method. Within a few days following transfection, dark pigmentation became visible in a small number of cells. Light microscopic observation revealed that the morphology of these transformed cells was comparable to that of normal melanophores in culture, and their proliferative activity was lower than that of amelanotic cells. Ultrastructural examination verified that amelanotic melanophores possessed a relatively small number of premelanosomes while the transformants contained numerous melanosomes at various stages of pigment deposition. The result indicated that tyrosinase cDNA of R. nigromaculata was expressed in amelanotic melanophores of R. brevipoda including the maturation of premelanosomes. It was also suggested that the expression of transfected tyrosinase cDNA had promoted differentiation of the amelanotic cells into fully developed melanophores.

Analysis of the tyrosinase gene of the Japanese pond frog, Rana nigromaculata: cloning and nucleotide sequence of the genomic DNA containing the tyrosinase gene and its flanking regions.

Three genomic DNA fragments containing the tyrosinase-encoding gene (TYR) of the Japanese pond frog, Rana nigromaculata, were cloned. The first, clone I, was isolated from a genomic library of sperm DNA using the mouse TYR cDNA as the probe and contained a DNA segment similar to exon 4 of the mouse TYR gene. Subsequently, the TYR cDNA was isolated by screening a frog embryo cDNA library using clone I as the probe. Two clones that contain genomic DNA of the TYR gene were isolated also from a blood cell DNA library using the frog TYR cDNA as the probe. Comparison of the nucleotide (nt) sequences of the genomic clone II DNA and the cDNA revealed that clone II contained a 3,140-bp DNA fragment consisting of the 5'-flanking region, the first exon, and a part of the first intron. The region upstream of the coding region contained the characteristic sequences for regulatory elements, including TATA- and CAAT-motifs, and also a pigment cell-specific promoter element, which is shared by the promoter regions of the vertebrate TYR genes. A 764-bp segment containing an upstream 748-bp non-coding region and 16-bp coding region was functional for expression of the promoter-less cat gene on a plasmid in the transiently transformed albino frog melanophore. The genomic clone III contained the 3'-untranslated region of the mRNA and its 3'-flanking region. Thus, the cDNA plus genomic DNA fragments isolated here cover the entire TYR gene and its flanking regions.

Accumulation of arsenic in blue-green alga, Phormidium sp.

The behavior of a blue-green alga, Phormidium sp., against inorganic arsenic dissolved in media was studied. The Phormidium sp. was shown to have capabilities of endurance against a high concentration stress of arsenic and of accumulation of arsenic. Studies on excretion of arsenic by the alga showed that there were two excretion modes, each of which had a characteristic rate constant and it could be attributed to two types of binding situations between arsenic and the tissues of the alga. The arsenate absorbed by the algae was readily reduced to arsenite within their tissues.