Recent gene conversion between genes encoding human red and green visual pigments

Nucleotide sequences of the human X-linked red and green pigment genes were compared, and the number of silent substitutions per site (KSc) between these genes was analysed in comparison with the corresponding values of primate genes. Taking the retarded mutation rate of X-linked genes into consideration (Miyata et al., 1987), the red and green pigment genes were shown to have undergone gene conversion at around the time of separation of African apes and orangutan. Thus the recent gene conversion and retarded mutation rate in these X-linked genes are probably responsible for the strong sequence similarity between these genes, which is likely to facilitate the occurrence of red-green color blindness in the human population. It was also shown that the red pigment gene evolved about five times more rapidly than the green pigment gene since the latest gene conversion.     

Bovine high molecular weight kininogen. The amino acid sequence, positions of carbohydrate chains and disulfide bridges in the heavy chain portion

The existence of two types of circulating bovine plasma high molecular weight kininogen (HMWK) was predicted from analyses of complementary DNAs coding for this protein (Kitamura, N., Takagaki, Y., Furuto, S., Tanaka, T., Nawa, H., and Nakanishi, S. (1983) Nature 305, 545-549). The present protein-based study provided evidence in support of the proposed amino acid sequence derived from analysis of the cDNA clone, and the results confirm the existence of two types of circulating HMWK. Type I HMWK contains a heavy chain composed of 361 residues, while the heavy chain of type II HMWK contains 359 residues. The amino acid sequences of type I and type II HMWK determined in this study were identical to that inferred from the cDNA sequence with the exception of microheterogeneity observed in the cDNA at position 87 (Glu/Gln) and 168 (Lys/Arg). The heavy chain of type I HMWK contains 4 asparagine-linked carbohydrate chains at Asn-69, -150 (or -151), -179, and -186, while the heavy chain of type II HMWK contains these and an additional carbohydrate chain at Asn-264. In addition, a carbohydrate chain was found to be O-glycosidically linked to Thr-118 in both chains. Among nine disulfide linkages found in HMWK, eight intrachain disulfide pairs were established in the heavy chain. One interchain disulfide bridge occurs between the heavy chain and the light chain. This disulfide pairing, as well as repeating amino acid sequences observed in the heavy chain, provides strong evidence for the existence of three homologous domains in the heavy chain of bovine HMWK.     

Early development of the laboratory-reared flounder,Pleuronichthys cornutus

Fertilized eggs ofPleuronichthys cornutus were obtained by both artificial fertilization and natural spawning of laboratory-reared fish. The present paper describes in detail the early development of the fish and the rearing methods employed to provide basic information for mass production of this species. Eggs and sperm for artificial fertilization were obtained from adult fish caught in the Ariake Sound, Kyushu in November and December of 1984. Their maturation was successfully induced by intermuscular injection of pituitary homogenate of the silver carp,Hypophthalmichthys molitrix. Fertilized eggs were also obtained in 1985 by natural spawning of a broodstock kept in a tank for a year. Hatched larvae were fed successively with rotifers,Artemia nauplii and the harpacticoid copepod,Tigriopus japonicus and reared for 80 days. Ten thousand young fish of about 33 mm TL were obtained in 1984 and 1985 with the survival rate of about 17%. Ten developmental stages were defined on the basis of the morphological characteristics: A) newly hatched to 4 day old larvae, 2.7 to 4.1 mm TL (2.6 to 3.9 mmNL), yolk sac present; B) 4 to 16 day old larvae, 3.8 to 5.9 mm (3.6 to 5.6 mm), yolk resorbed, actively feeding on rotifers; C) 15 to 30 day old larvae, 6.3 to 8.3 mm (6.0 to 7.9 mm), notochord straight, hypural fin ray visible; D) 24 to 40 day old larvae, 6.7 to 9.2 mm (6.4 to 8.8 mm), caudal notochord upturned (45°); E) 28 to 45 day old larvae, 7.9 to 10.8 mm (7.5 to 10.3 mm), caudal notochord upturned (45°–90°); F) 32 to 50 day old larvae, 10.8 to 15.7 mm (8.8 to 12.8 mm BL), eyes symmetrical; G) 35 to 66 day old larvae, 13.4 to 20.0 mm (10.9 to 16.3 mm), eyes asymmetrical, but left eye not visible from the right side; H) 40 to 75 day old larvae, 13.8 to 26.2 mm (11.3 to 21.4 mm), the upper edge of left eye visible over top of the head from the right side; I) 46 to 89 day old larvae, 20.1 to 27.4 mm (16.4 to 22.4mm), left eye on the edge of the head and pupil visible from the right side; and J) juveniles of 51 day old or over, 23.6 mm or more (19.3 mm or more), metamorphosis completed. One to three inflections were found for relative growth of total length, eye diameter, upper jaw length, preanal length, and distance between the base of the pectoral fin and the anus against the notochord length or body length. Two inflections were found for body length (or notochord length)-body weight relationship. Most inflections appeared at the stages of D, F and J, corresponding to the body length of 8, 9–12 and 18–22 mm respectively.     

Gene-specific transposon mutagenesis of the biphenyl/polychlorinated biphenyl-degradation-controlling bph operon in soil bacteria.

A transposon, Tn5-B21, was gene-specifically inserted into the chromosomal biphenyl/polychlorinated biphenyl-catabolic operon (bph operon) of soil bacteria. The cloned bphA, bphB and bphC genes of Pseudomonas pseudoalcaligenes KF707, coding for conversion of biphenyl into a ring meta-cleavage product (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid), carried random insertions of Tn5-B21. The mutagenized bphABC DNA, carried by a suicide plasmid, was introduced back into the parent strain KF707, resulting in the appearance of gene-specific transposon mutants by double crossover homologous recombination: the bphA::Tn5-B21 mutant did not attack 4-chlorobiphenyl, the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant produced dihydroxy compound. Gene-specific transposon mutants of the bph operon were also obtained for some other biphenyl-utilizing strains which possess bph operons nearly identical to that of KF707.     

Induction of ovarian maturation and development of eggs,larvae and juveniles of the tonguefish,Cynoglossus abbreviates,reared in the laboratory

Cynoglossus abbreviatus spawns from mid-March to mid-April in the Sea of Shimabara in Kyushu. During the spawning season ovarian maturation was successfully induced by injection of the pituitary homogenate ofHypophthalmichthys molitrix. The dose of the aceton-dried pituitary homogenate was 6.5 mg/kg body weight ofC. abbreviatus. It took about 2 days for ovulation after injection at a water temperature of 14 to 16°C. Artificial fertilizations were accomplished on March 29, 1974 and again on April 7, 1984, using the females matured by hormone injection in the latter case only. The larvae were reared on the rotifers,Artemia nauplii,Tigriopus japonicus and copepods collected from the sea over a period of 113 days in 1974 and 58 days in 1984. The eggs were pelagic, spherical, 1.19–1.23 mm in diameter and had 30–50 oilglobules of 0.068–0.095 mm in diameter, and the perivitelline space was narrow. The incubation period was 90–98 hours at a water temperature of 14 to 16°C. The newly hatched larvae were 3.18–3.45 mm TL and had 61–64 myomeres. The larvae had many melanophores and xanthophores on the body, forming three bands on the caudal region, but were lacking chromatophores on the finfolds. The yolk was completely absorbed when the larvae attained a size of 4.7–5.6 mm TL 8 days after hatching. A single elongated dosai fin ray developed on the head in the 8-day old larvae. The ray was reduced in size as long as the other rays 1 or 2 days after metamorphosis. The rudiment of pectoral fins were found on the both sides of the body in the 2-day old larvae, but two of them disappeared after metamorphosis. A pelvic fin first appeared as a ventral bud just anterior to the gut in the larva of 8.39 mm TL. The full count of 4 rays was observed on the larva of 10.83 mm TL. Metamorphosis began 22 days after hatching when the larvae were 11.20 mm TL. The right eye began to shift the left side of the head at night and reached to the final place after 8.5 hours. It took about 36 hours to complete the metamorphosis, including the eye movement and fusion of the hole in the rostral beak. At the last stage of metamorphosis, the dosal, caudal, anal and ventral fins became confluent. The larvae reached the juvenile stage at a size of 13.5–14.0 mm TL, approximately 28 days after hatchling. The growth of larvae reared in 1974 is expressed by the following equations: Y₁ = 3.448 · 1.0507^x (8≦X≦28) Y₂ = 6.3322 · 1.0275^x (28≦X≦75) where Y is the total length (mm) and X is the number of days after hatching. Growth rate changed after metamorphosis.     

Role of Tween 80 and Monoolein in a Lipid-Sterol-Protein Complex Which Enhances Ethanol Tolerance of Sake Yeasts

An exogenous ternary complex composed of Tween 80, ergosterol, and albumin increased the final ethanol concentration of fermentation by sake yeasts from 17.2 to 19.0% (vol/vol) and reduced the fermentation time from 30 to 25 days. Likewise, a complex of monoolein, albumin, and either ergosterol or ergosteryl oleate increased the final ethanol concentration of fermentation to 19.7 or 19.8% (vol/vol), respectively, and reduced the fermentation time to 25 days. Both Tween 80 and monoolein promoted the fermentative activity (Q_CO2) of cells, and the effect was enhanced by the presence of ergosterol.     

In vivo and in vitro synergistic antitumor effect of interleukin-2-cultured tumor-bearer spleen cells and immune fresh spleen cells

Summary The synergistic antitumor effect of interleukin-2(IL-2)-cultured tumor-bearer spleen cells (cultured lymphocytes) and immune fresh spleen cells was examined. Tumor-bearer cultured lymphocytes were obtained by culturing BALB/c spleen cells from syngeneic MOPC104E-tumor-bearing mice for 11 days with crude IL-2 and a soluble tumor extract. These cultured lymphocytes had weak antitumor activity when transferred i.p. into tumor-bearing mice that had been inoculated i.p. with 10⁵ tumor cells 5 days previously. Immune fresh spleen cells, obtained from mice in complete remission after the treatment with cyclophosphamide, also had weak antitumor activity when transferred at the same schedule. The cultured cells and the fresh cells, mixed together before transfer, significantly augmented the therapeutic effect. At least 1×10⁷ tumor-bearer cultured lymphocytes and 4×10⁷ immune cells were needed for the synergistic effect. A tumor-specific combination was needed for both cultured and fresh cells. The effective subpopulation of tumor-bearer cultured lymphocytes was a cytotoxic one from an Lyt2⁺ precursor, and that of the immune fresh spleen cells was noncytotoxic, Lytl⁺ and Lyt2⁺ T-cells.A similar synergistic effect was also observed during in vitro coculture of tumor-bearer and immune cells. Cytotoxicity, as assessed by the ⁵¹Cr-release test, of tumor-bearer IL-2-cultured lymphocytes was maintained most effectively after 3 or 4 days of culture without IL-2 when the lymphocytes were cocultured with immune fresh spleen cells and tumor cells.     

Interconversion of polyamines in wild-type strains and mutants of yeasts and the effects of polyamines on their growth

Yeasts of wild-type strains, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans were shown to have the ability to form aminopropylcadaverine and aminopropylhomospermidine from cadaverine and homospermidine, respectively. A polyamine autotroph S. cerevisiae 179-5, which lacks ornithine decarboxylase, produced both aminopropylcadaverine and aminopropylhomospermidine, while another mutant S. cerevisiae Y 260 A, which lacks spermine synthase, formed only aminopropylcadaverine. Naturally-occurring triamines and tetraamines except norspermidine and norspermine stimulated the growth of S. cerevisiae 179-5. All the six aliphatic diamines with carbon chain length ranging from one to six were effective in activating the growth of S. cerevisiae 179-5, though all of them were not converted to either triamines or tetraamines.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.