Acyl-coenzyme-A synthetase I from Candida lipolytica. Purification, properties and immunochemical studies.

Acyl-coenzyme-A synthetase I from Candida lipolytica has been purified to homogeneity as evidenced by polyacrylamide gel electrophoresis in the presence and absence of dodecylsulfate as well as by Ouchterlony double-diffusion analysis. The purification procedure involves resolution of cellular particles with Triton X-100 and chromatography on phosphocellulose, Blue-Sepharose and Sephadex G-100. The purified enzyme exhibits a specific activity of 20--24 U/mg protein at 25 degree C, which is about 100-fold higher than those of long-chain acyl-CoA synthetases hitherto reported. The molecular weight of the enzyme has been estimated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate to be approximately 84 000. The enzyme is specific for fatty acids with 14--18 carbon atoms regardless of the degree of unsaturation. Studies with the use of specific antibody to acyl-CoA synthetase I have indicated that this enzyme is immunochemically distinguishable from acyl-CoA synthetase II.     

Factors Affecting Party Size in Chimpanzees of the Mahale Mountains

We studied factors affecting party size and composition of wild chimpanzees at Mahale (M group) over an 11-month period. Parties with 1–5 individuals were most frequent (37.8%; 153/405 parties); they included 94.7% of all male parties (n = 76) and 81.3% of all female parties (n = 75). The median of monthly values was the standard for analysis. We divided the year into four periods based on the median size of monthly bisexual parties (30.9 individuals; includes both males and females): monthly bisexual party sizes were larger in May–June (period II) and October–January (period IV) and smaller in February–April (period I) and July–September (period III). Only bisexual parties changed in size with period. The number of fruit items (=species) eaten was fewer in periods II and IV when abundance per item appeared to be great. The sizes of bisexual parties, which included cycling females with maximal anogenital swelling, were larger, and their representation (%) in all bisexual parties was greater in periods III and IV. The numbers of both cycling females and cycling females with maximal anogenital swelling were also larger in periods III and IV. The percentage of cycling females with maximal anogenital swelling was greater in periods II and III. The results of this study and those of Nishida (1979) suggest that seasonal variation in party size of Mahale chimpanzees maintains a relatively consistent annual cycle. The factors assumed to affect party sizes are fruit availability and the presence of cycling females with maximal anogenital swelling.     

The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome

Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23S rRNA and plays a crucial role in its assembly. The three-dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3 A resolution by X-ray crystallography using the selenomethionyl MAD method. The RNA-binding domain of L2 consists of two recurring motifs of approximately 70 residues each. The N-terminal domain (positions 60-130) is homologous to the OB-fold, and the C-terminal domain (positions 131-201) is homologous to the SH3-like barrel. Residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains. The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid-binding proteins to create a 23S rRNA-binding domain in the very remote past.     

Langer-Giedion syndrome associated with submucous cleft palate

We report a 4-year-old girl with characteristic features of the Langer-Giedion syndrome (trichorhinophalangeal syndrome type II) who also had submucous cleft palate. When she underwent a palatoplasty, a diagnosis of Langer-Giedion syndrome was made because of the characteristic facial features, multiple exostoses, and partial deletion of the long arm of chromosome 8. This is the first case of trichorhinophalangeal syndrome associated with cleft palate. We review the clinical alterations of trichorhinophalangeal syndromes and differential diagnosis of Langer-Giedion syndrome from trichorhinophalangeal syndrome type I and hereditary multiple exostoses. We also describe the importance of trichorhinophalangeal syndrome in plastic surgery.     

Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae.

The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase. The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance.     

Nuclear and chloroplast DNA differentiation in Andean potatoes.

Over 3500 accessions of Andean landraces have been known in potato, classified into 7 cultivated species ranging from 2x to 5x (Hawkes 1990). Chloroplast DNA (ctDNA), distinguished into T, W, C, S, and A types, showed extensive overlaps in their frequencies among cultivated species and between cultivated and putative ancestral wild species. In this study, 76 accessions of cultivated and 19 accessions of wild species were evaluated for ctDNA types and examined by ctDNA high-resolution markers (ctDNA microsatellites and H3 marker) and nuclear DNA restriction fragment length polymorphisms (RFLPs). ctDNA high-resolution markers identified 25 different ctDNA haplotypes. The S- and A-type ctDNAs were discriminated as unique haplotypes from 12 haplotypes having C-type ctDNA and T-type ctDNA from 10 haplotypes having W-type ctDNA. Differences among ctDNA types were strongly correlated with those of ctDNA high-resolution markers (r = 0.822). Differentiation between W-type ctDNA and C-, S-, and A-type ctDNAs was supported by nDNA RFLPs in most species except for those of recent or immediate hybrid origin. However, differentiation among C-, S-, and A-type ctDNAs was not clearly supported by nDNA RFLPs, suggesting that frequent genetic exchange occurred among them and (or) they shared the same gene pool owing to common ancestry.     

Genetic diversity of the Andean tetraploid cultivated potato (Solanum tuberosum L. subsp. andigena Hawkes) evaluated by chloroplast and nuclear DNA markers.

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are native farmer-selected important cultivars that form a primary gene pool of the common potato (Solanum tuberosum L. subsp. tuberosum). The genetic diversity of 185 Andigena accessions and 6 Chilean native potatoes (S. tuberosum subsp. tuberosum) was studied using chloroplast DNA (ctDNA) microsatellites and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Andigena potatoes had 14 ctDNA haplotypes and showed higher variability in the central Andes, particularly in Bolivia, whereas those in the northern regions of the distribution area were remarkably uniform with A1 ctDNA and Chilean subsp. tuberosum with T ctDNA. Most of 123 clearly scored RFLP bands using 30 single-copy probes were randomly distributed throughout the distribution area and proved the same gene pool shared among these widely collected accessions. Nevertheless, the geographic trend of the nDNA differentiation from north to south along the Andes and the correlated differentiation between nDNA and ctDNA (r = 0.120) could also be revealed by canonical variates analysis. These results suggest that the genetic diversity in Andigena was brought about primarily from cultivated diploid species but considerably modified through sexual polyploidization and intervarietal and (or) introgressive hybridization and long-distance dispersal of seed tubers by humans.     

Innovative Approach for Improvement of an Antibiotic-Overproducing Industrial Strain of Streptomyces albus

Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, we demonstrated the efficacy of introducing drug resistance-producing mutations for further strain improvement. Mutants with enhanced salinomycin production were detected at a high incidence (7 to 12%) among spontaneous isolates resistant to streptomycin (Str^r), gentamicin, or rifampin (Rif^r). Finally, we successfully demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation. The Str^r mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12). Likewise, the Rif^r mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase β subunit). Increased productivity of salinomycin in the Str^r mutant (containing the K88R mutation in the S12 protein) may be a result of an aberrant protein synthesis mechanism. This aberration may manifest itself as enhanced translation activity in stationary-phase cells, as we have observed with the poly(U)-directed cell-free translation system. The K88R mutant ribosome was characterized by increased 70S complex stability in low Mg²⁺ concentrations. We conclude that this aberrant protein synthesis ability in the Str^r mutant, which is a result of increased stability of the 70S complex, is responsible for the remarkable salinomycin production enhancement obtained.     

Gonadotropin-releasing hormone increases melatonin release in the pineal gland of the female rat in vitro.

To evaluate the effect of gonadotropin-releasing hormone (GnRH) on melatonin ( N-acetyl-5-methoxytryptamine) release and its synthesizing enzyme activities in pineal glands, pineals from adult female rats during diestrus were organ-cultured in a medium containing 10 -12, 10 -10, or 10 -8 M GnRH for 6 h. Melatonin release increased significantly in pineals cultured with 10 -10 and 10 -8 M GnRH compared to controls. However, in pineal glands that were organ-cultured in a medium containing 10 -12 to 10 -8 M GnRH, the activity of arylalkylamine N-acetyltransferase, which is the key regulatory enzyme in melatonin biosynthesis, showed no significant difference from controls. Likewise, GnRH at these concentrations had no significant effect on the activity of pineal hydroxyindole- O-methyltransferase, which catalyzes the final step of melatonin biosynthesis. These results show that GnRH stimulates pineal melatonin release, but suggest that GnRH does not affect its melatonin synthesis.     

Biogeography of Hysterangiales (Phallomycetidae, Basidiomycota)

To understand the biogeography of truffle-like fungi, DNA sequences were analysed from representative taxa of Hysterangiales. Multigene phylogenies and the results of ancestral area reconstructions are consistent with the hypothesis of an Australian, or eastern Gondwanan, origin of Hysterangiales with subsequent range expansions to the Northern Hemisphere. However, neither Northern Hemisphere nor Southern Hemisphere taxa formed a monophyletic group, which is in conflict with a strictly vicariant scenario. Therefore, the occurrence and importance of long-distance dispersal could not be rejected. Although a pre-Gondwanan origin of Hysterangiales remains as a possibility, this hypothesis requires that Hysterangiales exist prior to the origin of the currently recognized ectomycorrhizal plants, as well as the arrival of mycophagous animals in Australia. This also requires that a basal paraphyletic assemblage represents parallel evolution of the ectomycorrhizal symbiosis, or that Hysterangiales was mycorrhizal with members of the extinct flora of Gondwana. Regardless, models for both ancient and more recent origins of Hysterangiales are consistent with truffle-like fungi being capable of transoceanic dispersal.