Mutations in {beta}-Tubulin Block Transhyphal Migration of Nuclei in Dikaryosis in the Homobasidiomycete Coprinus cinereus

ß-Tubulins from fourteen benomyl-resistant strainsof the homobasidiomycete Coprinus cinereus, which carry thebenA, benB, benC or benD mutations, were analyzed by urea SDS-PAGEor isoelectric focusing and subsequent immunoblot analysis.Electrophoretic aberrations in a major ß-tubulin isotype,denoted ß1 were found in two strains, BEN154 and BEN215,both of which carry benomyl resistance mutations in benA ⁺ Theaberrations of ß1 in BEN154 and BEN215 cosegregatedwith benomyl resistance among the progeny of outcrosses of BEN154 and BEN215 to wild type, indicating that the ß1aberrations were caused by the benA mutations. Both the mutantand wild-type ß1 tubulins were present in the heterozygousdikaryons, BEN 154/wild-type and BEN215/wild-type, ruling outpost-translational modification as a possible cause for theaberrations in ß1. Thus, we conclude that benA isa structural gene for ß1. Transhyphal migration ofnuclei in dikaryosis was blocked in the mycelia of BEN 154 andin its progeny that carried benA (ß1 mutation), demonstratingthat microtubules are involved in the migration process. Nuclearmigration in dikaryosis seems to differ in terms of mechanism,at least in part, from the migration of tetrad nuclei from basidiainto prespores during formation of basidiospores and from themigration of nuclei from basidiospores into hyphae during germination,because a benA mutation blocked the former without affectingthe latter two processes. (Received May 19, 1989; Accepted August 30, 1989)     

The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.     

cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a gtll library by plaque hybridization using this ³²P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.     

Synchronized endocytosis studied in the oocyte of a temperature-sensitive mutant of Drosophila melanogaster

Summary This study demonstrates that endocytosis in the oocyte of Drosophila melanogaster is reversibly blocked at the stage of pit formation by the temperature-sensitive, single-gene mutant, shibire ^ts1. Uptake of horseradish peroxidase conjugated with wheat-germ agglutinin was observed to be normal in mutant oocytes at 19°C, but was blocked at 29°C. After 10 min at 29°C, there was a build-up of coated pits along invaginations of the plasma membrane. Also, the endosomal compartment consisting of tubules, bulbs, and small yolk spheres, disappeared. Lowering the temperature to 19°C after 10 min at 29°C released a synchronized wave of endocytosis into a cytoplasm cleared of uptake-related organelles. By observing this synchronized wave after exposure to 19°C for varying durations, we determined that endocytosis proceeds as follows: coated pits/vesiclestubulessmall yolk spheresmature yolk spheres. The observations suggest that these organelles transform one into another within this sequence.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Three main components in plasma proteases and their relation to the renin-angiotensin system

The relation of plasma renin activity (PRA) and plasma levels of angiotensin I (AI) and II (AII) to those of various proteases, including eight endopeptidases and four aminopeptidases, was investigated in 51 normal control subjects. The multivariate study using factor analysis showed that the plasma proteases can be classified into three main components: the aminopeptidase, the plasmin, and the kinin-kallikrein. PRA and AI were related almost exclusively to the aminopeptidase component, while the AII level was related not only to the same component but also to the kallikrein-kinin component. This kind of multivariate study may help in the elucidation of the role of proteases and bioactive peptides, such as angiotensin derivatives, in essential hypertension through a comparison of multivariate relationships in controls and patients.     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

Constitution of Mitochondrial and Chloroplast Genomes in Male Sterile Tobacco Obtained by Protoplast Fusion of Nicotiana tabacum and N. debneyi

Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of malesterile tobacco plants obtained by fusion of Nicotiana tabacumprotoplasts and X-irradiated N. debneyi protoplasts were analyzed.Digestion of cpDNA isolated from ten male sterile lines withfour restriction endonucleases (EcoRI, XhoI, SmaI and HindIII)indicated that these lines possessed either one or the otherparental chloroplast genome. Neither mixture of two types ofcpDNA nor unique restriction fragments were detected in anyof the cases examined. The genetic constitution of chloroplastgenomes identified by restriction analysis of cpDNA showed goodagreement with that based on isoelectric focusing of the largesubunit of the Fraction I protein. The mtDNA from five fusion-derivedmale sterile plants showed banding patterns quite differentfrom each other and from the parental plants. Each plant exhibitednew restriction fragments not found in the parental species.These findings indicate that recombinational events in the mitochondrialgenomes take place rather frequently in the mixed cytoplasmsafter protoplast fusion, whereas the mixed chloroplasts becomesegregated to homogeneity. (Received June 19, 1987; Accepted October 5, 1987)