Identification of the structural gene and nonsense alleles for adenylate cyclase in Saccharomyces cerevisiae.

Tetraploid strains of Saccharomyces cerevisiae carrying different dosages of the CYR1+ gene have been constructed. Adenylate cyclase activity observed in these tetraploid strains was proportional to the dosage of the active CYR1+ gene. Of the 57 mutants requiring adenosine 3',5'-monophosphate for growth at 35 degrees C, two allelic temperature-sensitive cyr1 mutants produced thermolabile adenylate cyclase. Crude extract and plasma membrane fraction of cyr1 mutant cells had no adenylate cyclase activity when assayed with GTP or 5'-guanylyl imidodiphosphate in the presence of Mn2+ or Mg2+. Plasma membrane and Lubrol-soluble plasma membrane fractions obtained from the temperature-sensitive cyr1 mutant were thermolabile compared with those from the wild-type strain. Three cyr1 mutants carried nonsense mutations susceptible to ochre (UAA) suppressors, SUP3 and SUP-o, and had no detectable level of adenylate cyclase activity. It is concluded that the cyr1 mutants carry lesions in the structural gene for adenylate cyclase.     

Deep Supercooling in Most Tissues of Wintering Sasa senanensis and Its Mechanism in Leaf Blade Tissues

Cold hardiness of leaf blades, leaf sheaths, culms, rhizomes, and leaf buds in wintering Sasa senanensis (Fr. et Sav.) Rehder, a dwarf bamboo, was studied paying special attention to the types of resistance mechanisms which were determined with differential thermal analysis. Coincidence of LT₂₅ (lethal temperature at which 25% of the tissues are injured) with the initiation temperature of LTE (low temperature exotherm) suggested that all of these tissues described above owe their cold hardiness mechanism mostly to deep supercooling. Deep supercooling in leaf blades was also substantiated with microscopic observations, suggesting that the units of supercooling were minute tissues compartmentalized by longitudinal and cross veins. It was also shown that cooling rates and storage of shoots at −5°C for 1 to 5 days in the ice-inoculated state did not greatly affect the supercooling ability of leaf blades. Sasa senanensis seemed to exhibit a unique strategy against prolonged subzero temperature, and its leaves would be a good system for the study on mechanisms of deep undercooling in plants.     

Presence of functional receptors for atrial natriuretic peptide in human pheochromocytoma

Pheochromocytoma, a catecholamine-secreting adrenomedullary tumor, has been shown to contain the functional receptor for human atrial natriuretic peptide(h-ANP). Release of catecholamines from tissue slices of pheochromocytoma was inhibited by h-ANP in a dose-dependent manner. Binding assays using 125I-ANP revealed a single class of high affinity binding sites for ANP. When covalently tagged with 125I-ANP and electrophoresed under non-reducing and reducing conditions, the receptor migrated as a 140-kDa band and a 70-kDa band, respectively, reflecting its disulfide-linked subunit structure. The presence of ANP receptor in pheochromocytoma was further demonstrated by immunohistochemistry; the tumor was positively stained with an antireceptor antiserum. The antiserum was also useful to establish the zona glomerulosa localization of ANP receptor in the normal human adrenal gland.     

Rat ribosomal protein L35a multigene family: molecular structure and characterization of three L35a-related pseudogenes

The rat ribosomal protein L35a gene comprises a multigene family which contains 15-20 members as shown by the Southern blot analysis using L35a cDNA as a probe. We isolated 15 independent clones which contained distinct genes from a rat genomic library. Analysis of the restriction sites showed that all of them lacked the intervening sequences. Thermal stability of the hybrid molecules between these genes and the cDNA indicated that the similarity of the genes to the cDNA sequence varied. The nucleotide sequences of three genes gRL35a-A, gRL35a-B and gRL35a-G were determined. They shared some characteristics; namely: they lacked the intervening sequences, they contained (A)-rich tracts, and they were flanked by direct repeats. Two genes, gRL35a-A and gRL35a-B, contained a sequence completely identical to that of the cDNA. The nucleotide sequence of the 5' flanking region of gRL35a-B showed a significant homology with that of the same region of mouse ribosomal protein L32-related unmutated processed genes. Although this region of gRL35a-B contained the sequences homologous to the TATA box and the CCAAT box, gRL35a-B was not transcribed in an in vitro assay system. Thus, the L35a gene family comprises mostly processed pseudogenes. Further, Southern blot analysis in various animals indicated that the multigene construction of this ribosomal protein gene was a feature of mammalian genes. The origin and the evolutionary aspect of processed pseudogenes are discussed.     

The chromosomal normality of in vitro-fertilized rabbit oocytes

The chromosomal normality of rabbit oocytes fertilized in vitro was examined. Ovum donors were superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Follicular oocytes were collected laparoscopically 9-10 h after hCG treatment and incubated in vitro with spermatozoa capacitated in vivo. Of 267 aspirated oocytes, 191 (71.4%) were fertilized in vitro and developed to the 2- to 8-cell stage 24-48 h after insemination. In the chromosomal studies, 121 (63.4%) were examined. Of these, 94 (77.7%) had a normal diploid complement of chromosomes (2n = 44) and 6 (5%) showed aneuploidy. Of the remaining 21, 20 were triploid and one was tetraploid. The incidence of triploid oocytes after in vitro fertilization was higher than the rate in vivo (16.5% vs. 9.0%, respectively). These triploid oocytes were suspected to be the result of polyspermic fertilization in vitro. In addition, at the Metaphase II stage, 62 (89.9%) of 69 induced, preovulatory oocytes had a normal number of chromosomes.     

Changes in the chemical composition of carbohydrates and proteins in surface water during a bloom ofMicrocystis in Lake Suwa

Carbohydrates and proteins in surface water during a bloom ofMictrocystis, which is the dominant summer phytoplankton in Lake Suwa, were analyzed in order to evaluate the function ofMicrocystis in organic matter metabolism. Glucose was the predominant sugar constituent of the cellular carbohydrate fraction and decreased in quantity from inside towards the outside of the cell through the slime layer. Other constituent sugars, on the other hand, were present in larger proportions in the lake water. Although the sugar composition of the cells did not change in July and August, during the first period of theMicrocystis bloom, it changed appreciably in September when the water temperature decreased below 20°C accompanied by the decrease in solar radiation and a marked change in nutrient concentration. It appears that the sugar composition of the cells may change in response to some environmental stresses. In addition, a temporal change in the sugar composition was found, particularly in the fraction containing the slime extracted by shaking. Among the constituent amino acids of the cells, the percentage of arginine, aspartic acid and leucine decreased from inside toward the outside of the cell, while glutamic acid, threonine, serine and glycine showed an opposite trend. In contrast to the carbohydrates, the percentage composition of each amino acid varied little throughout the period of the bloom.     

Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon

Multiple isoforms of tropomyosin (TM) of rat cultured cells show differential effects on actin-severing activity of gelsolin. Flow birefringence measurements have revealed that tropomyosin isoforms with high Mr values (high Mr TMs) partially protect actin filaments from fragmentation by gelsolin, while tropomyosins with low Mr values (low Mr TMs) have no significant protection even when the actin filaments have been fully saturated with low Mr TMs. We have also examined effect of nonmuscle caldesmon on the severing activity of gelsolin because 83-kDa nonmuscle caldesmon stimulates actin binding of rat cell TMs (Yamashiro-Matsumura, S., and Matsumura, F. (1988) J. Cell Biol. 106, 1973-1983). While nonmuscle caldesmon alone or low Mr TMs alone show no significant protection against fragmentation by gelsolin, the low Mr TMs coupled with 83-kDa protein are able to protect actin filaments. Further, high Mr TMs together with 83-kDa protein appear to block the severing activity completely. Electron microscopic analyses of length distribution of actin filaments have confirmed the results. The average length of control actin filaments is measured as 1.46 +/- microns, and gelsolin shortens the average length to 0.084 +/- 0.039 micron. Similar short average lengths are obtained when gelsolin severs actin complexed with low Mr TMs (0.080 +/- 0.045 micron) or with nonmuscle caldesmon (0.11 +/- 0.072 micron) while longer average length (0.22 +/- 0.18 micron) is measured in the presence of high Mr TMs. The simultaneous addition of nonmuscle caldesmon makes the average length considerably longer, i.e. 0.61 +/- 0.37 micron in the presence of low Mr TMs and 1.57 +/- 0.97 micron in the presence of high Mr TMs. Furthermore, the actin binding of gelsolin is strongly inhibited by co-addition of high Mr TMs and nonmuscle caldesmon. These results suggest that TM and gelsolin share the same binding site on actin molecules and that the differences in the actin affinities between TMs are related to their abilities of protection against gelsolin.     

Increment in the Ta1+ cells in the peripheral blood and thyroid tissue of patients with Graves' disease

The present study utilized the anti-Ta1 mAb to characterize the cell surface phenotypes of peripheral blood and intrathyroidal lymphocytes in patients with Graves' disease. We found an increase in PBL bearing the Ta1 Ag in untreated patients. The euthyroid patients in remission, induced by antithyroidal drugs, radioisotope therapy, and subtotal thyroidectomy, had lower percentages of Ta1+ cells than did untreated patients. An increased percentage of Ta1+ cells in untreated patients was found in both CD4+ cells and CD8+ cells. The ratio of CD4+Ta1+ cells to CD8+Ta1+ cells in untreated patients was significantly higher than that of normal subjects. There was a positive correlation between the percentage of Ta1+ cells and the level of anti-TSH receptor antibody. In this prospective study, the proportion of Ta1+ cells was decreased in parallel with the reduction in anti-TSH receptor antibody and free T3 levels. In the chronically treated patients, the proportion of Ta1+ cells in the thyroid tissue was, yet similar to that in the peripheral blood, markedly increased in comparison to that of normal subjects. In contrast to Ta1+ cells, the thyroid tissue had a significantly higher percentage of HLA-DR+ T cells than did the paired peripheral blood. The proliferative responses of the Ta1+ cell-enriched population isolated from untreated patients toward thyroglobulin and microsomal Ag were markedly higher than those in a Ta1+ cell-depleted population, but both populations were able to respond equally to PHA. These results suggest that the Ta1+ cells may include Ag-triggered memory cells that are reactive with thyroid-specific Ag. Furthermore, monitoring such cells may provide an objective measure of abnormal immunologic activity.     

Isolation of a cDNA encoding the rat liver S-adenosylmethionine synthetase

We have isolated cDNA clones encoding the rat liver S-adenosylmethionine synthetase by means of immunological screening from a phage lambda gt 11 expression library containing cDNA synthesized from adult rat liver poly(A)-RNA. The amino acid sequence deduced from the cDNA indicates that the rat liver enzyme for this protein contains 397 amino acid residues and has a molecular mass of 43697 Da. The deduced amino acid sequence of rat liver S-adenosylmethionine synthetase was 68% similar to those of yeast S-adenosylmethionine synthetases encoded by two unlinked genes SAM1 and SAM2. The rat liver S-adenosylmethionine synthetase also shows 52% similarity with the deduced amino acid sequence of the MetK gene encoding the S-adenosylmethionine synthetase in Escherichia coli.