Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.     

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.     

Initial steps of phloroglucinol metabolism in Penicillium simplicissimum

The pathway for the aerobic catabolism of 1,3,5-trihydroxybenzene (phloroglucinol) by a new strain of Penicillium was investigated using both in vivo and in vitro cell-free systems. The fungal strain was isolated by enrichment on phloroglucinol and identified as P. simplicissimum (Oud) Thom. It grew optimally at pH 5.5 and 27°C with 119 mM (1.5%w/v) of phloroglucinol in a basal mineral salts medium. Vapours of the crystalline substrate placed in a Petri-plate lid supported the growth of the fungal colonies on the agar surface. Mycelia grown on phloroglucinol accumulated 1,2,4-trihydroxybenzene and resorcinol in the medium. Washed, resting mycelia grown on phloroglucinol, when resuspended in a buffer utilized oxygen in the presence of catechol, resorcinol, pyrogallol and phloroglucinol. A NADPH-dependent reductase in the cell-free extract reduced phloroglucinol to dihydrophloroglucinol. This electron donor could not be replaced by NADH. Resorcinol hydroxylase, phloroglucinol reductase, catechol-1,2-oxygenase, and catechol-2,3-oxygenase were detected in cell-free extracts of mycelia grown on phloroglucinol. The possible steps in the degradation of phloroglucinol are discussed.     

Cell kinetic disturbances induced by treatment of human diploid fibroblasts with 5-azacytidine indicate a major role for DNA methylation in the regulation of the chromosome cycle

Summary BrdU-Hoechst flow cytometry was used to investigate the effects of DNA hypomethylation, induced by treatment with 5-azacytidine (5AC), on cell proliferation. When human fibroblast-like cells derived from skin and amniotic fluid were exposed to 5AC during three successive cell cycles, their clone-forming ability was diminished after removal of the drug. Treated cells were rendered quiescent by culture with low serum in the absence of the drug. Upon serum stimulation, they showed a diminished fraction of proliferating cells, which exhibited a prolonged transit through the S and G2 phase of the cell cycle, and a permanent arrest within the G2 compartment. This pattern of disturbed cell proliferation may in part explain the changes in replication banding pattern reported in the literature. Cytogenetic analysis of 5AC-treated cells revealed numerous endomitoses and tetraploid metaphases indicating a disturbed chromosome cycle in association with these cell kinetic perturbations.     

Increased frequency of 6-thioguanine-resistant peripheral blood lymphocytes in Werner syndrome patients

Summary The frequency of spontaneous 6-thioguanine (TG)-resistant peripheral blood lymphocytes in five unrelated Werner syndrome (WS) patients was determined using an autoradiographic labeling assay. The average frequency of TG-resistant lymphocytes was eightfold higher in WS patients than in sex- and age-matched normal control donors. This finding and previous identification of increased spontaneous chromosomal rearrangements and deletions in WS cells or cell lines suggest that WS is a human genomic instability or mutator syndrome.     

Cytogenetic analysis of 400 sperm from three translocation heterozygotes

Summary Sperm chromosome complements were studied in three men who carried reciprocal translocations. A total of 400 sperm were karyotyped after in vitro penetration of hamster eggs: 217 sperm from t(2;9) (q21;p22), 164 from t(4;6) (q28;p23) and 19 from t(7;14) (q21;q13). All possible 22 and 31 meiotic segregations were observed for t(2;9) and t(4;6); for t(7;14) only 22 segregations were observed. For alternate segregations, the number of normal sperm was not significantly different from the number of sperm carrying a balanced form of the translocation in any of the translocations, as theoretically expected. The percentage of sperm with an unbalanced form of the translocation was 57% for t(2;9), 54% for t(4;6) and 47% for t(7;14). There was no evidence for an interchromosomal effect in any of the translocations since the frequencies of numerical abnormalities (unrelated to the translocation) were within the normal range of control donors. The frequencies of X- and Y-bearing sperm did not differ significantly from 50%. Results from a total of 17 reciprocal translocations studied by sperm chromosomal analysis were reviewed.     

Opacity factor from group A streptococci is an apoproteinase

Opacity factor (OF) is an enzyme, elaborated by certain serotypes of group A streptococci, which produces opalescence in mammalian sera. OF has been designated a lipoproteinase. Lipoproteins are complex structures and many enzymes are involved in their catalysis. We therefore set out to establish which of the many enzymes OF could be. Results showed that OF rendered high density lipoprotein (HDL) insoluble, accounting for the opalescence in serum, and altered its electrophoretic mobility. Electron microscopy revealed that OF caused an aggregation of HDL and an alteration in molecule shape. OF specifically split apoprotein AI of HDL into two fragments demonstrable by SDS-PAGE. We therefore designate OF as an apoproteinase.     

Indole-3-acetic acid content and glutamine synthetase activity in the pericarp, and peroxidase activity and isoenzymes in the meso- and exocarp of growing peach fruits

The indole-3-acetic acid (IAA) content in peach pericarp (Prunus persica L. Batsch cv. Merry) was highest at early stage I of development (200 ng/g fresh wt), decreased to the lowest level during stage II, and rose again at stage III to 60–70 ng/g fresh wt. High activity of glutamine synthetase was found in the pericarp during stage I. The soluble peroxidase activity was highest in the meso- and exocarp at stage II, and isoenzymatic changes in this fraction corresponded to the transition from cationic isoenzymes, predominant at stage I, to anionic isoenzymes at stage III. The ionically bound peroxidase activity in these tissues was highest at stage I. The three developmental stages showed marked differences in auxin content and enzyme activities; for peroxidases these changes reflect a developmental expression pattern for the isoenzymes.     

The control of megakaryocyte ploidy and platelet production: biology and pathology

Following experimental platelet destruction in animals, large platelets, which are more hemostatically active, are produced before any change in bone marrow megakaryocyte DNA content. When platelet production is stimulated by administration of i.v. vincristine in rats, megakaryocyte ploidy is increased, but mean platelet volume is unchanged. When platelet production and destruction are both stimulated by chronic hypoxia or administration of anti-platelet serum, mean platelet volume and megakaryocyte DNA content are both increased. Since platelet volume is determined primarily at thrombopoiesis, these results imply that mean platelet volume and megakaryocyte DNA content are under separate hormonal control. Therefore, it has been postulated that changes in mean platelet volume occur following changes in platelet production rate, whereas changes in megakaryocyte ploidy are associated with an increased rate of platelet production. In myocardial infarction, platelets have increased mean volume and reduced bleeding time more than in controls. In addition, men with myocardial infarction have increased megakaryocyte size and increased DNA content when compared to controls. These changes are similar to those observed in rabbits following cholesterol feeding. If megakaryocyte polyploidy and mean platelet volume are under separate hormonal control, this suggests that in myocardial infarction, both hormones are active--one stimulating an increased platelet size, the other stimulating the increased megakaryocyte DNA content. In contrast, patients with lymphoma exhibiting a secondary thrombocytosis have no change in mean platelet volume. However, these subjects also have larger bone marrow megakaryocytes when compared to controls. The relation between megakaryocyte size and ploidy implies that the DNA content of these cells is increased in lymphoma.(ABSTRACT TRUNCATED AT 250 WORDS)