Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins.

We have constructed and analyzed an in vitro system that will efficiently replicate plasmid RSF1010 and its derivatives. The system contains a partially purified extract from E.coli cells and three purified RSF1010-encoded proteins, the products of genes repA, repB (or mobA/repB), and repC. Replication in this system mimics the in vivo mechanism in that it (i) is initiated at oriV, the origin of vegetative DNA replication, (ii) proceeds in a population of plasmid molecules in both directions from this 396-base-pair origin region, and (iii) is absolutely dependent on the presence of each of the three rep gene products. In addition, we find that E.coli DNA gyrase, DnaZ protein (gamma subunit of poIIII holoenzyme) and SSB are required for in vitro plasmid synthesis. The bacterial RNA polymerase, the initiation protein DnaA, and the primosomal proteins DnaB, DnaC, DnaG and DnaT are not required. Furthermore, the replicative intermediates seen in the electron microscope suggest that replication in vitro begins with the simultaneous or non-simultaneous formation of two displacement loops that expand for a short stretch of DNA toward each other, and form a theta-type structure when the two displacing strands pass each other.     

Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

Linkage data suggesting allelic heterogeneity for paramyotonia congenita and hyperkalemic periodic paralysis on chromosome 17

Summary Paramyotonia congenita (PC), an autosomal dominant non-progressive muscle disorder, is characterised by cold-induced stiffness followed by muscle weakness. The weakness is caused by a dysfunction of the sodium channel in muscle fibre. Parts of the gene coding for the -subunit of the sodium channel of the adult human skeletal muscle (SCN4A) have been localised on chromosome 17. To investigate the role of this gene in the etiology of PC, a linkage analysis in 17 well-defined families was carried out. The results (z=20.61, =0.001) show that the mutant gene responsible for the disorder is indeed tightly linked to the SCN4A gene. The mutation causing hyperkalemic periodic paralysis (HyperPP) with myotonia has previously been mapped to this gene locus by the same candidate gene approach. Thus, our data suggest that PC and HyperPP are caused by allelic mutations at a single locus on chromosome 17.Dedicated to Professor P. E. Becker on the occasion of his 83rd birthday.     

The use of a resin adsorbent to isolate cytokinins from sea water

XAD-4 resin was used to isolate cytokinins present in Baltic sea water. Four compounds were tentatively identified as 6-(3-methylbut-2-enylamino) purine and zeatin, and their ribosides.     

Behaviour of drifting insect larvae

The larval drift behaviour of 23 species representing Ephemeroptera, Plecoptera and Trichoptera was investigated in the laboratory using different current regimes. Mayfly nymphs often performed swimming, while caddis larvae were reluctant to do so. Stonefly nymphs were intermediate. In mayflies swimming seemed to be used to reach the substrate as soon as possible. In contrast most stonefly nymphs by swimming prolonged the time spent in the water column. Modes of swimming and sinking posture differed markedly between the orders. Living passively sinking animals often reached bottom faster than dead control specimens, so consequently behaviour did not always express itself in activity. Some caddis larvae spun adherent anchor lines. Differences among taxa seemed more important in explaining swimming activity compared to preferred habitats (as stream, river and lake) in each species. However, observed differences among closely related species indicated subtle differences related to microhabitat to be of profound importance in explaining the alternative behavioural strategies used.     

The Molecular through Ecological Genetics of Abnormal Abdomen in DROSOPHILA MERCATORUM. I. Basic Genetics

The abnormal abdomen syndrome (aa) in Drosophila mercatorum is characterized by the persistence of juvenilized cuticle on the adult abdomen. The aa phenotype is shown to depend on at least two X-linked genetic elements that are about one map unit apart near the centromeric end of the X chromosome. These two genetic elements are necessary for aa expression; one behaves as a dominant element and the other as a recessive. Overlaying these genetic studies upon molecular work reported elsewhere, it is argued that the dominant element is the presence of a 5 kb insertion in a majority of the X-linked repeats coding for the 28S ribosomal RNA. The recessive element appears to be a locus controlling differential replication of noninserted over inserted 28S genes during polytenization. The aa syndrome requires both the presence of the inserted repeats and the failure to preferentially amplify noninserted repeats. Given the necessary X-linked elements for aa, a variety of modifiers are revealed. First, aa expression in males is Y-linked, apparently corresponding to a deletion of the 18S/28S rDNA gene cluster normally found on the Y. Moreover, all major autosomes can modify the penetrance of aa.     

Prolactin response to sulpiride in non insulin dependent diabetes mellitus

Blood glucose, insulin and prolactin concentrations were determined before and after sulpiride injection (50 mg i.m.) in 20 non-insulin-dependent diabetic patients (10 with retinopathy and 10 without evidence of retinal damage) and 10 subjects with normal glucose tolerance. Prolactin response to sulpiride was significantly higher in diabetics than in controls (at 20 min., p less than 0.01; at 30 and 60 min., p less than 0.005; at 90 min., p less than 0.01; at 120 min., p less than 0.05). The sulpiride induced hyperprolactinemia did not influence blood glucose and plasma insulin levels in controls as well as in diabetic patients. Prolactin response to sulpiride was the same in diabetics with and in those without retinal changes. We conclude that acute hyperprolactinemia seems to have no influence on glucose homeostasis in normal and non insulin-dependent diabetic subjects.     

Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomyces pombe

The subcellular localization of the enzyme invertase in Schizosaccharomyces pombe cells, both repressed and derepressed for synthesis of the enzyme, was studied. Most of the invertase was found to be located outside the plasma membrane and only a small percentage was found to be associated to membranes. A substantial portion of the external enzyme remained firmly bound to cell-wall material.All of the invertase recovered in soluble form from cellular extracts reacted with concanavalin A and with the lectin from Bandeiraea simplicifolia seeds, indicating the presence in the enzyme of a carbohydrate moiety which probably contains terminal mannosyl (or structurally related) and galactosyl residues.The possibility of the presence of two different forms of invertase in S. pombe was considered. An intracellular, soluble form of invertase, devoid of carbohydrate, similar to the small invertase of the budding yeast Saccharomyces cerevisiae, was not found in S. pombe. However, the Michaelis constant for sucrose of the enzyme present in repressed cells was smaller than that of the invertase synthesized under derepressing conditions, although this difference could also be the result of a different pattern of glycosylation of the invertase synthesized under different growth conditions.