Characterization of Six Bacteriophages of Serratia liquefaciens CP6 Isolated from the Sugar Beet Phytosphere

Six phages (ΦCP6-1 to ΦCP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 (ΦCP6-1 and ΦCP6-4). Like ΦCP6-2 and ΦCP6-5, ΦCP6-1 belonged to the family Siphoviridae, while ΦCP6-4 exhibited the morphology of the family Podoviridae. The other phages were members of the family Myoviridae. DNA-DNA cross-hybridization revealed that ΦCP6-1 and ΦCP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like ΦCP6-2, ΦCP6-3, and ΦCP6-5, ΦCP6-1 was capable of forming a lysogenic association with its host, while ΦCP6-4 and ΦCP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that ΦCP6-4 had a much shorter latent period and a smaller burst size than ΦCP6-1. Also, ΦCP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 × 10⁻⁹ to 3.9 × 10⁻⁷, whereas ΦCP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages. Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages.     

Glutamate Oxidation by Soybean Cotyledon and Leaf Mitochondria

Mitochondria purified from cotyledons of soybean seedlings fiveto ten days old have the capacity to rapidly oxidize glutamate(measured as glutamate dependent oxygen consumption). This capacitywas greatest at ten days after planting but was very low priorto emergence of cotyledons from the vermiculite and during senescence.Solubilized glutamate dehydrogenase activity, on the other hand,was substantial at two days after planting, peaked at sevendays, then declined and rose again during senescence. It issuggested that mitochondrial glutamate oxidation plays a rolein reserve mobilization and amino acid metabolism during seedlinggrowth. Leaf mitochondria and those from senescing cotyledonscould not sustain rapid rates of glutamate oxidation despiteready oxidation of other substrates and high solubilized glutamatedehydrogenase activity, suggesting an alternative role for theenzyme in these tissues. Possible controlling factors are discussed. ² Present address, Garvan Institute, Darlinghurst, N. S. W.,Australia. ³ Permanent address, Department de Biologia Vegetal, Facultatde Biologia, Universitat de Barcelona, Barcelona, Spain. (Received May 6, 1988; Accepted August 3, 1988)     

Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development

Actin is one of the major proteins in mammalian oocytes. Most developmental events are dependent on the normal distribution of filamentous (F-) actin. Polymerization of nonfilamentous (G-) actin into F-actin is important for both meiosis and mitosis. This study examined G- and F-actin distribution in pig oocytes and embryos by immunocytochemical staining and confocal microscopy. Actin protein was quantified by electrophoresis and immunoblotting. G-Actin was distributed in the whole cytoplasm of oocytes and embryos irrespective of their stages. F-Actin was distributed at the cortex of oocytes and embryos at all stages, at the joint of blastomeres in the embryos, in the cytoplasm around the germinal vesicle (GV), and in the perinuclear area of 2- to 4-cell-stage embryos. No differences in the amount of actin protein were found among oocytes and embryos. Oocytes cultured in medium with cytochalasin D (CD), an inhibitor of microfilament polymerization, underwent GV breakdown and reached metaphase I but did not proceed to metaphase II. Two- to 4-cell-stage embryos cultured in medium with CD did not develop to blastocysts. When GV-stage oocytes or 2- to 4-cell-stage embryos treated with CD for 6 h were re-cultured in media without CD, oocytes or embryos re-assembled actin filaments and underwent a meiotic maturation or blastocyst formation similar to that of controls. These results indicate that it is the polymerization of G-actin into F-actin, not actin protein synthesis, that is important for both meiosis and mitosis in pig oocytes and embryos.     

Intraspecific variation in the lens proteins

A review of the literature indicates that intraspecific variation among the lens proteins (crystallins) is almost invariably quantitative rather than qualitative. Lens extracts were examined from inbred strains of the laboratory mouse, rat, and guinea pig and from domestic breeds of sheep. Differences in the mobilities of protein bands after electrophoresis in polyacrylamide gels were never found to be greater than the variation observed when a sample was repeatedly subjected to electrophoresis. Differences, however, were observed between widely separated populations of the bank vole (Clethrionomys glareolus). In bank voles and mice, there were variations in the intensities of bands. Electrophoresis of extracts in the presence of 6 m urea indicated that there were no major differences between six mouse strains in the subunits constituting their - and -crystallins. More detailed examination of -crystallins from two mouse strains by isoelectric focusing and amino acid analysis supported this conclusion.This work was carried out while one of the authors (T.H.D.) was supported by a Medical Research Council Research Studentship and a European Molecular Biology Organisation Fellowship. A grant from the Edinburgh University Weir Memorial Fund allowed the collection of some of the Yugoslavian bank voles.     

Induction of alternative oxidase synthesis by herbicides inhibiting branched-chain amino acid synthesis

Sycamore suspension cells ( Acer pseudoplatanus L.) were incubated in the presence of sulfonylurea and imidazolinone herbicides. These inhibitors of acetolactate synthase (ALS), a key enzyme of branched-chain amino acid synthesis, triggered a dramatic induction of the alternative oxidase (AOX). AOX activity increased in treated cells, eventually exceeding cytochrome (cyt) pathway activity. This induction of AOX activity was correlated with the accumulation of a 35 kDa AOX protein in isolated mitochondria, detected by Western blotting with a monoclonal antibody against Sauromatum guttatum AOX. It was preceded by the accumulation of putative 1.6 kb AOX mRNA, detected using an Aox cDNA probe from soybean. The metabolic perturbations induced by the herbicides rather than the herbicide molecules themselves were responsible for this induction of AOX. However, α-oxobutyrate (one of the substrates of ALS) and its transamination product, α-aminobutyrate, which accumulated after herbicide treatment, were not involved. The inhibition of branched-chain amino acid synthesis was probably somehow responsible for the AOX induction since: (i) a mixture of those amino acids (leucine, isoleucine, valine) prevented AOX induction by ALS inhibitors; (ii) the herbicide Hoe 704, a potent inhibitor of acetolactate reducto-isomerase (the enzyme following ALS in the branched-chain amino acid pathway), also triggered AOX induction.     

Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements

The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity. We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis. Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity). On the other hand, the aspartic acid mutant (ED144) retained in vivo activity. Substrate binding and catalytic studies were done with purified ED144 enzyme. The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage. Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive. The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex.     

The transition from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: quantitative and qualitative aspects of protein synthesis

A study was conducted to identify the embryonic stage when the zygotic genome begins to direct development and to characterize protein synthesis in pig oocytes and embryos. Reproductive tracts of gilts were flushed to obtain unfertilized oocytes (UFO), zygotes (Z), 2-, 4-, and 8-cell embryos, compact morulae (M), initial blastocysts (IB), blastocysts (B), and hatched blastocysts (HB). Pig eggs and embryos were cultured in medium containing 1 microM L-[35S]methionine and evaluated for amino acid uptake, incorporation of the radiolabel into protein, and qualitative changes in protein profiles specific to each cleavage stage. Unfertilized oocytes sequestered 65.7 fmol methionine/4 h/embryo. Uptake of methionine decreased (p less than 0.05) from the Z (49.4), 2-cell (41.8), and 4-cell (37.6) embryonic stages to the M (8.97 fmol/4 h/embryo) stage. This downward trend was reversed at the IB, B, and HB stages when uptake increased to 37.3, 50.3, and 84.2 fmol/4 h/embryo, respectively. Incorporation of methionine into protein followed a similar pattern, being relatively higher in the UFO (21.0), Z (20.5), and 2-cell stages (16.0); decreased (p less than 0.05) at the 4-cell (6.67), 8-cell (6.84), and M (6.16) stages; and increased (p less than 0.05) at the IB (28.0), B (41.5), and HB (69.6 fmol/4 h/embryo) stages. Differences in protein profiles were observed for UFO, Z, 4-cell, and M stages using lysates of single embryos, one-dimensional SDS-PAGE, and fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)