Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca²⁺-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca²⁺ inhibited, while ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca²⁺-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.     

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.     

Comparison of the effect of temperature on kidney cortex mitochondria from rabbit,dog, pig,and human: Arrhenius plots of ADP-stimulated respiration

The effect of temperature on the rate of ADP-stimulated respiration of mitochondria from dog, rabbit, pig, and human kidney cortex mitochondria was plotted according to the Arrhenius relationship. The temperature at which the plot demonstrated a break was at 15 °C for mitochondria from dog, pig, and human kidneys. The discontinuity occurred at 10 °C or less for mitochondria from rabbit kidneys. This difference suggests that mitochondria from rabbit kidneys undergo a lipid-phase transition at lower temperatures than for other species commonly used in experimental renal preservation. The implications of this difference suggest caution in using results obtained with rabbit kidneys for comparison to results obtained from hypothermic renal preservation of other species kidneys. Apparent fluidization of dog kidney mitochondrial membranes with adamantine abolished the discontinuity in the Arrhenius plot.     

Identification of a gene required for maturation of an extracellular lactococcal serine proteinase.

Directly upstream of the Lactococcus lactis subsp. cremoris Wg2 proteinase gene is an oppositely directed open reading frame (ORF1). The complete nucleotide sequence of ORF1, encoding a 33-kilodalton protein, was determined. A protein of approximately 32 kilodaltons was synthesized when ORF1 was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter. L. lactis subsp. lactis MG1363 transformants carrying the proteinase gene but lacking ORF1 were phenotypically proteinase deficient, unlike transformants carrying both the proteinase gene and ORF1. Synthesis and secretion of proteinase antigen by L. lactis could be detected with proteinase-directed monoclonal antibodies regardless of whether ORF1 was present. The requirement of ORF1 for proteinase activation was reflected in a reduction in the molecular weight of the secreted proteinase. Furthermore, deletion of the 130 C-terminal amino acids of the Wg2 proteinase prevented attachment of the enzyme to lactococcal cells.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Correlation between rainfall in the dry season and the occurrence of the white rice borer (Scirpophaga innotata Wlk.) in Java

The amount of rain which falls sporadically in the relatively dry season at the time that the white rice borer is diapausing as a full grown larva in the rice stubble, has a very important influence on the size of population of the borers in the following wet season when rice is grown. If this dry season is wet or very wet (according to our standard, based on the figures of rainfall and borer damage over 26 years in five regions of Eastern Java), no damage can be expected in the following planting season. If the dry season is really dry or very dry, outbreaks of borers may occur (in the period under survey this occurred in nine out of fourteen years). We have tried to give an outline of a method of prediction of borer damage in the planting season, based on the rainfall dates of the foregoing dry season.

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Zusammenfassung Der Weisse Reisbohrer (Scirpophaga innotata Wlk.) kommt nur in Gebieten vor, wo jährlich eine ausgeprägte Regen- und Trockenzeit (Monsun) auftritt. Am Ende der Regenzeit gehen die vollerwachsenen Raupen, die im Stengel der reifenden Reispflanze leben, in Diapause. Während der folgenden relativ trockenen Jahreszeit, wenn nur gelegentlich Regenschauer fallen, ruhen die Raupen in der Stoppel. Bald nachdem die ersten heftigen Regenfälle der neuen Regenzeit einsetzen, verpuppen sich die Larven und alle Falter schlüpfen gemeinsam zum Stoppelflug.Dieser Zünsler ist in gewissen Gebieten Javas einer der Hauptschädlinge des Reises, obwohl sein Auftreten sehr unregelmäßig ist. Jahrelang war man allgemein der Auffassung, daß relativ reichlicher Regenfall während der Trockenzeit die Reisbohrerpopulation erheblich vermindere und daß in diesem Falle keine schweren Schäden durch Reisbohrerbefall erwartet werden können.Während eines Zeitraums von 26 Jahren (1915–1940) wurden in fünf Gebieten Ost-Javas sowolhl der Niederschlag wie der Bohrerschaden untersucht und Berechnungen der jährlichen Niederschlagsmenge in der Trockenzeit und des Bohrerschadens verglichen. Dies erhärtete die alte Auffassung, daß nach einer relativ feuchten oder sehr feuchten Trockenzeit kein Bohrerbefall von Bedeutung während des Beobachtungszeitraumes auftritt. Dagegen folgten in neun von 14 Jahren mit einer trockenen oder sehr trockenen Trockenzeit schwere oder ziemlich schwere Ausbrüche der Weißen Reisstengelbohrer während der folgenden feuchten Jahreszeiten. Obwohl auch andere Faktoren von einiger Bedeutung sein dürften, wird geschlossen, daß die Niederschlagsmenge während der Trockenzeit einen sehr entscheidenden Einfluß auf die Möglichkeit des Auftrétens einer Reisbohrer Kalamität im darauffolgenden Jahre hat. Diesen Daten entsprechend wird auf der Grundlage der Niederschlagszahlen in der vorangegangenen Trockenzeit ein Prognose-Schema entwickelt, wann Bohrerschaden zu erwarten ist.

     

Proposal for adoption of the base of the Undulograptus austrodentatus Biozone as a global Ordovician stage and series boundary level

The base of the Undulograptus austrodentatus Biozone appears to be a synchronous event that is widely recognizable within graptolitic facies around the world. It occurs within an interval in which graptolite species ranges are now well known and in which there is a rapid turnover in the composition of graptolite faunas. This turnover reflects the rapid evolutionary radiation of the Diplograptacea simultaneously with the appearance of several distinctive pseudisograptid and glossograptid species. These events provide the basis for the recognition of two thin but widely applicable subzones; a lower Arienigraptus zhejiangensis Subzone and an upper U. sinicus Subzone. The occurrence of the lower boundary of the U. austrodentatus Biozone within a succession of first appearances also permits accurate and reliable identification of the boundary as well as assessment of stratigraphic completeness across the boundary interval in correlated sections. Diverse graptolite faunas of late Yapeenian and early Darriwilian age occur in association with the Histiodella altifrons Biozone of the North American midcontinent conodont zonation and the Paroistodus originalis and Microzarkodina parva biozones of the North Atlantic conodont zonation. They also occur in association with the shelly-fossil zonations developed for several different continents. These features of the base of the U. austrodentatus Biozone make it a suitable level for use as the boundary level for a global stage. Its stratigraphic position within the Ordovician System relative to other likely global stages as well as its coincidence with one of the major events in graptolite evolutionary history suggest that this level also may be a suitable level for the base of a global Middle Ordovician Series.Ordovician System, Ordovician stages, graptolite zonation, chronostratigraphy, international correlation. Charles E. Mitchell and Jörg Maletz, Department of Geology, State University of New York at Buffalo, Buffalo, New York 14260-1550, USA; 13th July, 1994; revised 22nd May, 1995.     

Use of a synthetic DNA oligonucleotide to probe the precision of RNA splicing in a yeast mitochondrial petite mutant.

In some strains of Saccharomyces cerevisiae the mitochondrial gene coding for 21S rRNA is interrupted by an intron of 1143 bp. This intron contains a reading frame for 235 amino acids: Unassigned Reading Frame (URF). In order to check whether expression of this URF is required for proper splicing of precursors to 21S rRNA, the precision of RNA splicing was analysed in a petite mutant, where no mitochondrial protein synthesis is possible anymore. We have devised a new assay to monitor the precision of the splicing event. The method is of general application, provided that the sequence of the splice boundaries is known. In the case of the 21S rRNA it involves the synthesis of the DNA oligonucleotide d(CGATCCCTATTGTC( complementary to the 5' d(CGATCCCTAT) and 3' d(TGTC) borders flanking the intron in the 21S rRNA gene. The oligonucleotide is labelled with 32p at the 5'-end, hybridised to RNA and subsequently subjected to digestion with S1 nuclease. Resistance to digestion will only be observed if the correct splice-junction is made. The petite mutant we have studied contains a 21S rRNA with the same migration behaviour as wildtype 21S rRNA. In RNA blotting experiments, using an intron specific hybridisation probe, the same intermediates in splicing are found both in wild type and petite mutant. Finally the synthetic oligonucleotide hybridises to petite 21S rRNA and its thermal dissociation behaviour is indistinguishable from a hybrid formed with wildtype 21S rRNA. We conclude that expression of the URF, present in the intron of the 21S rRNA gene, is not required for processing and correct splicing of 21S ribosomal precursor RNA.