Construction of improved bacteriophage phi 105 vectors for cloning by transfection in Bacillus subtilis

A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage phi 105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated phi 105J23, phi 105J24, phi 105J27 and phi 105J28, show frequencies of plaque formation that are equal to those of wild-type phi 105. This represents at least a 10-fold improvement over phi 105J9, the vector used in previous cloning experiments. Two of the new vectors phi 105J27 and phi 105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.     

Exemplars and the reproduction of everyday life: The Amherst affair

Frederick Errington is Aaaociate Professor of Anthropology, at Keene State College, New Hampshire. Deborah Gewertz is Professor of Anthropology, at Amherst College, Massachusetts.     

Chicken lens delta-crystallin gene expression and methylation in several non-lens tissues.

RNA sequences coding for the most abundant chicken lens proteins, delta-crystallin, were detected at very low levels in day old post hatch chick lung, heart, kidney and liver, and in 6 day embryo headless bodies. The pattern of cytosine methylation within the CCGG sequences of the delta-crystallin genes was also examined and shown to vary in several non-lens tissues, from several stages of development. Embryonic neural retina, which expresses a higher level of delta-crystallin RNA than the above tissues, is no less methylated in the sites studied than the tissues which have no association with the eye, and is actually more heavily methylated than the kidney. Thus no obvious correlation was found between undermethylation and gene expression.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation.

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.