Distribution of pelagic larvae of benthic marine invertebrates in the Bellingshausen Sea

During November and December 1992, plankton samples were collected using a ring net of mesh size 200 m vertically hauled through a 600 m water column, at five stations along a transect running north from the Allison Peninsula in the Bellingshausen Sea. Three stations were located over the continental shelf; two of these were ice bound, whilst the third was at the ice edge. Two other stations were in deeper, ice-free water. Sixteen different larval and juvenile types were found representing seven phyla: Echinodermata, Nemertea, Coelenterata, Mollusca, Annelida, Arthropoda and Bryozoa, of which the first two were the most abundant. Larval numbers and types decreased with distance offshore and away from permanent sea ice. The presence of many stages of nemertean larval development within a short time scale, in an area where developmental tends to be slow, suggests that reproduction occurs over an extended period and that the larvae have a long planktonic phase. The increased size of later developmental stages of the nemertean larvae indicates they obtain nutrition within the water column during winter, when little particulate food is present.     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system.

Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.     

Temporal appearance and distribution of the Ca2+ + Mg2+ ATPase of the sarcoplasmic reticulum in developing chick myocardium as determined by immunofluorescence labeling

The temporal appearance and distribution of the Ca2+ + Mg2+ ATPase of the sarcoplasmic reticulum were determined in the developing chick heart (stage 9 to stage 16) by indirect immunofluorescence labeling. The results obtained showed that the Ca2+ + Mg2+ ATPase was first observed in the bulbus ventricular region of the single tubular heart at stage 9 to 10 of development, when these myocardial cells first contract. As the atrial and later the sinus venosus tissues became incorporated into the single tubular heart the Ca2+ + Mg2+ ATPase was also observed in these regions, however, the highest density of Ca2+ + Mg2+ ATPase labeling was generally observed in the region of the heart most recently incorporated. These results suggest that the sarcoplasmic reticulum is present and perhaps functional in the regulation of the cytoplasmic Ca2+ concentration and thereby the contraction-relaxation cycle in myocardial cells when the first contraction occurs, as well as throughout all subsequent stages of development. Furthermore comparison between the relative density and intensity of the Ca2+ + Mg2+ ATPase labeling and the intrinsic rate of contraction of the myocardial cells in the various regions of the heart (A. Barry, 1942, J. Exp. Zool. 91, 119-130) supports the possibility that a positive correlation exists between these two characteristics of the myocardial cells.     

The inverted repeats of Tn5 are functionally different

The inverted repeats of Tn5, which have identical restriction endonuclease cleavage patterns, have different functional properties. They differ with respect to RNA polymerase binding, full promotion of neomycin resistance, the polypeptides coded for by the repeats and their function in the transposition process. There is a week RNA polymerase binding site present in one repeat and not in the other which seems to be important for neomycin resistance. The two inverted repeats code for polypeptides of different molecular weights, with each repeat appearing to encode two polypeptides. The polypeptides from only one of the repeats of Tn5 appear to be absolutely required for Tn5 transposition.     

Analysis of the composition of samples of Babesia bovis and the influence of different environmental conditions on genetically distinct subpopulations.

A recombinant DNA probe specific for a tandemly repeated sequence located within the BoVA1 gene of Babesia bovis was used to analyse 10 independent samples of B. bovis. Twelve different alleles of the BoVA1 gene and flanking regions were identified in the 18 different subpopulations analysed. Most samples of B. bovis originally derived from single animals contained more than one genetically distinct subpopulation. However, only one population of parasites was identified in samples of the Ka line used in Australia from 1979 until 1990 as the live attenuated vaccine strain. In contrast, the replacement attenuated vaccine line, Ta, contained two genetically distinct subpopulations of parasites. Changes in the ratios of subpopulations of parasites were identified during attenuation and under different culture conditions. Batch-to-batch variation in the composition of doses of the live attenuated vaccine may lead to differences in efficacy and in severity of the infection associated with vaccination.     

Identification, characterization, and molecular cloning of a novel transporter-like protein localized to the central nervous system.

During the course of large scale purification of the D1 dopamine receptor from rat brain, a protein of approximately 87,000 daltons (p87) was observed to copurify with the D1 receptor through four chromatographic steps. To characterize the nature of this protein, bovine and rat cDNA clones were isolated and sequenced. The bovine and rat clones were highly conserved (98.5% identity). Each clone possessed an open reading frame of 2226 base pairs encoding a protein of 742 amino acids (calculated MW of 82,500), containing three stretches of peptide sequence obtained from p87 sequence analysis. Comparison of the deduced peptide sequence of this protein with those found in available databanks revealed that it was a novel protein related to the family of nutrient transport proteins from eukaryotes and bacteria, including, the mammalian facilitated glucose transporters, the yeast transporters for maltose, lactose, and glucose, and the proton-driven bacterial transporters for arabinose, xylose, and citrate. In addition p87 also shares with these transporters a similar hydropathicity profile that suggests the presence of 12 transmembrane segments. The mRNA for p87 appears to be localized primarily, if not exclusively, to the central nervous system. Northern blot analysis reveals a message of approximately 4.8 kb in cortex, hippocampus, brain stem, and cerebellum, but no detectable signal in peripheral tissues such as spleen, liver, kidney, lung, heart, or skeletal muscle. Evidence form Western blot analysis and immunohistochemistry suggests that this protein may be expressed in intracellular organelles or the membrane of synaptosomes rather than plasma membrane. Based on its structure and properties, p87 appears to define a new class of transporter-like proteins.     

Improved RNA isolation from cells in tissue culture using a commercial nucleic acid extractor.

The Applied Biosystems 340A Nucleic Acid Extractor automates isolation of either DNA or RNA from tissue or cells in culture. We have found that several modifications to the manufacturer's recommended protocol greatly improve the quality of RNA that can be routinely isolated from cells in culture. These modifications include lysis of monolayer cells directly on plates, centrifuging samples after homogenization to remove precipitable RNase contaminants and purging the instrument's reagent lines with 0.1% diethyl pyrocarbonate. These simple modifications enhance both RNA quality and reproducibility of yield.     

Genetic and Molecular Organization of Ribosomal DNA (Rdna) Variants in Wild and Cultivated Barley

Twenty rDNA spacer-length variants (slvs) have been identified in barley. These slvs form a ladder in which each variant (with one exception) differs from its immediate neighbors by a 115-bp subrepeat. The 20 slvs are organized in two families, one forming an eight-step ladder (slvs 100-107) in the nucleolus organizer region (NOR) of chromosome 7 and the other a 12-step ladder (slvs 108a-118) in the NOR of chromosome 6. The eight shorter slvs (100-107) segregate and serve as markers of eight alleles of Mendelian locus Rrn2 and the 12 longer slvs (108a-118) segregate and serve as markers of 12 alleles of Mendelian locus Rrn1. Most barley plants (90%) are homozygous for two alleles, including one from each the 100-107 and the 108a-118 series. Two types of departures from this typical pattern of molecular and genetic organization were identified, one featuring compound alleles marked by two slvs of Rrn1 or of Rrn2, and the other featuring presence in Rrn1 of alleles normally found in Rrn2, and vice versa. The individual and joint effects on adaptedness of the rDNA alleles are discussed. It was concluded that selection acting on specific genotypes plays a major role in molding the strikingly different allelic and genotypic frequency distributions seen in populations of wild and cultivated barley from different ecogeographical regions.