Dsi-1, a region with frequent proviral insertions in Moloney murine leukemia virus-induced rat thymomas.

Dsi-1 is a region of chromosomal DNA that underwent proviral insertion in 3 of 24 Moloney murine leukemia virus-induced rat thymomas. In one of these tumors, a provirus is also integrated adjacent to the proto-oncogene c-myc. The proviruses in Dsi-1 have been characterized and appear to be complete. The proviruses were located within a 2-kilobase region that contained four prominent DNase I-hypersensitive sites. These hypersensitive sites were observed in Moloney murine leukemia virus-induced thymomas but not in NRK cells. The region of Dsi-1 immediately 3' to the insertions cross-hybridized with human and chicken DNA, indicating that it contains highly conserved sequences. No evidence could be found for the expression of this highly conserved region. Dsi-1 was mapped to mouse chromosome 4. This location demonstrates that Dsi-1 is different from 16 of the known proto-oncogenes (c-abl, c-erbA c-erbB, c-ets-1, c-ets-2, c-fes, c-fos, c-myb, c-myc, c-raf, A-raf, c-Ha-ras, c-Ki-ras, N-ras, c-sis, and c-src) and 12 cellular regions of tumor-associated integrations in retrovirus-induced tumors (c-erbB, Fis-1, int-1, int-2, Mis-1/pvt-1, Mlvi-1, Mlvi-2, c-mos, c-myb, c-myc, Pim-1, and c-Ha-ras). Hybridization experiments indicated that Dsi-1 is probably different from five additional proto-oncogenes (c-fgr, c-fms, c-mos, neu, and c-yes) and from two additional frequent integration regions (lck and Mlvi-3).     

Regulation of Protein Synthesis during Photomorphogenesis of Gametophytes of the Fern Onoclea sensibilis

Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with [³⁵S]methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation.     

Elastase activities of human bladder cancer cell lines derived from high grade invasive tumours

Elastase activities in intact human bladder cancer cell lines, established from three patients, were measured using a fluorogenic substrate highly specific for elastase, under conditions of physiological pH and ionic strength. This method allowed separation of cell-associated from secreted enzyme activity. As secreted elastase accounted for only 8% of the total, we concluded that the elastases were present at the cell surface. Inhibition studies using extracts of cell-surface elastases showed them to be serine proteinases which were also inhibited by alpha 1-antitrypsin. Partially purified fractions showing the highest specific activity towards the fluorogenic substrate hydrolysed insoluble elastin thus confirming the presence of elastases. This is the first time that elastase activity has been demonstrated in human bladder cancer cells and may represent a mechanism involved in tumour invasion.     

Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.     

Abortion in Canada: religious and ideological dimension's of women's attitudes

This paper examines a number of demographic and sociocultural factors (e.g., age, marital status, family size, religion, religious assiduity, sex-role ideology) as predictors of women's attitudes toward abortion, using data from the Canadian Fertility Survey of 1984. The findings suggest that women's abortion attitudes are to a greater extent based on ideological positions. It appears that anti-abortion stance affects those women who are religious, presumably by increasing the relationship between their general sex-role ideological stances and abortion attitudes. Abortion attitudes also vary according to a woman's education, her size, and province/region of residence.     

Oligosaccharides from placenta: early diagnosis of feline mannosidosis

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.     

Two abnormalities of hexosaminidase A in clinically normal individuals

Two abnormalities of beta-hexosaminidase A (HEX A) activity are described. One, found in two unrelated Jewish children, was characterized by the complete absence of HEX A activity in serum, but low levels of activity in leukocytes and fibroblasts using artificial substrate. The other, found in a non-Jewish man, was characterized by uniformly low levels of HEX A activity in leukocytes, fibroblasts, and serum against artificial substrate. In all cases, the pH optimum of HEX A was normal, there was no increased lability at 37 degrees C, and no inhibitor was detected to account for the deficiency of activity. Cultured fibroblasts of these individuals were capable of synthesizing and processing alpha- and beta-subunits of HEX A and capable of cleaving GM2 ganglioside. The patients, ranging in age from 6 to 30 years, are clinically normal. They are probably genetic compounds carrying the classical Tay-Sachs gene and a differently mutated allele that imparts the anomalous phenotypic features observed.     

Direct fermentation of cassava starch to ethanol by mixed cultures of Endomycopsis fibuligera and Zymomonas mobilis: Synergism and limitations

Summary A mixed culture of Endomycopsis fibuligera NRRL 76 and Zymomonas mobilis ZM4 could directly and more efficiently ferment cassava starch (22.5% w/v) to ethanol (10.5% v/v) than the monocultures. The combination of culture filtrate of E.fibuligera containing amylases and Z.mobilis simultaneously saccharified and fermented the cassava starch to ethanol equally well. Glucoamylase (0.01%) added to the fermenting medium improved ethanol (13.2% v/v) production by the above mixed culture to almost the theoretical level (98%) indicating that this enzyme is a rate-limiting factor in E.fibuligera. Z. mobilis alone converted the enzymehydrolyzed starch only to almost theoretical level (98%).     

Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was Isolated as an Intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd30₁. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPH1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex plants in two such constructs BSL 12–1 and BSL 12–3.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.