Structural relatedness of lysis proteins from colicinogenic plasmids and icosahedral coliphages

The host-lysis-inducing functions of phi X174 protein E and MS2 protein L were recently shown to reside on the N-terminal and C-terminal halves of the two respective lysis proteins. In the present study it is shown that the small lysis proteins encoded in various colicinogenic plasmids share local sequence similarities and certain structural characteristics with the essential peptides of their coliphage-coded counterparts. Despite their dissimilar sizes and origins, it is suggested that the colicinogenic lysis proteins are functionally analogous and evolutionarily related to those of icosahedral single- stranded DNA and RNA phages.      

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle

Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.     

Analysis of Pteridium ribosomal RNA sequences by rapid direct sequencing

A total of 864 bases from 5 regions interspersed in the 18S and 26S rRNA molecules from various clones of Pteridium covering the general geographical distribution of the genus was analysed using a rapid rRNA sequencing technique. No base difference has been detected amongst the three major lineages, two of which apparently separated before the breakup of the ancient supercontinent, Pangaea. These regions of the rRNA sequences have thus been conserved for at least 160 million years and are here compared with other eukaryotic, especially plant rRNAs.     

Comparison of male adult population densities of the oriental and artocarpus fruit flies,Dacus spp. (Diptera: Tephritidae), in two nearby villages in Penang,Malaysia

The populations of native male adult oriental fruit fly Dacus dorsalis (Hendel ) and artocarpus fruit fly D. umbrosus (F.) in two selected site (BU and SD) were estimated weekly by the capture-recapture technique using live traps baited with methyl eugenol. In BU where many varieties of fruit trees were grown, the estimated population densities of D. dorsalis were between 980 and 3100 male flies per ha between May and July, 1984. During the same period, in SD where there were fewer number and varieties of fruit trees, the estimated population densities were between 300 and 1000 flies per ha. The estimated population densities of D. umbrosus over the same period were between 570 and 1290 flies per ha in BU; and between 5 and 95 flies per ha in SD. Of a total 6828 marked D. dorsalis flies released only one fly (released 6 weeks earlier in BU) was caught in a different site.     

Lymphoma models for B-cell activation and tolerance. II. Growth inhibition by anti-mu of WEHI-231 and the selection and properties of resistant mutants

Regulation of the growth of murine B-cell lymphomas has been used as a model for tolerance induction. The inhibition by anti-immunoglobulin reagents of the growth of WEHI-231 and several variant clones has now been studied. The parental line is exquisitely sensitive to growth inhibition by heterologous or monoclonal anti-mu or anti-k reagents and ceases to incorporate thymidine within 24-48 hr of exposure to anti-immunoglobulin reagents. Growth inhibition is initially reversible, but prolonged exposure to anti-mu results in cell death. This inhibition is specific for immunoglobulin light and heavy chains since growth is not inhibited by antibodies directed at either class I or class II histocompatibility antigens. In order to study the mechanism of growth inhibition, we have mutagenized WEHI-231 with ethylmethane sulfonate and cloned the surviving colonies in the presence of anti-mu. Such variants, which have been repeatedly recloned, are able to grow normally in the presence of anti-mu up to 100 micrograms/ml. These "resistant" clones, while expressing amounts of surface IgM similar to that observed on WEHI-231, do not differ markedly in their ability to cap their immunoglobulin receptors compared to the parental line but appear to have lost class II antigens. Cell cycle analysis revealed that anti-mu causes a block in the transition of WEHI-231 from G1 to S phase. The relevance of these processes to models of B-cell tolerance induction are discussed.     

Production, Purification, and Characterization of Recombinant Human Hemoglobin Rainier Expressed inSaccharomyces cerevisiae

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the β145 tyrosine is substituted with cysteine. The α and β^Rainierglobin cDNAs were cloned in a high copy number vector and expressed inSaccharomyces cerevisiaeunder the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual α and β^Rainierglobin chains. Coexpression of both α and β^RainiercDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the α and β chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A₀. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.     

Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.     

Preliminary results on the effects of salinity and settling conditions on megalopal metamorphosis of fiddler crab Ilyoplax pusilla

Metamorphosis of fiddler crab Ilyoplax pusilla larvaefrom megalopal to first crab stage wasstudied in laboratory experiments under variousconditions of salinity and substratum. The hatchedzoea metamorphose through five stages before reachingmegalopal stage. The megalops were placed in a 20 mlPetri dishes (2–3 megalops/dish), with salinities of10, 20 or 30 . Each salinity level was tested eitherwith or without sandy mud substratum. Thirty to 35megalops were used in each of the six treatments. Theexperiment was carried out at a water temperature of28 ±0.5°C and with daily feeding of the diatomChaetoceros gracilis. Treatment of 20 salinity andsandy mud substratum revealed the highest metamorphicrate (87%), while in contrast no metamorphosis wasobserved at 30 salinity withoutsandy mud substratum. Duration of the metamorphosiswas about 16 days. Numerous malformed juvenile crabswere observed in treatments conductedwithout sandy mud substratum.