Mutation as an error in base pairing

Summary The model of mutation by transitional change (Freese 1959) predicts that a heritable change in genotype is established when two replications of DNA succeed the initial incorporation of an analogue. The model was tested in populations ofSalmonella typhimurium strainstryD-10 andtryD-79 whose division had been synchronized by fractional filtration. Mutation from auxotrophy to prototrophy (try ^–try ⁺) induced by 5-bromodeoxyuridine (BUDR) and 2-aminopurine (AP) occurred in accordance with DNA replication. Two subsequent DNA replications were necessary to obtain BUDR-induced prototrophs inD-79, one subsequent DNA replication was required for AP-induced prototrophs inD-79, while no subsequent DNA replication was necessary for AP-induced prototrophs inD-10. This was observed whether the mutagens were present continuously or during only the first replication and also when the cells were allowed to replicate their DNA without cell division in the presence of inhibitory concentrations of the base analogue or when protein synthesis was blocked in the presence of chloramphenicol. A statistical analysis of the patterns of mutant increase observed for six mutant strains was used to distinguish between errors in replication and errors in incorporation induced by the base analogues and thereby the base pair at the mutant site was identified.With 10 Figures in the TextSupported in part by grants from the American Cancer Society the U.S. Public Health Service and the National Science Foundation administered by ProfessorF. J. Ryan.     

Developmental and organ-specific expression of an ABA- and stress-induced protein in barley

An mRNA species, HVA1, has been shown to be rapidly induced by abscisic acid (ABA) in barley aleurone layers (Hong, Uknes and Ho, Plant Mol Biol 11: 495–506, 1988). In the current work we have investigated the expression of HVA1 in other organs of barley plants. In developing seeds, HVA1 mRNA is not detected in starchy endosperm cells, yet it accumulates in aleurone layers and embryo starting 25 days after anthesis, and its level remains high in these organs in dry seeds. Although the levels of HVA1 mRNA are equivalent in the dry embryos of dormant and nondormant barley seeds, upon imbibition HVA1 mRNA declines much slower in the dormant than in the nondormant embryos. The HVA1 mRNA and protein levels are highly induced by ABA treatment in all organs of 3-day-old seedlings. However, the induction in the leaf of 7-day-old seedlings is less than one tenth the level observed in the leaf of 3-day-old seedlings. In the leaf, HVA1 mRNA and protein are induced mainly at the base. These observations indicate that the expression of HVA1 is under developmental regulation. Besides the HVA1 protein, a smaller protein (p20) of approximately 20 kDa cross-reacting with anti-HVA1 polyclonal antibodies, is induced by ABA in barley seedlings but not in seeds. HVA1 mRNA is induced by drought, NaCl, cold or heat treatment. Similar to ABA treatment, the drought induction of HVA1 occurs in all the tissues of 3-day-old seedling, but the induction decreases dramatically in the leaf of 7-day-old plants. The significance of organ-specific, developmentally regulated, and stress-induced expression of HVA1 is discussed.     

Cellulolytic Actitivity of Penicillium digitatum and P. italicum Related to Fungal Growth and to Pathogenesis in Citrus Fruits

Cultural conditions under which Penicillium digitatum and P. italicum develop were an important factor in determining the extent of their cellulolytic activity. At pH 5.5–7.5 of the culture medium, Cx-cellulase activity was correlated with mycelial dry weight. However, at pH 4.5 and more so at pH 3.5, activity was markedly reduced while fungal growth was not affected. Cx-cellulases of both species were not induced by the presence of carboxymethyl cellulose as a carbon source and were defined as constituent enzymes. Cellulase activity of the two Penicillia on different carbon sources was detected prior to the initiation of sporulation. A sporeless mutant of P. digitatum exhibited cellulolytic activity similar to that of the normal strain, suggesting no role for sporulation in the Cx-cellulase synthesis. Cx-cellulase activity in Valencia oranges started during the early stages of pathogenesis, before the appearance of disease symptoms. Correlation between the cellulase activity and the severity of the disease symptoms was apparent during the first three days after inoculation. At the end of the incubation period both fungi almost reached their maximum enzymatic activity, whereas the disease index continued to rise gradually until total fruit rot was achieved. A possible role of Cx-cellulases of the two Penicillia in the early stages of pathogenesis was suggested.     

Effects of cycloheximide on virus RNA replication in an inducible line of polyoma-transformed rat cells.

In this article, we describe two distinct effects of cycloheximide (CH), a potent inhibitior of protein synthesis, on the replication of polyoma virus (PV) DNA in an inducible line of PV-transformed rat cells (LPT cells). Exposure of LPT cells to CH causes up to an 8 fold increase in the cellular concentration of PV DNA determined by molecular hybridization. The same treatment inhibits cell division and chromosomal DNA replication. However, the amount of chromosomal DNA per cell is not affected by the drug. In LPT cells treated with mitomycin C (MMC), PV DNA replication is enhanced after 7 hr. During the period extending from 7 hr to 24 hr, the concentration of virus DNA increases at least 100 fold. CH added to the cells 0-7 hr after treatment with MMC inhibits the replication of PV DNA by 90-100%. The inhibition is less effective in cells exposed to CH from 7 hr and on. The inhibitory effect is reversible: virus DNA synthesis is resumed after removal of CH from the growth medium. Thus CH acts as an inducer of virus DNA synthesis in cells whose resident viral genome is repressed, but inhibits the autonomous replication of the activated genome following induction with MMC.     

Chromatographically Fractionated Complementary Strands of Bacillus subtilis Deoxyribonucleic Acid: Biological Properties

Biological, physical, and chromatographic properties of methylated albuminkieselguhr (MAK)-fractionated complementary strands, designated as light (L) and heavy (H), of Bacillus subtilis deoxyribonucleic acid (DNA) are presented. The pattern of transforming activity along the MAK elution profile of alkilidenatured DNA shows that the residually active molecules selectively fractionated ahead of the L strand fraction, whereas the most active self-annealed molecules fractionated preferentially at the trailing end of the H strand fraction. The restoration rate of transforming activity in the late-eluting H molecules was rapid and independent of concentration during the annealing reaction. The data suggest that the self-annealing activity in the H strand is due in part to the formation of intrastrand secondary structures. Hydroxyapatite chromatography of self-annealed L and H strands yielded a major fraction (I) of highly purified strand preparations devoid of transforming activity and hypochromicity, and a minor "nativelike" fraction (II). Sedimentation velocity measurements show that, in addition to the mutual complementary nature of the L and H fractions, they differ in molecular size and possibly configuration.     

The killer phenomenon in Ustilago: Electron microscopy of the dsRNA encapsidated in individual virus particles

From earlier studies with the Ustilago maydis virus and other dsRNA viruses it is known that discrete dsRNA segments typical of each virus are obtained by extraction. A variation exists with respect to the encapsidation of these segments among different viruses. The encapsidation of the genome in individual particles of the Ustilago virus was examined by electron microscopy after disruption of virus particles. The study included the P6 wild-type and 2 mutants containing only part of the genome. The results indicate that most virus particles of the wild-type and the mutants contain up to 12–14×10⁶ daltons of dsRNA. Since the largest extracted molecule is 3.2×10⁶D these findings suggest that an individual particle may contain more than one segment of dsRNA. Free linear molecules that exceed in size the extracted segments were also found following the disruption of each of the 3 virus types examined. Thus, the viral genome seen segmented after extraction is organized as a concatamer in the capsid and each virus particle can contain an entire viral genome consisting of each type of the segments seen after extraction, a repeat of a single segment or a random assortment. In each case the information may be organized as a concatamer.     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

Conformational changes induced in the human protein translin and in the single-stranded oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5) upon binding of these oligodeoxynucleotides by translin

Translin is a human single-stranded DNA and RNA binding protein that has been highly conserved in eukaryotic evolution. It consists of eight subunits having a highly helical secondary structure that assemble into a ring. The DNA and the RNA are bound inside the ring. Recently, some of us demonstrated that the human translin specifically binds the single-stranded microsatellite repeats, d(GT)(n), the human telomeric repeats, d(TTAGGG)(n), and the Tetrahymena telomeric repeats, d(GGGGTT)(n). These data suggested that translin might be involved in recombination at d(GT)(n).d(AC)(n) microsatellites and in telomere metabolism. Other data indicated that translin might stimulate binding of telomerase to single-stranded telomeric overhangs by unwinding secondary structures formed by the telomeric repeats. Here we present a circular dichroism (CD) analysis of complexes formed between the human translin and the microsatellite and telomeric oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5). We report that conformational changes occur in both the translin and the oligodeoxynucleotides upon formation of the complexes. In translin octamers bound to the oligodeoxynucleotide d(GT)(12), the fraction of alpha-helices decreases from approximately 67% to approximately 50%, while the fraction of turns and of the unordered structure increases from approximately 11% to approximately 17% and from approximately 19% to approximately 24%, respectively. In the bound oligodeoxynucleotide d(GT)(12), we observed CD shifts which are consistent with a decrease of base stacking and a putative anti-syn switch of some guanines. The oligodeoxynucleotide d(TTAGGG)(5) formed intramolecular quadruplexes under the conditions of our assays and translin was found to unfold the quadruplexes into structures consisting of a single hairpin and three unwound single-stranded d(TTAGGG) repeats. We suggest that such unfolding could account for the stimulation of telomerase activity by translin mentioned above.     

Fourth meeting of the European Network of Research Tissue Banks – Future strategy to increase collaborations in the supply of human tissue for biomedical research

This report records the Fourth meeting of the European Network of Research Tissue Bank (Brussels, 18th March 2004) which was attended by Mel Read MEP. The existing membership of this informal group represents European Human Research Tissue Bankers, biomedical researchers seeking access to human tissue and allied groups including animal welfare representatives. This Fourth meeting provided a forum to update members on individual activity in this area. A particular focus of this meeting was to consider the status of this group and future affiliations to increase the profile and activity of this Network. This meeting addressed differences in legislative and ethical requirements governing the use of human tissue in biomedical research in the different countries represented. Future activity of the ENRTB, planned at this meeting, will target harmonisation of current differences which are currently barriers to increased access to human tissue for biomedical research. Through the harmonisation of procurement, processing and distribution of human tissue specimens the ENRTB will provide a mechanism to benefit human health through increased use of human tissue in pharmacotoxicological studies and the associated replacement of animal tests.