Amplification of bacterial plasmids without blocking protein biosynthesis

The effect of amino acids (presence or absence from the growth media) and metal ions on the replication of Escherichia coli plasmids in rel A+ strains was studied. It was found that: (i) The absence of one amino acid from the growth media had no effect on the plasmid copy number in prototrophic E. coli strains: (ii) The presence of only one amino acid in artificial media free of amino acids had a negligible effect on the plasmid copy number for the amino acids Ala, Arg, Glu, His, Leu, Phe, Thr, Trp, and Tyr: (iii) The combination of Met and Thr caused a rise in pBR322 plasmid copy number up to 90-100 plasmid copies per cell: (iv) The Fe3+ concentration had an amplification effect on E. coli plasmids. The pBR322 plasmid copy number for media free of amino acids and supplemented with 0.2-0.4 mM FeCl3 was 60-80 plasmid copies per cell: (v) The combination of Fe3+ with certain amino acids (Ala, Arg, Glu, Leu, Thr, and Trp) leads to a dramatic increase in the plasmid copy number reaching 180-270 plasmid copies per cell for the plasmid pBR322 and 20-24 for the plasmid pR100.     

Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions.

UV laser irradiation has been used to covalently crosslink histones to DNA in nuclei, chromatin and core particles and the presence of the different histone species in the covalently linked material was detected immunochemically. When nuclei were irradiated and then trypsinized to cleave the N- and C- terminal histone tails, no histones have been found covalently linked to DNA. This finding shows that UV laser-induced crosslinking of histones to DNA is accomplished via the non-structured domains only. This unexpected way of crosslinking operated in chromatin, H1-depleted chromatin and core particles, i.e. independently of the chromatin structure. The efficiency of crosslinking, however, showed such a dependence: whilst the yield of crosslinks was similar in total and H1-depleted chromatin, in core particles the efficiency was 3-4 times lower for H2A, H2B and H4 and 10-12 times lower for H3. The decreased crosslinking efficiency, especially dramatic in the case of H3, is attributed to a reduced number of binding sites, and, respectively, is considered as a direct evidence for interaction of nonstructured tails of core histones with linker DNA.     

Investigation of Lactobacilli Strains during Lyophilization

The Freezing Points of three lactobacilli strains cultivated in skim milk and MRS-broth were determined. Their survival was analyzed after lyophilization. A maximal preservation of the cell number of the R1 strain was found. The fermentation activity of the laclobacilli was investigated before and after lyophilization in order to characterize their species identification. Copyright 1993, 1999 Academic Press.     

Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains

Summary In submerged cultures of Claviceps sp. CP II, elymoclavine was synthesized only by the growing mycelium (phase P1), whereas cultures of C. purpurea strain 129 produced agroclavine after vegetative growth had also ceased (phase P2). In strain CP II, the peak of activity of malate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphatases was related to the time of maximum growth rate and alkaloid production. Citrate synthase activity paralleled the course of alkaloid synthesis. Strain 129 exhibited a further activity peak of the same magnitude during phase P2. ATP levels in both cultures corresponded to the pattern of change in enzyme activities. Strain CP II contained roughly twice as much orthophosphate and ATP in its cells as strain 129 and exhibited higher average activity of glucose-6-phosphate dehydrogenase. It follows from these results that alkaloid synthesis requires the processes of primary metabolism, even when it occurs after active growth of the culture has ceased. Cultures producing alkaloids oxidized at C-8 exhibit higher glucose-6-phosphate dehydrogenase activity, probably because of a higher NADPH consumption.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Identification of an in vivo inhibitor of Bacillus anthracis spore germination

Germination of Bacillus anthracis spores into the vegetative form is an essential step in anthrax pathogenicity. This process can be triggered in vitro by the common germinants inosine and alanine. Kinetic analysis of B. anthracis spore germination revealed synergy and a sequential mechanism between inosine and alanine binding to their cognate receptors. Because inosine is a critical germinant in vitro, we screened inosine analogs for the ability to block in vitro germination of B. anthracis spores. Seven analogs efficiently blocked this process in vitro. This led to the identification of 6-thioguanosine, which also efficiently blocked spore germination in macrophages and prevented killing of these cells mediated by B. anthracis spores. 6-Thioguanosine shows potential as an anti-anthrax therapeutic agent.     

Temperature-dependence of UV laser one-electron oxidative guanine modifications as a probe of local stacking fluctuations and conformational transitions

By monitoring R(pip)/R(Fpg), i.e. the relative sensitivity to hot piperidine and to formamidopyrimidine DNA glycosylase (Fpg protein) of the guanine lesions induced in DNA exposed to UV laser irradiation, we have previously observed that the formation of the two major types of one-electron oxidative guanine modifications, oxazolone and 7,8-dihydro-8-oxoguanine (8-oxodG), depends on DNA conformational features. While oxazolone is largely predominant at each site of single-stranded DNA (R(pip)>R(Fpg)), 8-oxodG is the major lesion at most of the sites of double-stranded DNA (R(pip)R(Fpg) at 20 degrees C and the ratio R(pip)/R(Fpg) does not vary significantly during the melting process. Interestingly, these guanine residues display a high sensitivity to dimethyl sulfoxide methylation while the opposite cytosine residues are unsensitive, suggesting that the prevalence of R(pip) over R(Fpg) is related not to base-pairing disruption but rather to the local helical alteration of the B-DNA stacking geometry. This leads us to propose that the slight variations in the ratios R(pip)/R(Fpg) observed, at individual sites, at temperatures below the helix-coil transition reflect local small-scale breathing motions, unstacking single dinucleotide steps prior to opening. Our results thus support the view that the temperature dependence of the ratio of R(pip)/R(Fpg) at sites of B-DNA provides a sensitive probe of the DNA internal local thermal stability and are discussed in relation with the mechanisms proposed for the intramolecular rearrangement of the guanyl radical.     

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.