Lyophilization of the spores and vegetative cells of Bacillus brevis var. G-B.--producer of gramicidin S

Viability, antibiotic properties and variation of 4 variants of Bac. brevis var. G.-B. were studied after lyophilization and storage for a year in the lyophilized state. It was shown that the spores and vegetative cells of S and P- variants not synthesizing gramicidin S were somewhat more stable than the spores and cells of R and P+ variants producing the antibiotic. The latter dissociated by 10 per cent towards the cells producing and not producing gramicidin. The developmental rate of the lyophilized vegetative cells was higher than that of the lyophilized spores. Under analogous cultivation conditions they produced higher amounts of the biomass and antibiotic. The lyophilization method described may be recommended for the maintenance of viability and stability of the spores and vegetative cells of Bacillus brevis var. G.-B. producing gramicidin S.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

DNA cytosine methylation in plant development

Cytosine bases of the nuclear genome in higher plants are often extensively methylated.Cytosine methylation has been implicated in the silencing of both transposable elements (TEs) and endogenous genes,and loss of methylation may have severe functional consequences.The recent methylation profiling of the entire Arabidopsis genome has provided novel insights into the extent and pattern of cytosine methylation and its relationships with gene activity.In addition,the fresh studies also revealed the more dynami...     

湿地松×洪都拉斯加勒比松及亲本DNA胞嘧啶甲基化的初步研究

以湿地松×洪都拉斯加勒比松（Pinus elliottii×P.caribaea var.hondurensis）及亲本为实验材料,采用甲基化敏感扩增多态性技术对其基因组中CCGG位点的甲基化相对水平及遗传变异模式进行了初步分析。结果表明,杂种及亲本CCGG总甲基化相对水平介于77.74%~81.75%,CG甲基化相对水平略低于CNG甲基化水平,CG/CNG甲基化相对水平高于亲本。杂种遗传自亲本的CG与CNG甲基化位点数之比接近1：1,遗传自母本的甲基化位点数与遗传自父本的CCGG甲基化位点数比例为1：1;杂种产生的全新甲基化与完全去甲基化位点数之比接近7：1,初步推测大量甲基化变异促进了杂合体的生长发育。     

DNA cytosine methylation and heat-induced deamination

The heat-induced conversion of 5-methylcytosine (m⁵C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied. The calculated rates for deamination at 37°C and pH 7.4 were 9.5×10^–10 and 2.1×10^–10 sec^–1, respectively. N⁴-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine. Thermophilic bacteria which contain N⁴-methylcytosine rather than m⁵C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m⁵C in their DNA.     

Determination of the role of DNA cytosine methylation in human genetic individuality]

By the restriction analysis method we established that methylation of the 5'-end cytosine in 5'-m5CC-3' duplexes had individual specific features. This genetic peculiarity did not change even in DNA from human stomach carcinomas.     

Non-symmetrical cytosine methylation in tobacco pollen DNA

We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region, 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1×m⁵CpTpC, 1×m⁵CpGpT, 1×m⁵CpCpT, 2×m⁵CpTpT, 2×m⁵CpGpG, and 3×m⁵CpApT of which only m⁵CpGpG and m⁵CpGpT fitted the consensus sequence for symmetrical methylation in plants.     

Various characteristics of lipid metabolism of Bacillus brevis var. G.-B

Some characteristic features of the lipid metabolism of Bacillus brevis var. G.-B. natural variants and Bacillus brevis mutant 101 were studied. The authors found that upon submerged cultivation gramicidine S-producing P+-variant and B. brevis mutant 101 synthesized higher amounts of tocopherols as compared to other colonial-morphological variants. The highest tocopherol content was observed in P+-variant, whose cells contained the highest amount of total lipids as compared to other gramicidine S-producers.     

Growth limitations and biosynthesis of gramicidin in Bacillus brevis var. G.-B

Gramicidin S biosynthesis was studied in Bacillus brevis var. G.-B. during its batch and continuous cultivation when the culture growth was limited with nutrient sources (glycerol, ammonium nitrogen, phosphate), oxygen deficiency and the action of a physical factor (a low temperature). The antibiotic biosynthesis was shown to be induced by a change in the growth rate caused by the action of any factor decelerating the growth. The authors propose a mathematical model for the antibiotic synthesis, biomass accumulation and the utilization of a substrate limiting the growth. The model is based on the age separation of cells. The model is analyzed in terms of optimizing the one-stage continuous cultivation process. The model allows one to calculate optimal conditions of the antibiotic synthesis in the process of one-stage continuous cultivation.     

High-resolution analysis of cytosine methylation in ancient DNA

Epigenetic changes to gene expression can result in heritable phenotypic characteristics that are not encoded in the DNA itself, but rather by biochemical modifications to the DNA or associated chromatin proteins. Interposed between genes and environment, these epigenetic modifications can be influenced by environmental factors to affect phenotype for multiple generations. This raises the possibility that epigenetic states provide a substrate for natural selection, with the potential to participate in the rapid adaptation of species to changes in environment. Any direct test of this hypothesis would require the ability to measure epigenetic states over evolutionary timescales. Here we describe the first single-base resolution of cytosine methylation patterns in an ancient mammalian genome, by bisulphite allelic sequencing of loci from late Pleistocene Bison priscus remains. Retrotransposons and the differentially methylated regions of imprinted loci displayed methylation patterns identical to those derived from fresh bovine tissue, indicating that methylation patterns are preserved in the ancient DNA. Our findings establish the biochemical stability of methylated cytosines over extensive time frames, and provide the first direct evidence that cytosine methylation patterns are retained in DNA from ancient specimens. The ability to resolve cytosine methylation in ancient DNA provides a powerful means to study the role of epigenetics in evolution.     

Common methods for cytosine methylation analysis in DNA

The review considers the methods most commonly used to detect DNA methylation, their advantages, potential limitations, and selection for various purposes. A detailed protocol is described for bisulfite treatment, which is used as a preliminary step in the majority of DNA methylation assays.