Vitrification Has an Effect like Culture on Gene Expression and Histone Modification In Mouse Embryos.

BACKGROUND: OBJECTIVE: This paper reports results from studies on modifications of histone marks in transcriptional regulatory regions of H19, Igf2 and Mest genes in vitrified-warmed and cultured mouse embryos. MATERIALS AND METHODS: Non vitrified and vitrified-warmed 8-cell embryos were cultured in Ham's F10+10% BSA into blastocyst stage in two experimental groups. Gene expression level was assessed by quantitative PCR and histone modifications were evaluated by ChIP assay method. RESULTS: The expression levels of the selected genes in vitrified embryos were found to be markedly reduced compared to the control group. Significant increase in H3K9me2 as a gene silencer was seen in the regulatory regions of all genes in vitrified group. Furthermore, H3K4me3 and H3K9ac decreased in some regulatory regions but not in all of them. Additionally, H3K9me2 in regulatory regions of Igf2 and H19 significantly increased in group I, whereas, there was no change in Mest compared to the control group. CONCLUSIONS: Vitrification and culture condition affects gene down regulation of Igf2 and H19. Although changes in each histone mark are not always the same as gene expression, the total pattern and sum of their changes are in line with gene down regulation upon vitrification and culture.

A natural variant of Bacillus licheniformis alpha-amylase isolated from flour mill wastewaters sheds light on the origin of high thermostability.

AIMS: Understanding the origin of high thermostability exhibited by the alpha-amylase produced by a natural strain of Bacillus licheniformis. METHODS AND RESULTS: The MSH320 alpha-amylase gene has been cloned from a native strain of B. licheniformis isolated from flour mill wastewaters in Kashan, central Iran, and its nucleotide sequence was determined (GenBank Accession Number AF438149). Whereas previously cloned B. licheniformisalpha-amylase (BLA) genes are nearly identical, the MSH320 gene coding sequence presents only 93% identity with the reference 'wild-type' BLA gene, most of the nucleotide changes leading to silent mutations. Amino acid substitutions occurred at 19 of the 483 residues of the matured protein, distributed all along the protein sequence. Nevertheless, the natural BLA variant presents thermoinactivation kinetics similar to that of the reference BLA. Protein modelling and structural predictions at the substitution sites suggest that half of the mutations may have a significant stabilizing or destabilizing effect on the protein structure. Compensatory mutations thus occurred in the natural variant in order to maintain thermostability to the level of the reference enzyme. CONCLUSIONS: The exceptional high thermostability of BLA, although produced by a nonthermophilic organism, is not fortuitous but subject to a selective pressure still at work in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: BLA thermal performances are not naturally maximized and can be substantially improved by protein engineering.

Expression and secretion of an alpha-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene.

The gene encoding a hyperthermostable alpha-amylase from a Bacillus licheniformis native strain was cloned in pET24d transcription vector containing T7 promoter, and expressed in Escherichia coli BL21(DE3) cells. Having confirmed the alpha-amylase activity through activity staining method on SDS-PAGE gel, the yields of production were determined in two separated intra and inter-cellular phases and compared using enzymatic assay methods. Extracellular production of the active recombinant enzyme implies the recognition of the putative signal peptide of this Bacillus sp. by E. coli secretory system. This may be because of the amino acid sequence of this signal peptide which covers all the structural parameters of a standard signal peptide processed by Lep B, the major signal peptidase in E. coli secretory system. This study recommends the use of this signal peptide for extracellular production of other foreign proteins in E. coli.