The gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli. The synthase was purified to homogeneity and crystallized. The enzyme carried only two cysteine residues in contrast to its counterparts from other sources, which have four to six cysteine residues. Either or both of the cysteine residues can be replaced with serine without causing a loss of the catalytic activity. The conserved arginine residue that occupies the third position from the C-terminus was also replaced with valine without significant loss of activity, but the valine mutant showed a weakened affinity for isopentenyl diphosphate.
The traditional varieties of chick pea have low potentiality and restricted variability with respect to economic characters. Broadening the genetic base for crop improvement can be quickly achieved through induced mutagenesis. The present study was undertaken in order to comparing the effectiveness and efficiency of mutagens on Cicer arietinum. In this regard, Co – 4 variety of chick pea was subjected to different dose/concentration of Gamma rays (20, 30, 40, 50 and 60 kR) and Ethyl Methane Sulphonate (10, 20, 30, 40 and 50 mM) for inducing mutation. Mutagenic effectiveness and efficiency was calculated based on biological damage in M1 and chlorophyll mutations in M2. The results indicated that, mutagenic effectiveness increased with the increase in dose/concentration of mutagen. Intermediate treatments in general were found more efficient in causing less biological damage and inducing maximum amount of mutations. It shows that the chemical mutagens are more effective and efficient than physical mutagen for inducing mutation in Chick pea.
Astaxanthin is a xanthophill pigment with commercial application in the aquaculture, pharmaceutical, food and cosmetic industries. The red yeast Xanthophyllomyces dendrorhous is one of the most promising microorganisms for its industrial production. However, astaxanthin content in wild yeast strains is low. Pigment production by X. dendrorhous can be improved by mutagenesis. The aim of the study was to assess the efficiency of four mutagens: UV radiation, benomyl, ethyl methanesulfonate and ethidium bromide in generating asthaxanthinhyperproducing strains of the yeast Xanthophyllomyces dendrorhous DSM 5626. Mutations with benomyl, ethidium bromide and UV radiation generated a group of hyperpigmented mutants exhibiting increases up to 100% in astaxanthin content. Ethyl methanesulfonate turned out to be useless in this respect.
The Swc4p protein, encoded by an essential gene, is shared by two chromatin-remodeling complexes in Saccharomyces cerevisiae cells: NuA4 (nucleosome acetyltransferase of H4) and SWR1. The SWR1 complex catalyzes ATP-dependent exchange of the nucleosomal histone H2A for H2AZ (Htz1p). The activity of NuA4 is responsible mainly for the acetylation of the H4 histone but also for the acetylation of H2A and H2AZ. In this work we investigated the role of the Swc4p protein. Using random mutagenesis we isolated a collection of swc4mutants and showed that the essential function of Swc4p resides in its N-terminal part, within the first 269 amino acids of the 476-amino acid-long protein. We also demonstrated that Swc4p is able to accommodate numerous mutations without losing its functionality under standard growth conditions. However, when swc4mutants were exposed to methyl methanesulfonate (MMS), hydroxyurea or benomyl, severe growth deficiencies appeared, pointing to an involvement of Swc4p in many chromatin-based processes. The mutants’ phenotypes did not result from an impairment of histone acetylation, as in the mutant which bears the shortest isolated variant of truncated Swc4p, the level of overall H4 acetylation was unchanged.
Seeds of Lathyrus sativus cv. Derek and Krab were used as biological material for induced mutagenesis. Three mutant lines were obtained from seeds of grass pea cv. Derek and 15 lines from mutagenised seeds of cv. Krab. Twelve ethanol-soluble carbohydrates were identified in the seeds. We have selected grass pea mutant lines with high oligosaccharides content (lines D4, K56, K25, and K7) and lines with low raffinose family oligosaccharides (RFO) content (lines K12, K29 and K13). Mutations changing the levels of RFO have not affected the contents of galactosyl cyclitols.
The Escherichia coli Umu proteins are best characterized by their role in damage inducible mutagenesis. Recently, we discovered that the intracellular levels of the UmuD and UmuC proteins are kept to a minimum by the Lon serine protease. Studies with the Salmonella typhimurium UmuD protein (which is 73% homologous with its E. coli counterpart) revealed that it too is degraded by Lon, suggesting that both UmuD proteins share conserved structural motifs. In contrast, E. coli UmuD' is removed from the cell by the ClpXP serine protease, but only when it is in a heterodimer complex with UmuD. We have generated deletion mutants of UmuD' and have co-expressed the mutant proteins with UmuD1 (a non-cleavable UmuD protein). By assaying the sensitivity of the mutant UmuD'-UmuD1 complex to ClpXP, we have been able to map regions of UmuD' that appear essential for efficient UmuD'-UmuD heterodimer formation. Previous experiments have suggested that the in vivo posttranslational processing of UmuD to UmuD' is inefficient. We have, however, discovered that limited cleavage occurs in an undamaged cell, but that these small amounts of UmuD' are rapidly degraded by ClpXP, thus giving rise to the appearance of inefficient cleavage. The ClpXP protease therefore plays dual roles in regulating SOS mutagenesis: it keeps the basal levels of UmuD' to a minimum in undamaged cells but it also acts in damaged cells to reduce the elevated levels of mutagenically active UmuD' protein, thereby returning the cell to a resting non-mutable state.
We report the solution structure of two heptanucleotides each containing a central N4-methoxycytosine, in one case with adenine on the opposite strand and in the other with guanine. For the N4 -methoxycytosine-adenine pair only the imino form of the N4 -methoxycytosine residue is observed and base pairing is in Watson-Crick geometry. However, rotation of the methoxy group about the N-OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of .V,-methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the syn conformation of the OCH3 group in the single strand which inhibits base pair formation. For N4 -methoxycytosine base paired with guanosine we observe the N4 -methoxycytosine base in the amino form in Watson-Crick geometry and a slow exchange of this species with an imino form base paired in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 40°C. Our results point to preferential replacement of dTTP by N4 -methoxycytosine in primer elongation.
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