The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.     

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.     

Effect of Arbuscular Mycorrhizal Inoculation on Salt-induced Nodule Senescence in <Emphasis Type="Italic">Cajanus cajan</Emphasis> (Pigeonpea)

Many physiological and biochemical plant processes affected by salt stress trigger premature nodule senescence and decrease their ability to fix nitrogen. The objective of this study was to evaluate the role of arbuscular mycorrhiza (AM) in moderating salt-induced premature nodule senescence in Cajanus cajan (L.) Millsp. Greenhouse experiments were conducted in which the plants were exposed to salinity stress of 4, 6, and 8 dSm⁻¹. Various parameters linked to nodule senescence were assessed at 80 days after sowing. Nodulation, leghemoglobin content, and nitrogenase enzyme activity measured as acetylene-reducing activity (ARA) were evaluated. Two groups of antioxidant enzymes were studied: (1) enzymes involved in the detoxification of O₂⁻ radicals and H₂O₂, namely, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), and (2) enzymes that are important components of the ascorbate glutathione pathway responsible for the removal of H₂O₂, namely, glutathione reductase (GR) and ascorbate peroxidase (APOX). Exposure of plants to salinity stress enhanced nodule formation; however, nodule growth suffered remarkably and a marked decline in nodule biomass, relative permeability, and lipid peroxidation was observed. Leghemoglobin content and ARA were reduced under saline conditions. AM significantly improved nodulation, leghemoglobin content, and nitrogenase activity under salt stress. Activities of SOD, CAT, APOX, POX, and GR increased markedly in mycorrhizal-stressed plants. A synthesis of the evidence obtained in this study suggests a correlation between enhanced levels of antioxidant enzyme activities, reduced membrane permeability, reduced lipid peroxidation, and improved nitrogen-fixing efficiency of AM plants under stressed and unstressed conditions. These factors could be responsible for the protective effects of mycorrhiza against stress-induced premature nodule senescence.     

Determination of protein synthesis in vivo using labeling from deuterated water and analysis of MALDI-TOF spectrum.

This paper describes a method of determining protein synthesis and turnover using in vivo labeling of protein with deuterated water and analysis of matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) spectrum. Protein synthesis is calculated using mass isotopomer distribution analysis instead of precursor to product amino acid enrichment ratio. During protein synthesis, the incorporation of deuterium from water changes the mass isotopomer distribution (isotope envelop) according to the number of deuterium atoms (0, 1, 2, 3, etc.) incorporated, and the distribution of the protein with 0, 1, 2, 3,... atoms of deuterium follows a binomial distribution. A mathematical algorithm by which the distribution of deuterium isotopomers can be extracted from the observed MALDI-TOF spectrum is presented. Since deuterium isotopomers are unique to newly synthesized proteins, the quantitation of their distribution provides a method for the quantitation of newly synthesized proteins. The combined use of postsource decay sequence identification and mass isotopomer distribution analysis makes the use of in vivo labeling with deuterated water a precise method to determine specific protein synthesis.     

Feasibility of utilization of horse dung spiked filter cake in vermicomposters using exotic earthworm Eisenia foetida

This contribution reports the potential of vermicomposting technology in the management of horse dung (HD) spiked sugar mill filter cake (SMFC) using an epigeic earthworm Eisenia foetida under laboratory conditions. A total of six vermicomposters filled with different ratios of HD and SMFC were maintained for this study. The growth and fecundity of E. foetida was monitored for 12 weeks. Maximum growth was recorded in 90% HD+10% SMFC feed mixture containing vermicomposter. Earthworms' biomass gain and reproduction was favorably up to 50% HD+50% SMFC feed composition. Maximum cocoons were also recorded in 90% HD+10% SMFC feed mixtures, however increasing proportions of SMFC in different vermicomposters affected the growth and fecundity of worms. A significant decrease in C:N ratio and increase in total kjeldahl nitrogen, total available phosphorus and calcium contents was recorded. The heavy metals content was higher in the vermicompost obtained in all the reactors than initial feed substrates. Based on investigations it is concluded that vermicomposting could be an alternative technology for the management of filter cake if it is mixed in 1:1 ratio with horse dung.     

DNA polymerases that propagate the eukaryotic DNA replication fork

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase ot-primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase ca and recruits DNA polymerase S and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase 8, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase E normally replicates this strand,but under conditions of dysfunction, DNA polymerase 8 may substitute.     

Synthesis of a novel plant growth promoter from gallic acid

Gallic acid has been modified to naphthophenone derivatives with esterified fatty acid side chain. Compound 12, an ethyl crotonate ester of naphthophenone derivative has shown potent auxin like growth promoter activity. This is the first example of naphthophenone derivatives with plant growth promoting activity.     

Time dependent study to evaluate the efficacy of zinc on hepatic marker enzymes and elemental profile in serum and liver of protein deficient rats

This study was designed to determine the time dependent protective effects of zinc sulfate on the serum and liver marker enzymes along with elemental profile in protein deficient Sprauge Dawley (S.D.) female rats. Zinc sulfate in the dose of 227 mg/l in drinking water was administrated to normal control as well as protein deficient rats for a total duration of 8 weeks. The effects of different treatments were studied on enzymes like alkaline phosphatase (ALP), aspartate aminotransferases (AST) and alanine aminotransferases (ALT) in rat serum at different time intervals of 1, 2, 4 and 8 weeks and in the rat liver at the end of study. The status of different essential elements in liver was also studied. The serum ALP activity got significantly depressed when estimated at the intervals of 4 and 8 weeks. Activity of serum ALT was significantly increased after 4 weeks interval in protein deficient rats and the increasing trend continued upto 8 weeks of protein deficiency. On the other hand, activity of AST showed a significant increase just after 2 weeks and activity continued to be increased up to 8 weeks. Moreover activities of all the hepato marker enzymes showed a significant increase in liver of protein deficient rats. Interestingly, supplementation of Zn to protein deficient rats helped in regulating the altered activities of ALP, AST and ALT both in serum and liver. However, zinc treatment alone to normal rats did not indicate any significant change in the activities of all the enzymes in liver as well serum except at the interval of 2 weeks where a marginal increase in the activity of AST was seen. It has also been observed that concentrations of zinc, copper, iron and selenium were found to be decreased significantly in protein deficient animals. However, the levels of these elements came back to within normal limits when zinc was administrated to protein deficient rats. Published online December 2004