Differentiation of the American and Oriental Maydeae Accessions Using the Maize Simple Sequence Repeat (SSR) Markers

The present study evaluated the utility of maize microsatellite markers in genotype differentiation and analysis of genetic diversity among the Oriental Maydeae accessions (Coix aquatica and Chionachne koenigii) vis-à-vis the American Maydeae (maize, teosintes and maize-teosinte derivatives). Analysis of 27 microsatellite loci distributed throughout the maize genome led to the detection of 89 SSR alleles at average of 3.30 per locus. Polymorphism information content (PIC) values and observed genetic distances showed the existence of large variability among the various accessions. The maize SSR markers, particularly nc013, phi059, bnlg1523 and umc1014, were highly effective in differentiating the Oriental Maydeae accessions from the American Maydeae. Cluster analysis and Principal Component Analysis based on the SSR data clearly indicated significant genetic divergence between the Oriental Maydeae and the American Maydeae accessions, as well as between Coix aquatica and Chionachne koenigii. Molecular analyses would be valuable in gaining a better understanding of genera belonging to the Oriental Maydeae, the lesser known wild relatives of maize.     

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.     

Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.     

Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis

The plant signaling hormones salicylic acid (SA) and jasmonic acid (JA) are regulators of inducible defenses that are activated upon pathogen or insect attack. Cross-talk between SA- and JA-dependent signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. SA-mediated suppression of the JA-responsive PDF1.2 promoter was exploited for setting up a genetic screen aiming at the isolation of signal transduction mutants that are impaired in this cross-talk mechanism. The PDF1.2 promoter was fused to the herbicide resistance gene BAR to allow for life/death screening of a population of mutagenized transgenic plants. Non-mutant plants should survive herbicide treatment when methyl jasmonate (MeJA) is applied, but suppression of the JA response by SA should be lethal in combination with the herbicide. Conversely, crucial SA/JA cross-talk mutants should survive the combination treatment. SA effectively suppressed the expression of the PDF1.2::BAR transgene. However, suppression of the BAR gene did not result in suppression of herbicide resistance. Hence, a screening method based on quantitative differences in the expression of a reporter gene may be better suited to identify SA/JA cross-talk mutants. Here, we demonstrate that the PDF1.2::GUS reporter will be excellently suited in this respect.Key words: plant defense, salicylic acid, jasmonic acid, cross-talk, mutant screen, Arabidopsis     

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.