Site-specific integration in Streptomyces ambofaciens: localization of integration functions in S. ambofaciens plasmid pSAM2.

In Streptomyces ambofaciens ATCC 15154, an 11.1-kilobase element, pSAM2, exists as a single integrated copy in the chromosome. In S. ambofaciens 3212 (a derivative of ATCC 15154), pSAM2 exists as a free, circular plasmid as well as an integrated element. BclI fragments from the free form of pSAM2 were cloned into an Escherichia coli plasmid vector. By using gene transplacement methods, the chromosomally integrated form of pSAM2 was marked with a gene coding for apramycin resistance. This enabled us to isolate both a segregant that had lost the integrated pSAM2 element and a cosmid clone containing integrated pSAM2 along with the flanking chromosomal sequences. One of the BclI fragments derived from free pSAM2 was shown to contain all the plasmid-specified information required to direct site-specific recombination in a derivative of S. ambofaciens lacking the resident pSAM2 element as well as in a number of other Streptomyces strains. The attachment sites used by the plasmid and the chromosome in site-specific recombination and the junctions created after integration were cloned and sequenced. Certain structural features in common with other integrating elements in actinomycetes were noted.     

Conformations of deoxydinucleoside phosphates containing pyrimidine-pyrimidine photodimers

Conformational analysis of deoxydinucleoside monophosphates with the sequences TpT and CpC have been carried out with the incorporation of both cyclobutane type pyrimidine dimers and 6-4 photoadducts using the methods of molecular mechanics energy minimization. The effect of flexibility with respect to sugar geometries and glycosidic torsions have been studied and the relative energies of a large variety of structures have been compared. The salient features obtained from these calculations have been compared with the crystallographic and spectroscopic data on pyrimidine dimer incorporated deoxydinucleoside monophosphates. Effects of "inserting" the energetically favourable conformations of such structures into B-DNA helices have been discussed in terms of the distortions in helical structures.     

Directed metabolic flow with high butanol yield and selectivity in continuous cultures ofClostridium acetobutylicum

Summary This work addresses the problem of stable butanol formation byClostridium acetobutylicum in continuous culture. Sustained altered electron flow was observed in the presence of benzyl viologen which serves to redirect carbon flow towards primarily butanol formation. A yield of butanol of over 0.28 g.g^–1 glucose was obtained and butanol comprised over 90% of the total solvents formed. Additionally, acid formation decreased significantly with butyric acid as the dominant acid end product.     

X-linkage of malic enzyme in Anopheles culicifacies species B

A survey for malic enzyme (Me) in laboratory strains of species A and species B of Anopheles culicifacies had uncovered two electrophoretic variants, slow and fast, in two strains of species B. Genetic analysis revealed the two variants to be codominant alleles segregating at a locus, Me, which is sex linked. Because of the XX-XY sex determining mechanism, in F1 females, two electromorphs, viz., slow and fast, were observed, whereas in males only one electromorph of maternal origin was seen. Linkage experiments with another X-linked mutant, white eye (w), indicated the map distance between the two loci to be 9.52 +/- 0.86.     

Modes of binding of 2'-AMP to RNase T1. A computer modeling study.

The modes of binding of adenosine 2'-monophosphate (2'-AMP) to the enzyme ribonuclease (RNase) T1 were determined by computer modelling studies. The phosphate moiety of 2'-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites--(1) The primary base binding site where the guanine of 2'-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3'-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2'-AMP to RNase T1 were found to be of nearly the same energy implying that in solution 2'-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T1-2'-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T1 catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2'-AMP and 2'-GMP with RNase T1 reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme-2'-GMP complex.     

Molecular dynamics studies on nucleoside 2',3'-cyclic phosphates.

2',3'-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3'-GMP,3'-UMP, A greater than p, G greater than p, U greater than p, C greater than p, GpA greater than p and UpA greater than p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.     

The HRAS1 gene cluster: two upstream regions recognizing transcripts and a third encoding a gene with a leucine zipper domain.

We have cloned and characterized a 55-kb region of DNA surrounding HRAS1. It contains a cluster of two, and possibly three, genes associated with CpG islands within the 32 kb immediately upstream of HRAS1. We have sequenced cDNAs representing one of these genes, provisionally designated HRC1. The locus, which is located 29 kb upstream of HRAS1, is divergently transcribed. HRC1 cDNA probe recognizes fragments on Southern blots of DNA from other vertebrate species. In human DNA, multiple homologous fragments are detected in addition to the predicted ones containing HRC1. Therefore, this locus may represent a member of an evolutionarily conserved gene family. HRC1 expression is upregulated with HRAS1 in the EJ bladder carcinoma cell line, suggesting the possibility of coordinate regulation. The deduced translational product of the longest open reading frame (1119 nucleotides, 373 amino acids) predicts a protein with regions rich in glutamine and proline and a region similar to the helix-loop-helix motif adjacent to a carboxy-terminal leucine zipper dimerization motif with four heptad repeats. Alternate splicing of terminal exons occurs, resulting in the truncation of one proline-rich domain and preservation of the leucine zipper. Thus, a biologically important region of chromosome 11p consists of a gene cluster. At least one of these genes, in addition to HRAS1, may be involved in regulation of cell growth or differentiation.     

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I gene

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.     

Induction of rat liver DNA alterations by chronic administration of peroxisome proliferators as detected by 32P-postlabeling

The mechanisms of the hepatocarcinogenicity of non-mutagenic peroxisome proliferators, i.e. compounds used as hypolipidemic drugs and industrial plasticizers, are not sufficiently understood. To gain more information on the mechanism of their action, the chronic effects of two structurally diverse peroxisome proliferators on rat-liver DNA were investigated by the 32P-postlabeling assay. Male F-344 rats (1.5 month old) were fed ciprofibrate (0.025%) in the diet for 2, 5, 8, and 16 months or Wy-14643 (0.1%) for 18 months. Liver DNA from individual treated animals (3-4 per group) and age-matched controls was analyzed by the nuclease P1/bisphosphate version of the 32P-postlabeling assay. Three distinct types of exposure-related DNA alterations were observed: (i) A significant reduction of the age-dependent accumulation of I-compounds (putative indigenous DNA modifications) (type 1), (ii) adduct-like DNA derivatives induced by the treatments (type 2), and (iii) as yet structurally uncharacterized radiolabeled material occupying substantial areas of DNA adduct maps and accumulating in an exposure time-dependent manner (type 3). DNA from liver tumors generated by these agents displayed only traces of I-compounds, lacked all but one adduct-like derivatives, and had no type 3 alterations. Thus, in contrast to the non-mutagenicity of peroxisome proliferators in short-term assays, chronic administration of these compounds led to DNA alterations that were detectable by 32P-postlabeling assay.     

Phosphamidon, methylparathion and dichlorvos impact on tissue oxidative metabolism in penaeid prawn, Metapenaeus monoceros

Changes in oxidative metabolism of hepatopancreas and muscle tissues of penaeid prawn, Metapenaeus monoceros was studied, following its exposure to selected organophosphorous insecticides phosphamidon, dichlorovos and methylparathion. The OPI are found to inhibit the activity levels of acetylcholinesterase, succinate dehydrogenase, isocitrate dehydrogenase, pyruvate dehydrogenase, lactate dehydrogenase and cytochrome-c-oxidase and cause accumulation of acetylcholine in the hepatopancreas and muscle tissues. These changes in the activity levels of selected oxidative enzymes during insecticide exposure in these tissues of prawn indicates the shift in the metabolic emphasis from aerobic to anaerobic conditions and is interpreted as a functional adaptation to insecticide induced metabolic stress. These observed changes at cellular level pave way for successful survival of prawns in insecticide polluted environ.