Hybrid plasmid with bacterial and fungal markers carrying the denV gene of T4 phage and restoring the UV-resistance of E. coli uvrA

The hybrid plasmid pYBP2 with bacterial (ampR), yeast (LEU2) and bacteriophage T4 (denV) genes has been constructed. The plasmid transformed Escherichia coli CSR603 uvrA recA ampS leuA phr- to ampicillin resistance, leucine independence, UV-resistance similar to the one of uvrA+ recA strain. Cell-free extracts of transformed Escherichia coli cells contain low level of ultraviolet-endonuclease activity in contrast to nontransformed cells containing no enzyme.     

Subfamilies and nearest-neighbour dendrogram for the LTRs of human endogenous retroviruses HERV-K mapped on human chromosome 19: physical neighbourhood does not correlate with identity level

Sequences of 45 long terminal repeats (LTRs) of the human endogenous retroviruses HERV-K family, precisely mapped by us earlier on human chromosome 19, were determined and a nearest-neighbour dendrogram was constructed. No correlation was observed between the degree of identity of the LTR pairs and their relative positions on the chromosome. Thus, sequences of distantly located LTRs, even positioned on different chromosome arms, could be highly similar to each other, whereas those of closely located LTRs could differ significantly. We conclude that the LTRs have randomly transposed across the chromosome in the course of evolution. The alignment of the LTR sequences allowed us to assign most of the LTRs to two major subfamilies. The LTRs belonging to the first subfamily (LTR-I) are characterised by higher intrasubfamily sequence divergence than those of the second subfamily (LTR-II). The two subfamilies are easily distinguished by the presence of characteristic deletions/insertions in the LTR sequences. The higher divergence of the first subfamily members suggests that their propagation started at earlier stages of evolution, probably soon after the insertion of their ancestral sequence into the primate genome. In turn, each of the subfamilies includes several distinct branches with various degrees of intragroup divergence and with characteristic diagnostic features, suggesting that the members of the branches represent amplified copies of particular master genes which had appeared at different periods of evolution. The sequences of the LTRs demonstrate a characteristic distribution of conservative and variable regions, indicating that the LTRs might have some sequence-dependent functions in the primate genome. Received: 11 August 1997 / Accepted: 22 September 1997     

Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus.

Deoxyribonucleic acid polymerase I was purified from Bacillus stearothermophilus to 50 to 70% homogeneity. Its molecular weight was 76,000. The enzyme was insensitive to sulfhydryl blocking agents and showed maximal activity at 60 degrees C, pH 8 to 9, 0.25 M KCl, and 0.02 M MgSO4. The rate of heat inactivation of the deoxyribonucleic acid polymerase followed first-order kinetics with a half-life of 90 min at 60 degrees C; the addition of 0.05% bovine serum albumin protected the enzyme, which could be heated for 180 min without loss of activity. The ratios of polymerase to nuclease activities were about 20 for 5'-3' exonuclease and more than 500 for 3'-5' exonuclease. The Km for deoxyribonucleoside-5'-triphosphates was 7 microM.     

Ultraviolet-endonuclease activity in cell extracts of Saccharomyces cerevisiae mutants defective in excision of pyrimidine dimers.

Cell-free extracts of ultraviolet-sensitive mutants of Saccharomyces cerevisiae defective in excision of pyrimidine dimers, rad1, rad2, rad3, rad4, rad10, and rad16, as well as the extracts of the wild-type strain RAD+, display ultraviolet-endonuclease activity.     

Purification and characterization of glycolate oxidase from pumpkin cotyledons

Glycolate oxidase was purified and crystallized from cotyledons of germinating pumpkin seedlings. The molecular weight of the enzyme was determined to be 280,000-320,000, consisting of 8 identical subunits with molecular weight of 38,000. There are two absorption peaks at 340 and 450 nm, indicating the glycolate oxidase is a flavin protein. Several kinetic parameters were determined, Km (glycolate) 0.33 mM and Km (O2) 76.2 microM at pH 8.0. Oxalate and oxalacetate were found to be potent competitive inhibitors against glycolate; the Ki values for oxalate and oxalacetate were 4.5 and 7.8 mM, respectively. Fatty acids such as linoleic acid inhibited the enzyme noncompetitively; the Km for linoleic acid was 0.63 mM. The regulation of glycolate oxidase in the glycolate pathway occurring in leaf peroxisomes is discussed.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Synapse Formation Activates a Transcriptional Program for Persistent Enhancement in the Bi-directional Transport of Mitochondria

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On the nature of hybrid dwarfness

In studies of the hybrid combination ((F₄ Khar’kovskaya 46 × Triticum dicoccum) × Khar’kovskaya 46), a short-stemmed line, whose progeny exhibited traits of hybrid dwarfism, segregated with a frequency close to 1: 15. Analysis of this line in F₃ and hybrids from crosses of the short-stemmed line from this combination with the long-stemmed accession T. turgidum L. (c-38097) showed that the stem size variability curves of the both lines have two peaks. Since these results cannot be explained by complementary interaction of D genes, hybrid dwarfism is probably controlled by pleiotropic action of two genes that have multiple pleiotropic effect on the D ₂ D ₂ D ₃ D ₃ genes. Two or three rounds of selection performed on the progeny of plants higher than 80–100 cm produces stable lines without hybrid dwarfism traits, i.e., the pleiotropic effect disappears.     

Structural organization of alpha-melanotropin molecules

Using a semi-empirical method, and a priori conformational analysis of the tridecapeptide alpha-melanocyte-stimulating (alpha-MSH) was carried out. The spatial structure of alpha-MSH can be described by ten low-energy conformations. Calculations produced the values of all dihedral angles of the backbones and side chains of these forms as well as intra- and inter-residue interaction energies.