Status and reaction to anoxia of rat fetuses developing in conditions of placental insufficiency

Significant adverse correlation of the fetus mass and the degree of its hydration was noted at term in rats with placental insufficiency induced by ligation of about 40% of preplacental vessels on the 16th day of gestation. Disorders revealed in transplacental water-salt metabolism are apparently of some importance for the pathogenesis of fetal growth retardation. Even moderate retardation alters fetal respiratory activity in anoxia, which is associated with the changes in the function of respiratory centres and neuromuscular abnormalities.     

A model system for the study of repair of DNA double-strand breaks in Saccharomyces cerevisiae

The efficiency of "LiCl transformation" in Saccharomyces cerevisiae haploid cells by an autonomously replicating pLL12 plasmid carrying yeast LEU2 and LYS2 genes is increased (by an order or more) when the plasmid is linearized by the restriction endonuclease XhoI cleavage of a unique site in LYS2 gene. Transformants were selected on the medium lacking leucine. This phenomenon has been shown to be a result of recombinational repair of double-strand breaks (DSB) of plasmid DNA stimulated by a restriction endonuclease. The kinetic data have shown the process of plasmid DNA DSB repair to consist of two phases. The completion of the first phase occurs during an hour and the second phase occurs in 14-18 hours. DNA double-strand gaps (the deleted sequences of plasmid LYS2 gene in DSB region) with maximal length of 2-2.5 kb are repaired with the same efficiency as DSB. The genetic control of the recombinational repair of plasmid DNA DSB has been studied.     

The complete amino-acid sequence of the alpha and beta subunits of B-phycoerythrin from the rhodophytan alga Porphyridium cruentum

Determination of the complete amino-acid sequence of the subunits of B-phycoerythrin from Porphyridium cruentum has shown that the alpha subunit contains 164 amino-acid residues and the beta subunit contains 177 residues. When the sequences of B- and C-phycoerythrins are aligned with those of other phycobiliproteins, it is obvious that B-phycoerythrin lacks a deletion at beta-21-22 present in C-phycoerythrin. However, relative to C-phycoerythrin from Fremyella diplosiphon (Calothrix) (Sidler, W., Kumpf, B., Rüdiger, W. and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-642), B-phycoerythrin has deletions at beta-141k-o, beta-142, beta-143, beta-147 and beta-148. The four singly-linked phycoerythrobilins at positions alpha-84, alpha-143a, beta-84 and beta-155, and the doubly-linked phycoerythrobilin at position beta-50/61 are at sites homologous to the attachment sites in C-phycoerythrin. The aspartyl residues (alpha-87, beta-87, and beta-39), that interact with the bilins at alpha-84, beta-84, and beta-155 in C-phycocyanin, are found in the homologous positions in B-phycoerythrin. B-Phycoerythrin, in common with other phycobiliproteins, contains a N gamma-methylasparagine residue at position beta-72.     

In vitro attachment of bilins to apophycocyanin. III. Properties of the phycoerythrobilin adduct

Addition of phycoerythrobilin (PEB) to apophycocyanin at pH 7.0 resulted in covalent adduct formation. The adduct showed absorbance maxima at 575 and 605 nm and fluorescence emission maxima at 582 and 619 nm. Analysis of bilin peptides obtained upon tryptic digestion of the adduct showed residues alpha-Cys-84 and beta-Cys-82 to be the sites of bilin addition. The product of PEB addition at the alpha-Cys-84 site was shown by 1H NMR analysis to be a dihydrobiliviolinoid peptide-linked pigment differing in structure from that of the naturally occurring PEB-adduct by the presence of a double bond in between C2 and C3 of ring A. At the beta-Cys-82 site both a dihydrobiliviolinoid and a PEB adduct were obtained. Biliverdin also formed a covalent adduct with apophycocyanin with a lambda max of 669 nm. These results show that the spontaneous in vitro addition of bilins to apophycocyanin does not exhibit the site selectivity of bilin addition observed in vivo. This offers the opportunity to form novel semisynthetic phycobiliproteins.     

Mutability of the LYS2 gene in diploid saccharomycete yeasts. I. The occurrence of spontaneous mutants and mutants induced by x-ray and ultraviolet radiation

More than 3000 spontaneous and induced lys2 mutants were obtained in haploid and diploid strains of yeast Saccharomyces. The ability to utilize alpha-aminoadipate was used for lys2 mutant screening. The spontaneous and induced mutation rates were measured in haploid and diploid strains. Mitotic segregation of pho1 marker linked to LYS2 was studied in lys2 mutants obtained in diploid strains. Fertility of diploid lys2 mutants was tested. The conclusion to be drawn from the data presented is that mutations appeared in one of two homologous chromosomes and then segregated by mitotic homozygotization.     

Incorporation of [6-3H] glucose into lipid, protein, RNA and DNA of slices of differentiating rat cerebral cortex

Abstract— An assay in vitro utilizing [6^?3H)glucose as precursor for synthesis of lipids, proteins, RNA and DNA was developed for incubated slices of rat cerebral cortex. The developmental changes in synthesis of macromolecules were followed during differentiation of rat cerebral cortex. The incorporation of glucose into lipids and proteins decreased 10-fold in incubated slices of cerebral cortex during progression from foetal to adult ages. In contrast, the specific radioactivities of RNA and DNA in incubated slices increased from the values at 3 days prepartum to peaks at 2–4 weeks postpartum.     

Construction of a cDNA for the human c-fes protooncogene protein-tyrosine kinase and its expression in a baculovirus system.

Previous studies have established that the 93-kDa protein-tyrosine kinase (PTK) encoded by the human c-fes protooncogene plays an active role in the induction of terminal myeloid differentiation. However, this enzyme is expressed at very low levels in myeloid cells, making isolation of sufficient quantities for detailed biochemical analysis difficult. To overcome this problem, we used the polymerase chain reaction to construct a full-length c-fes cDNA from overlapping 5' and 3' partial cDNA sequences. The c-fes cDNA was expressed at high levels in a baculovirus system, and the catalytically active recombinant c-fes gene product p93c-fes was partially purified by DEAE-Sepharose and tyrosine-agarose chromatography. Recombinant p93c-fes was indistinguishable from the native protein in terms of its apparent molecular weight following SDS-PAGE, catalytic activity, Km for poly(Glu,Tyr)4:1, antigenicity, and phosphopeptide pattern generated with Staphylococcus aureus protease.     

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.     

Genes of the R-phycocyanin II locus of marine Synechococcus spp., and comparison of protein-chromophore interactions in phycocyanins differing in bilin composition

R-phycocyanin II (RPCII) is a recently discovered member of the phycocyanin family of photosynthetic light-harvesting proteins. Genes encoding the and subunits of RPCII were cloned and sequenced from marine Synechococcus sp. strains WH8020 and WH8103. The deduced amino acid sequences of RPCII were compared to two other types of phycocyanin, C-phycocyanin (CPC) and phycoerythrocyanin (PEC). These three types vary in the composition of their covalently bound bilin prosthetic groups. In terms of amino acid sequence identity RPCII is highly homologous to CPC and PEC, suggesting that the known three-dimensional structures of the latter two are representative of RPCII. Thus the amino acid residues contacting the three bilins of RPCII could be inferred and compared to those in CPC and PEC. Certain residues were identified among the three phycocyanins as possibly correlating with specific bilin isomers. In overall sequence RPCII and CPC are more homologous to one another than either is to PEC. This probably reflects functional homology in the roles of RPCII and CPC in the transfer of light energy to the core of the phycobilisome, a function not attributed to PEC. The genomes of Synechococcus sp. strains WH8020, WH8103 and WH7803 share homologous open reading frames in the vicinity of RPCII genes. The nucleotide sequence extending 3 from RPCII genes in strain WH8020 revealed two open reading frames homologous to components of an CPC phycocyanobilin lyase. These open reading frames may encode a lyase specific for the attachment of phycoerythrobilin to RPCII.