Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.      

Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster

A plasmid library of Acinetobacter calcoaceticus HindIII fragments was constructed, and clones that complemented an Escherichia coli pabA mutant were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were obtained. We infer that complementation of E. coli pabA mutants was the result of the expression of the amphibolic anthranilate- synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that the plasmid insert carried the entire trpGDC gene cluster. In E. coli minicells, the plasmid insert directed the synthesis of polypeptides of 44,000, 33,000, and 20,000 daltons, molecular masses that are consistent with the reported molecular masses of phosphoribosylanthranilate transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase component II, respectively. A 3,105- bp nucleotide sequence was determined. Comparison of the A. calcoaceticus trpGDC sequences with other known trp gene sequences has allowed insight into (1) the evolution of the amphibolic trpG gene, (2) varied strategies for coordinate expression of trp genes, and (3) mechanisms of gene fusions in the trp operon.      

Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli

A combination of site-specific mutagenesis and 19F nuclear magnetic resonance has been used to investigate the structural properties of D-lactate dehydrogenase, a membrane-associated enzyme of Escherichia coli. The protein (65,000 Da) has been labeled with 5-fluorotryptophan for 19F nuclear magnetic resonance studies. Tryptophan has been substituted for individual phenylalanine, tyrosine, isoleucine, and leucine residues at various positions throughout the enzyme molecule, and the fluorinated native and substituted tryptophan residues have been used as probes of the local environment. All 24 mutants thus generated are expressed in E. coli. Ten are fully active and purfiable following the usual procedure, while 14 either are inactive or produce low levels of activity. The amount of active enzyme produced from the low-yield mutants is dependent on the temperature at which synthesis is carried out, with more active enzyme produced at 18 degrees C than at 27, 35, or 42 degrees C. Cells grown at 27 degrees C and then incubated at 42 degrees C retain 90-100% of their activity. All of the expressed protein from the inactive mutants is Triton-insoluble, aggregated, and not readily purfiable; the inactive mutant protein appears to be improperly folded. Most of the expressed D-lactate dehydrogenase from the partially active mutants is also Triton-insoluble; a small fraction, however, is soluble in Triton and can be purified to yield active enzyme. All the purified enzymes from these low-yield mutants of D-lactate dehydrogenase have essentially normal VmaxS, and all but two have normal KmS. Once purified, the low-yield mutant enzymes are stable at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distributions of two recently inserted long interspersed elements of the L1 repetitive family at the Alb and beta h3 loci in wild mice populations

The presence of the L1 sequences, L1Md4 next to the pseudogene beta h3 and I12 found in the twelfth intron of the albumin gene, in certain strains of laboratory mice but not of others has led to the suggestion that these sequences were recent insertions into the Mus mus domesticus genome. To be sure that they are really recent insertions and not relics of an ancestral chromosome, we investigated the presence or absence of these sequences in populations of wild mice belonging to the semispecies M. m. domesticus and M. m. musculus as well as in other species of the genus Mus and in related murids. The sequence I12 in the albumin gene was found in 34% of the chromosomes of the wild mice belonging to M. m. domesticus and to a lesser extent (6%) in M. m. musculus. Of 114 M. m. domesticus chromosomes, L1Md4 was found in only nine, seven of which came from the same locality. Its presence was associated with the haplotype Hbbp, which is relatively rare in European populations of M. musculus. Since there was no evidence for the presence of these two L1 sequences in more distantly related species, we conclude that they are recent insertions in the M. musculus genome.      

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells.

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37 degrees C to 22 degrees C with an activation energy of 11.8 +/- 0.2 kcal/mol in this region. There is a sharp decrease in agglutination rate below 22 degrees C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 (2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.     

Protein and m-RNA expression of farnesyl-transferases,RhoA and RhoB in rat liver hepatocytes: action of perillyl alcohol and vitamin A <Emphasis Type="BoldItalic">in vivo</Emphasis>.

Summary We analysed the action, in rats in vivo, of the protein isoprenylation inhibitor perillyl alcohol (POH) and that of vitamin A, alone or in association, on m-RNA and protein expression of farnesyltransferases (FTases α and β subunits) and their protein substrates RhoA and RhoB, in isolated hepatocytes. Combined administration of POH and vitamin A induced a sharp decrease in FTase α protein after 96 h, suggesting an involvement not only of farnesyltransferases but also of geranylgeranyltransferases, which share the FTase α protein. FTase β protein did not decrease. POH plus vitamin A, in contrast with POH or vitamin A alone, induced a decrease in RhoB protein, probably because of different cleavages. No modification was observed in RhoA protein. Vitamin A alone increased RhoB m-RNA and protein expression. As one of the functions of RhoB is cell polarisation, these data support our previous hypothesis of a polarised transport of vitamin A from hepatocytes to hepatic stellate cells. As the behaviours of m-RNAs and proteins in this study were often different, cytoplasmic metabolic pathways must be considered for the parameters studied. The behaviour of Rho B, which is thought to have an antioncogene function, is discussed in view of its isoprenylated forms in the membranes. These preliminary findings stress the need, when studying the association of two isoprenoids in cancer therapy, to consider normal as well as tumour-bearing animals.     

1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: Presence of an amino-terminal helix in solution

Summary ¹H, ¹⁵N and ¹³C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007–1010]. In the group I enzyme from porcine pancreas, the amino-terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G.H., Kaptein, R. and Verheij, H.M. (1995) Nature Struct. Biol., 2, 402–406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well-positioned aminoterminal helix in the absence of substrate. Therefore, there is a clear difference between the conformations of group I and group II enzymes in solution. These conformational differences suggest that formation of the amino-terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases.Abbreviations PLA2 phospholipase A2 - App-D49 phospholipase from Agkistrodon piscivorus piscivorus - NOE nuclear Overhauser effect     

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.