The UL2 Open Reading Frame of Bovine Herpesvirus 1 Encodes a Uracil-DNA Glycosylase

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence downstream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl β-D -thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.     

Sequence analysis of the 5′-flanking region of the gene encoding human N-acetylglucosaminyltransferase III

We have isolated and characterized the immediate (1651 bp) 5′-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5′-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2-binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.     

Synthesis of biodegradable emulsifier using lipase in anhydrous pyridine

Summary Synthesis of a bioemulsifier using a lipase from Pseudomonas sp. with fructose and vinyl laurate was carried out in anhydrous pyridine. The synthetic product was identified as laurylfructose with an emulsifying activity on various hydrocarbons, edible oils and petroleum oils. The compound reduced the surface tension of distilled water from 72 mN/m to 29 mN/m and the interfacial tension of water/n-hexadecane from 50 mN/m to 6 mN/m.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Genetic and clonal diversity in Korean populations ofVitex rotundifolia (Verbenaceae)

Vitex rotundifolia L.f. is a woody perennial and has sexual and asexual modes of reproduction. Allozyme study was conducted on 550 plants in 13 Korean populations. The levels of genetic variability and divergence within and among populations, respectively, are considerably lower and higher than the mean values for woody plants with similar life history tralts. Mean percentage of polymorphic loci (P _P), mean number of alleles per locus (A _P), and mean genetic diversity (He _P) within populations ofV. rotundifolia were: 16.7%, 1.21, and 0.047. On average, about 79% of the total variation inV. rotundifolia was common to all populations (meanG _ST=0.208). In addition, significant differences in allele frequencies among populations were found in all polymorphic loci examined (P<0.001). On the other hand, levels of genotypic diversity within and among populations were moderate. About 44% (18/41) of multilocus genotypes were “local genotypes” (genotypes occurring in only one population), whereas only one “widespread genotype” (genotypes occurring in more than 75% of the populations) were detected. The mean number of multilocus genotypes per population (G) and mean genotypic diversity index (D _G) were 8.4 and 0.74, respectively. Most common multilocus genotypes found in populations were homozygous for five polymorphic loci. The abundance of ramets of these genets is responsible for the low levels of expected heterozygosity within populations. The results indicate that clonal reproduction may act as an enhancer of genetic drift by reducing effective size of local populations ofV. rotundifolia.     

Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of theras-Gene-Encoded p21 protein with the structure of the normal (non-transforming) wild-type protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.     

Cell aggregation and neurite growth in gels of extracellular matrix molecules

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three-dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 mug/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (>/=0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four- to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. (c) 1994 John Wiley & Sons, Inc.     

Expression and secretion of human lipocortin-1 by promoter and signal sequence of STA1 from Saccharomyces diastaticus

Summary For the secretion of human lipocortin-1 (LC-1) in yeast, a expression and secretion vector was constructed by using the promoter and signal sequence of glucoamylase gene (STA1) of Saccharomyces diastaticus. After the cDNA of human LC-1 was ligated with the secretion vector, the resulting hybrid plasmid was transformed into S. diastaticus. When the recombinant S. diastaticus was cultivated in YPD medium, LC-1 was expressed and secreted into the extracellular medium, yielding LC-1 protein at a concentration of 2.5 g/mL.