UDP-GalNAc : GalNAc-mucin α-N-acetylgalactosamine transferase activity in human intestinal cancerous tissues

GalNAc transferase activities of 6 human intestinal cancerous tissues were examined using bovine submaxillary gland mucin and its desialylated derivative, asialomucin, as acceptors. A Triton X-100 extract of these tissues was used as an enzyme source. All the tissues examined had GalNAc transferase that catalyzes the transfer of GalNAc from UDP-GalNAc to serine or threonine residues of the polypeptide chain. One of 6 specimens showed in addition UDP-GalNAc:GalNAc-mucin α-GalNAc transferase activity, synthesizing a disaccharide unit, GalNAcα→ GalNAc, when asialomucin was used as an acceptor. This carbohydrate structure was deduced on the basis of results of gel filtration, exoglycosidase digestion, and high-voltage paper electrophoresis.GalNAc transferaseHuman intestinal cancerous tissueBovine submaxillary gland mucin O-Glycosidically linked sugar chain     

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5 and 3 splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg²⁺- and ATP-dependent manner, and this activity was inhibited by 4-(9-acridinylamino)-3-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII ^ts yeast mutant.     

Molecular cloning,characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis)

The gene and the RNA from Arabidopsis thaliana for the plastid-located glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) and their encoded product have been studied. The gene (designated ATS1) was isolated by screening a DASH genomic library for cross-hybridization with a radiolabeled probe prepared from cDNA for GPAT from squash. cDNA clones representing the mRNA were isolated by screening a ZAPII cDNA library for hybridization with a radiolabeled probe prepared from a DNA fragment of ATS1. The nucleotide sequences of the gene and the cDNA were determined, and the 5 end of the RNA was mapped by primer extension. Sequences similar to the TATA box, polyadenylation sequences and intron-splicing sequences were found at the expected locations. The pre-mRNA was 3288 nucleotides long and contained 5 and 3-untranslated sequences of 57 and 442 nucleotides, respectively. The coding sequence of 1377 nucleotides was interrupted by 11 introns of 1412 nucleotides in total and the 3-untranslated sequence contained another intron of 94 nucleotides. The open-reading frame encoded a polypeptide of 459 amino acid residues, the amino acid sequence of which was highly homologous to those of precursors to plastid-located GPATs from squash and pea. The enzymatic activity of a gene product that was over-produced in Escherichia coli confirmed the indentity of the gene.Abbreviations ACP acyl carrier protein - GPAT glycerol-3-phosphate acyltransferase - IPTG isopropyl--thiogalactopyranoside.     

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Summary Previous genetic analyses indicated that translational frameshifting in the –1 direction occurs within the run of six adenines in the sequence 5-TTAAAAAACTC-3 at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5-CTTAAAAACTC-3 at positions 305–315 carrying the termination codon TAA in the B-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The -galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5-TTAAAAAACTC-3, indicating that –1 frameshifting does occur within the run of adenines.     

Localization and nucleotide sequence of the gene for the membrane polypeptide D2 from pea chloroplast DNA

Summary The gene for the membrane polypeptide D2 has been mapped on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of MW 39.5 kD. A potential ribosome binding site is located 6 nucleotides upstream from the initiation codon and there are two sets of putative promotor sequences in the 5 flanking region. The polypeptide has a high content of hydrophobic amino acids, predominatly grouped in clusters of 20 or more residues. The 3 end of the D2 gene is overlapped by 50 nucleotides of a second open reading frame (UORF I) which is at least 369 nucleotides long. Based on current data we suggest the D2 polypeptide to be a constituent of photosystem II (PSII).     

Characterization of a class Ib gene of the rat major histocompatibility complex

The cDNA and a partial genomic sequence of a rat class I major histocompatibility (RT1) gene, 11/3R, is reported here. The sequence contains several unique amino acid residues at certain positions, mutations in exon 7 (which is not expressed), a mutation of the canonical exon 8 stop codon to a sense codon, and includes a long 3 unstranslated region (utr). The structure of exon 7 differs from that found in most rat class I genes and resembles exon 7 of most H-2K,D,L.Q genes. Parts of the 3 noncoding region are homologous to the RT1.A-4 and certain H-2 genes. Expression is detectable by northern blot analysis in mitogen-stimulated lymphocytes only, by polymerase chain reaction (PCR) in each tissue tested. After transfection into L cells 11/3R can be shown to be expressible at the cell surface. Probes derived from the 3 noncoding part crosshybridize with a number of restriction fragments which map to the RT1.C region, thus defining a subfamily of RT1.C region genes. Several members of this subfamily are deleted in the M1 RT1 mutant. The 11/3R gene presents typical features of a class Ib gene. Aspects of evolution and the potential of the gene are discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank molecule sequence data base and have been assigned the accession numbers X67503 ande X67504.     

Characterization of the genes and attachment sites for site-specific integration of plasmid pSE101 in Saccharopolyspora erythraea and Streptomyces lividans

The 11.3 kb plasmid pSE101 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site and into the chromosome of Streptomyces lividans at many sites. Multisite integration in S. lividans was also observed when a 1.9 kb segment of pSE101 containing attP and adjacent plasmid sequence was used to transform a pSE101^– S. lividans host. Nucleotide sequencing of this segment revealed the presence of a complete open reading frame (ORF) designated int, encoding a putative polypeptide of 448 amino acids that shows similarities to site-specific recombinases of the integrase family. Sequencing of the 1.3 kb segment upstream of int revealed the presence of three additional ORFs: the one most distal to int encodes a putative 76 amino acid basic polypeptide analogous to the Xis proteins of a number of bacteriophages. Nucleotide sequencing of attP, and the attB, attL and attR sites from Sac. erythraea revealed a 46 by sequence common to all sites with no duplications of chromosomal sequences in the integrated state. A putative structural gene for a tRNA^Thr was found to overlap the 46 by common sequence at attB. Sequencing of four pSE101 integration sites (attB) and corresponding attL and attR sites in S. lividans showed that the 46 by sequence was present at each attR site, whereas only the first three bases, CTT, were retained at each attL and attB site. A feature common to the four attB sites and to attB is a highly conserved 21 by segment with inverted repeats flanking the CTT sequence. This indicates that crossover at each attB site in S. lividans employed attP and a site within a 5 by sequence in attB and suggests that the secondary structure of the 21 by sequence is important for site-specific integration at attB or attB.     

A single-base deletion in soybean flavonoid 3′-hydroxylase gene is associated with gray pubescence color

The T locus of soybean (Glycine max (L.) Merr.) controls pubescence and seed coat color and is presumed to encode flavonoid 3-hydroxylase (F3H). The dominant T and the recessive t allele of the locus produce brown and gray pubescence, respectively. PCR primers were constructed based on the sequence of a soybean EST clone homologous to the F3H gene. A putative full-length cDNA, sf3h1 was isolated by 3 and 5 RACE. Sequence analysis revealed that sf3h1 consists of 1690 nucleotides encoding 513 amino acids. It had 68% and 66% homology with corresponding F3H protein sequences of petunia and Arabidopsis, respectively. A conserved amino acid sequence of F3H proteins, GGEK, was found in the deduced polypeptide. Sequence analysis of the gene from a pair of near-isogenic lines for T, To7B (TT, brown) and To7G (tt, gray) revealed that they differed by a single C deletion in the coding region of To7G. The deletion changed the subsequent reading frame resulting in a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain. Genomic Southern analysis probed by sf3h1 revealed restriction fragment length polymorphisms between cultivars with different pubescence color. Further, sf3h1 was mapped at the same position with T locus on LG3(c2). PCR-RFLP analysis was performed to detect the single-base deletion. To7B and three cultivars with brown pubescence exhibited shorter fragments, while To7G and three cultivars with gray pubescence had longer fragments due to the single-base deletion. The PCR-RFLP marker co-segregated with genotypes at the Tlocus in a F₂ population segregating for the T locus. The above results strongly suggest that sf3h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.     

The malate synthase gene of cucumber

The complete sequences of a full-length cDNA clone and a genomic clone encoding the Cucumis sativus glyoxysomal enzyme malate synthase, have been determined. The sequences have enabled us to identify putative control regions at the 5 end of the gene, three introns, and possible alternative polyadenylation sites at the 3 end. The deduced amino acid sequence predicts a polypeptide of 64961 molecular weight, which has 48% identity with that of Escherichia coli. Comparison of the sequence of malate synthase from cucumber with that from E. coli and with other glyoxysomal and peroxisomal enzymes, shows that a conserved C-terminal tripeptide is a common feature of those enzymes imported into microbodies.     

Evolutionary conservation of the 3′ ends of members of a family of giant secretory protein genes inChironomus pallidivittatus

Summary Two cDNA clones representing the 3-end regions of BR1 and BR2 75S mRNA were obtained fromChironomus pallidivittatus. The regular structure characterizing the core of these genes, consisting of tandemly arranged repeat units, changes into a more irregular structure toward the 3 end. Distal to a standard type of repeat unit with a characteristic excess of positive charges, a new type of repeat with a high, negative charge density is interspersed among parts of the standard unit. The last 111 amino acids before the stop codon represent a unique region distinctly different in amino acid composition from upstream regions, and include two partially homologous hydrophobic regions. Sequence comparison of 3-end regions from clones representing BR1 and BR2 genes indicates striking sequence conservation in the unique part of the region. Analysis of the level of silent site divergence shows that the homology increases in the 3 direction up to the polyadenylation site. That the unique region is retained as a part of the secreted protein is shown by Western blotting.     

Characterization ofVitis vinifera L. glutamine synthetase and molecular cloning of cDNAs for the cytosolic enzyme

Grapevine (Vitis vinifera L.) glutamine synthetase (GS) was analysed into two distinct classes of isoforms; one of them was present in both leaf and root tissues while the other one showed leaf specificity. Western blot analysis revealed that grapevine GS consists of three types of polypeptides of distinct size and differential tissue specificity. Two structurally distinct cDNA clones, pGS1;1 and pGS1;2, encoding grapevine GS were isolated from a cell suspension library and characterized. Both clones contained open reading frames encoding for polypeptides of 356 amino acids with a predicted molecular mass of about 39 kDa. Although the coding sequences of pGS1;1 and pGS1;2 were 84% similar, their 5-and 3-untranslated sequences showed only 40% similarity. The coding sequences of the two clones and the derived amino acid sequences showed higher homology to cytosolic than to chloroplastic GSs of other higher plants indicating that the cDNAs isolated encode for cytosolic isoforms of grapevine GS. Southern blot analysis suggested the existence of more than two GS genes in the grapevine genome. In northern blots both clones were hybridized to mRNAs of about 1.4 kb that are differentially expressed in the various tissues. Supply of nitrate or ammonium in the cell suspension culture medium, as a sole nitrogen source, resulted in differential response of the pGS1;1-and pGS1;2-related genes.     

Chicken deoxyribonuclease: purification,characterization, gene cloning and gene expression

Chicken DNase was purified to apparent homogeneity from the pancreas extract. It showed two isoforms, A and B forms, on cation-exchange chromatography. On SDS-PAGE it was a 30-kDa protein. When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882. The enzyme was bound to concanavalin A, indicating its glycoprotein nature. The carbohydrate side chain could be removed by endoglycosidase F. Chicken DNase was activated by metal ions and for half-maximum activation, Mn²⁺ and Mg²⁺ required were 1 mM and 4 mM, respectively. The pH optimum was between 7 and 8 depending on the metal ions used. In the presence of Cu²⁺, it was almost completely inactivated by 0.1 M iodoacetate within 1 min. In the absence of Ca²⁺ at pH 8, chicken DNase resisted to the trypsin or -mercaptoethenol inactivation. When the purified enzyme was subjected to protein sequencing, 93% of the sequence was established. Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced. The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5-untranslated region and 166 of the 3 and, in the 5-untranslated region, two types of sequences occurred. The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids. As compared with mammalian DNases, chicken DNase had an overall 58 ± 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide. The cDNA was cloned into the pET15b expression vector. When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates.     

Nucleotide sequence analysis of a cDNA clone encoding the 34K movement protein gene of odontoglossum ringspot virus,ORSV-Cy,the Korean isolate

The partial nucleotide sequence of the 3-terminal region of the Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) from cool-growing Cymbidium was determined. The sequence contained a full length open reading frame (ORF) coding for the viral cell-to-cell movement protein (MP). The ORF was located upstream of the coat protein gene and 105 nucleotides longer than that of tobacco mosaic virus (TMV). The ORF predicts a polypeptide chain of 303 amino acids with a molecular weight of 33573. The ORF contained a similar region of conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA was located at about 1,100 nucleotides away from the 3 end. The predicted amino acid sequence for the MP gene of ORSV-Cy is more closely related to pepper mild mottle virus (PMMV), TMV-vulgare and TMV-Rakkyo than to tobacco mild green mosaic virus (TMGMV), TMV-L, cowpea strain of TMV (SHMV), and cucumber green mottle mosaic virus (CGMMV).