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PubMed2006

Detection of a new SHV-type extended-spectrum beta-lactamase, SHV-31, in a Klebsiella pneumoniae strain causing a large nosocomial outbreak in The Netherlands.

A Klebsiella pneumoniae strain resistant to third-generation cephalosporins was isolated in the eastern Netherlands. The strain was found to carry a novel extended-spectrum beta-lactamase, namely, SHV-31. The combination of the two mutations by which SHV-31 differs from SHV-1, namely, L35Q and E240K, had previously only been described in association with one or more additional mutations.

Anti-Bacterial Agents↗
PubMed2002

Immunological characterization of 5-HT3 receptor transmembrane topology.

The 5-hydroxytryptamine3 (5-HT3) receptor is a member of the Cys-loop family of ligand-gated ion channels. These receptors are pentamers with the greatest homology to nicotinic acetylcholine (nACh) receptors. The proposed topological organization of a 5-HT3 receptor subunit is based largely on hydropathy profiles and by homology to nACh receptors, and indicates a large N-terminal extracellular domain and four transmembrane regions. There is, however, little direct evidence for this model. We therefore investigated the topology of the 5-HT3A receptor subunit using a panel of 5-HT3 receptor-specific antisera that interact with defined regions of the receptor. An antiserum generated against a short peptide from the N-terminal domain of the 5-HT3A receptor subunit, pAb120, was shown to bind to 5-HT3 receptor-expressing cells with intact cell membranes, indicating that the N-terminal end of the subunit is extracellular. Two antisera generated against regions of the loop between predicted transmembrane regions three and four did not bind to cells with intact membranes. However on membrane permeabilization these antibodies both bound to the receptor in intracellular areas, thus indicating that the loop between transmembrane domains three and four is intracellular. These data therefore provide direct evidence for an extracellular N-terminal domain and an intracellular loop between the third and fourth transmembrane domains, thus supporting the conventional ligand-gated ion channel subunit topological model.

Amino Acid Sequence↗
PubMed1995

Population dynamics of a meiotic/mitotic expansion model for the fragile X syndrome.

A model to explain the mutational process and population dynamics of the fragile X syndrome is presented. The mutational mechanism was assumed to be a multipathway, multistep process. Expansion of CGG repeats was based on an underlying biological process and was assumed to occur at two time points: meiosis and early embryonic development (mitosis). Meiotic expansion was assumed to occur equally in oogenesis and spermatogenesis, while mitotic expansion was restricted to somatic, or constitutional, alleles of maternal origin. Testable hypotheses were predicted by this meiotic/mitotic model. First, parental origin of mutation is predicted to be associated with the risk of a woman to have a full mutation child. Second, "contractions" seen in premutation male transmissions are predicted not to be true contractions in repeat size, but a consequence of the lack of mitotic expansion in paternally derived alleles. Third, a portion of full-mutation males should have full-mutation alleles in their sperm, due to the lack of complete selection against the full-mutation female. Fourth, a specific premutation-allele frequency distribution is predicted and differs from that based on models assuming only meiotic expansion. Last, it is predicted that approximately 65 generations are required to achieve equilibrium, but this depends greatly on the expansion probabilities.

Female↗
PubMed1984

Isolation and nucleotide sequence analysis of the beta-type globin pseudogene from human, gorilla and chimpanzee.

The beta-globin gene cluster of human, gorilla and chimpanzee contain the same number and organization of beta-type globin genes: 5'-epsilon (embryonic)-G gamma and A gamma (fetal)-psi beta (inactive)-delta and beta (adult)-3'. We have isolated the psi beta-globin gene regions from the three species and determined their nucleotide sequences. These three pseudogenes each share the same substitutions in the initiator codon (ATG----GTA), a substitution in codon 15 which generates a termination signal TGG----TGA, nucleotide deletion in codon 20 and the resulting frame shift which yields many termination signals in exons 2 and 3. The basic structure of these psi beta-globin genes, however, remains consistent with that found for functional beta-globin genes: their coding regions are split by two introns, IVS 1 (which splits codon 30, 121 base-pairs in length) and IVS 2 (which splits codon 104, 840 to 844 base-pairs in length). These introns retain the normal splice junctions found in other eukaryotic split genes. The three hominoid psi beta-globin genes show a high degree of sequence correspondence, with the number of differences found among them being only about one-third of that predicted for DNA sites evolving at the neutral rate (i.e. for sites evolving in the absence of purifying selection). Thus, there appears to be a deceleration in the rate of evolution of the psi beta-globin locus in higher primates.

Animals↗
PubMed1992

RNA progeny of an infectious two-base deletion cDNA mutant of potato spindle tuber viroid (PSTV) acquire two nucleotides in Planta.

Deletion mutations were generated in four structural domains of a potato spindle tuber viroid (PSTV) complementary DNA (cDNA) clone. Deletions of 3 to 5 nucleotides at the central conserved domain (CCGGG, positions 94 to 98), variable domain (GCCG, positions 146 to 149) and pathogenicity domain (CGA, positions 286 to 288) abolished infectivity of dimeric or trimeric cDNA constructs, or their in vitro transcripts. By contrast, a clone (St4) with a deletion of two nucleotides (UU, positions 339 and 340), located at the left terminal domain, retained infectivity when DNA or in vitro transcribed (+)RNA was used as inoculum. Sequencing of four cDNA clones of such viroid progeny demonstrated that two nucleotides were added at the deletion site. Two of the viroid progeny contained a CG addition. A third clone possessed a GU addition, whereas the fourth clone had a UU addition which represents a true reversion to full-length wild-type PSTV RNA. Ribonuclease protection assay of viroid progeny from St4-infected tomato plants suggested that only a negligible proportion of the St4 progeny were true revertants.

Base Sequence↗
PubMed1993

Use of new Escherichia coli/Streptomyces conjugative vectors to probe the functions of the two groEL-like genes of Streptomyces albus G by gene disruption.

Streptomyces albus G contains two groEL-like genes encoding three related proteins [Guglielmi et al., J. Bacteriol. 173 (1991) 7374-7381; Mazodier et al., J. Bacteriol. 173 (1991) 7382-7386]. Two proteins, HSP58 and HSP18, are synthesized from a single start codon site in groEL1. HSP18 may be a processed form of HSP58 or the result of early termination after frameshifting. The third protein, HSP56 is encoded by groEL2. In order to determine the physiological roles of these different proteins, both groEL genes were mutagenized by using a new approach for obtaining insertions in the streptomycete chromosome. Escherichia coli plasmids containing fragments homologous to groEL1 or groEL2 are unable to replicate in Streptomyces. They were introduced into S. albus by conjugation with E. coli. We then screened for mutants in which groEL1 or groEL2 had been disrupted due to recombination events (single or double crossover) at specific sites. Using this approach, the functionally indispensable domain of HSP58 was localized to within 249 amino acids of the N-terminus. HSP58 was not detected in the mutant generated by the most upstream insertion into the groEL1 coding sequence. However, HSP18 was synthesized in this mutant after heat shock. This groEL1 mutant was not impaired in growth in the 30-41 degrees C temperature range and SDS-PAGE analysis showed its overall pattern of gene expression to be indistinguishable from the parental strain. The inability to generate strains containing groEL2 disruptions strongly suggests that HSP56 is indispensable for growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2006

Expression cloning of three Rhizobium leguminosarum lipopolysaccharide core galacturonosyltransferases.

The lipid A and core regions of the lipopolysaccharide in Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, are strikingly different from those of Escherichia coli. In R. leguminosarum lipopolysaccharide, the inner core is modified with three galacturonic acid (GalA) moieties, two on the distal 3-deoxy-D-manno-octulosonic acid (Kdo) unit and one on the mannose residue. Here we describe the expression cloning of three novel GalA transferases from a 22-kb R. leguminosarum genomic DNA insert-containing cosmid (pSGAT). Two of these enzymes modify the substrate, Kdo2-[4'-(32)P]lipid IV(A) and its 1-dephosphorylated derivative on the distal Kdo residue, as indicated by mild acid hydrolysis. The third enzyme modifies the mannose unit of the substrate mannosyl-Kdo2-1-dephospho-[4'-(32)P]lipid IV(A). Sequencing of a 7-kb subclone derived from pSGAT revealed three putative membrane-bound glycosyltransferases, now designated RgtA, RgtB, and RgtC. Transfer by tri-parental mating of these genes into Sinorhizobium meliloti 1021, a strain that lacks these particular GalA residues, results in the heterologous expression of the GalA transferase activities seen in membranes of cells expressing pSGAT. Reconstitution experiments with the individual genes demonstrated that the activity of RgtA precedes and is necessary for the subsequent activity of RgtB, which is followed by the activity of RgtC. Electrospray ionization-tandem mass spectrometry and gas-liquid chromatography of the product generated in vitro by RgtA confirmed the presence of a GalA moiety. No in vitro activity was detected when RgtA was expressed in Escherichia coli unless Rhizobiaceae membranes were also included.

Carbohydrate Sequence↗
PubMed1998

Mutation in P0, a dual function ribosomal protein/apurinic/apyrimidinic endonuclease, modifies gene expression and position effect variegation in Drosophila.

In a search for modifiers of gene expression with the white eye color gene as a target, a third chromosomal P-element insertion mutant l(3)01544 has been identified that exhibits a strong pigment increase in a white-apricot background. Molecular analysis shows that the P-element insertion is found in the first intron of the gene surrounding the insertion site. Sequencing both the cDNA and genomic fragments revealed that the identified gene is identical to one encoding ribosomal protein P0/apurinic/apyrimidinic endonuclease. The P-element-induced mutation, l(3)01544, affects the steady-state level of white transcripts and transcripts of some other genes. In addition, l(3)01544 suppresses the variegated phenotypes of In(1)wm4h and In(1)y3P, suggesting a potential involvement of the P0 protein in modifying position effect variegation. The revertant generated by the precise excision of the P element has lost all mutant phenotypes. Recent work revealed that Drosophila ribosomal protein P0 contains an apurinic/apyrimidinic endonuclease activity. Our results suggest that this multifunctional protein is also involved in regulation of gene expression in Drosophila.

Alleles↗
PubMed1998

Characterization of Trichomonas vaginalis AP33 adhesin and cell surface interactive domains.

Adherence to host target cells is a critical step in establishing infection with the sexually transmitted pathogen Trichomonas vaginalis. Four parasite surface proteins mediating attachment to vaginal epithelial cells have been identified. One surface protein, termed AP33, was characterized further to identify domains interactive with previously generated antibodies and with host surface sites. N- and C-terminal deletion subclones were generated and tested for reactivity with both mAb and rabbit antiserum against AP33, and were also examined for their ability to bind to host cells. Surprisingly, the rabbit antiserum known to inhibit cytoadherence recognized an epitope(s) contained within 72 residues in the N-terminal half of the protein. However, the mAb epitope was immunoreactive with a 28-amino-acid region near the C-terminus. Subsequent mapping of this region with overlapping peptides identified a nine-amino-acid sequence reactive with the mAb. Equally surprising, two domains interactive with host cell surfaces were identified at distinct parts of AP33: one in the N-terminal half of the protein, and the other within 24 residues in the C-terminal third. Further analysis of the C-terminal binding domain revealed that a peptide representing this area could inhibit T. vaginalis cytoadherence by 40%.

Amino Acid Sequence↗
PubMed1996

The gene cluster directing O-antigen biosynthesis in Yersinia enterocolitica serotype 0:8: identification of the genes for mannose and galactose biosynthesis and the gene for the O-antigen polymerase.

The rfb gene cluster of Yersinia enterocolitica serotype O:8 (YeO8) strain 8081-c was cloned by cosmid cloning. Restriction mapping, deletion analysis and transposon mutagenesis showed that about 19 kb of the cloned DNA is essential for the synthesis and expression of the YeO8 O-side-chain in Escherichia coli. Deletion analysis generated a derivative that expressed semi-rough LPS, a phenotype typical of an rfc mutant lacking the O-antigen polymerase. The deletions and transcomplementation experiments allowed localization of the rfc gene to the 3'-end of the rfb gene cluster. The deduced YeO8 Rfc did not share significant amino acid sequence similarity with any other protein, but its amino acid composition and hydrophobicity profile are similar to those of identified Rfc proteins. In addition, the codon usage of the rfc gene is similar to other rfc genes. Nucleotide sequence analysis identified three other genes upstream of rfc. Two of the gene products showed 60-70% identity to the RfbM and RfbK proteins that are biosynthetic enzymes for the GDPmannose pathway of enterobacteria. The third gene product was about 50-80% identical to the bacterial GalE protein, UDPglucose 4-epimerase, which catalyses the epimerization of UDPglucose to UDPgalactose. Since mannose and galactose are both present in the YeO8 O-antigen repeat unit, the above three genes are likely to belong to the rfb gene cluster. A gene similar to the gsk gene downstream of rfc, and genes similar to adk and hemH upstream of the rfb gene cluster, were recognized. Thus the rfb gene cluster of YeO8 is located between the adk-hemH and gsk loci, and the order is adk-hemH-rfb-rfc-gsk in the chromosome. Also in other Yersinia spp., the locus downstream of the hemH gene is occupied by gene clusters associated with LPS biosynthesis.

Amino Acid Sequence↗
PubMed1992

Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan.

Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan.

Amino Acid Sequence↗
PubMed1990

Interaction of a nuclear protein with a palindromic sequence of the mouse immunoglobulin lambda 2-chain gene promoter is important for its transcription.

By using a gel mobility retardation assay, we detected the formation of three major complexes from the binding of nuclear proteins to the promoter of the immunoglobulin lambda 2-chain gene. Two of the complexes were generated by the presence of an unidentified nuclear factor(s) called herein NF-lambda 2. Although the sequences between lambda 2- and lambda 1-chain gene promoters are very similar, the lambda 1-chain promoter did not compete for the binding of NF-lambda 2 efficiently. The binding site of NF-lambda 2 was localized by DNase I footprinting to a 14-bp region which is about 30 bp upstream of the immunoglobulin octamer motif. This region, referred to as the NF-lambda 2 motif, is within an 18-bp region of twofold rotational symmetry. Experiments with oligomers containing either the NF-lambda 2 or the octamer motifs as competitors for binding and DNase I footprinting, showed that the third complex is the product of the simultaneous binding of an octamer-binding protein and NF-lambda 2. Changing the sequence of the NF-lambda 2 motif to that of the lambda 1-chain counterpart abolished the binding ability of NF-lambda 2. Concomitantly, the level of chloramphenicol acetyltransferase expression driven by the mutated lambda 2 promoter decreased by two- to fivefold when compared with that of the wild-type promoter. It is therefore concluded that the interaction of NF-lambda 2 with the NF-lambda 2 motif stimulates transcription of the lambda 2-chain gene.

Animals↗
PubMed2000

Carboxy terminal region of the Fanconi anemia protein, FANCG/XRCC9, is required for functional activity.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with eight complementation groups. Four of the FA genes have been cloned, and at least three of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a nuclear complex, required for the maintenance of normal chromosome stability. In the current study, mutant forms of the FANCA and FANCG proteins have been generated and analyzed with respect to protein complex formation, nuclear translocation, and functional activity. The results demonstrate that the amino terminal two-thirds of FANCG (FANCG amino acids 1-428) binds to the amino terminal nuclear localization signal (NLS) of the FANCA protein. On the basis of 2-hybrid analysis, the FANCA/FANCG binding is a direct protein-protein interaction. Interestingly, a truncated mutant form of the FANCG protein, lacking the carboxy terminus, binds in a complex with FANCA and translocates to the nucleus; however, this mutant protein fails to bind to FANCC and fails to correct the mitomycin C sensitivity of an FA-G cell line. Taken together, these results demonstrate that binding of FANCG to the amino terminal FANCA NLS sequence is necessary but not sufficient for the functional activity of FANCG. Additional amino acid sequences at the carboxy terminus of FANCG are required for the binding of FANCC in the complex. (Blood. 2000;96:1625-1632)

Animals↗
PubMed1975

Human anaphylatoxin (C3a) from the third component of complement. Primary structure.

C3a anaphylatoxin is derived from the third component (C3) of the blood complement system. Selective proteolysis of C3 by activated proenzymes indigenous to blood generates the C3a fragment. Human C3a was isolated from inulin-activated serum containing 6-aminohexanoic acid, according to recently published procedures (Hugli, T. E., Vallota, E., and Müller-Eberhard, H. J. (1975) J. Biol. Chem. 250, 1472-1498). The human C3a fragment is a highly cationic molecule exhibiting an approximate molecular weight of 9000 and composed of 77 amino acid residues. It consists of a single polypeptide chain containing 8% cysteine and lacks both tryptophan and carbohydrate. A tentative primary structure for the human C3a molecule, deduced from overlapping peptides obtained after cyanogen bromide cleavage, tryptic and chymotryptic digestion, is: See article. Two cystelhylcysteine sequences were established at positions 22, 23 and 56, 57 in human C3a. The 6 half-cystine residues in C3a are all interconnected through three disulfide linkages intersecting in a disulfide knot. The functionally amino acid residues distributed among 14 residues at the COOH-terminal end of C3a. This unusually cationic COOH-terminal region of C3a is presumed to play an important role in the interaction of this protein molecule with cellular receptors. A comparison between the linear sequence of human C3a and the NH2-terminal sequences of light and heavy chains of human immunoglobulin indicates that limited identity exists.

Amino Acid Sequence↗
PubMed1991

Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.

cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of tomato spotted Wilt virus defective interfering RNAs and detection of truncated L proteins.

Junction sites of 25 different defective interfering (DI) RNAs of tomato spotted wilt virus (TSWV) were characterized. The DI RNAs varied in size from 2.0 to 5.2 kilobases (kb) and contained a single internal deletion. The absence of DI RNAs smaller than 2 kb suggested a size constraint for the survival of TSWV DI RNAs. This hypothesis was reinforced by the finding of a dimeric DI RNA formed by two 1.6-long monomers linked head to tail. Three types of junction sites were found, one type originating from a simple deletion; the second type contained a few extra nucleotides of unknown origin; and the third type contained a stretch of three to five nucleotides, originally occurring at both sides of the deletion and of which one was deleted. In 19 of the 25 DI RNAs studied, the original reading frame was maintained, suggesting a selective preference of DI RNAs with translational potency. Truncated proteins encoded by these DI RNAs were indeed detected in the nucleocapsid preparations. Folding studies of the complete L RNA revealed that the calculated minimal energy of folding was at 16 degreesC lower than at 23 degrees, indicating a higher stability of this molecule at low temperatures. The results suggest an involvement of locally folded secondary structures in the process of deletion, rather than the requirement of certain sequences around the deletion point. The DI RNA generation in TSWV is essentially, as discussed, similar to the process of RNA recombination described in many viruses.

Base Sequence↗
PubMed1986

Genetic and molecular characterization of argB+ transformants of Aspergillus nidulans.

Thirty-three argB- to argB+ transformants of Aspergillus nidulans have been subjected to genetic and molecular analysis. Two showed high levels of mitotic instability although it is suggested that this is a consequence of heterokaryosis rather than instability of the transformation event. Most transformants resulted from the integration of the transforming DNA in tandem with the chromosomal argB locus. The maximum number of inserted sequences was two, to generate three copies of the argB locus. The other main transformant type showed replacement of the argB- mutation by the wild-type allele present on the transforming plasmid. Transformants were also recovered in which the transforming DNA had integrated into non-homologous chromosomal regions. Selfed or hybrid cleistothetica from all transformants, except the gene replacement types gave arginine requiring recombinants. Most transformants showed low levels of meiotic instability. Others displayed varying levels which in some cases differed between selfed and hybrid cleistotheticia. There was some correlation between meiotic instability and the nature of the transformation event. Diploid parasexual and aneuploid analysis located the integrated DNA in each transformant to chromosome III. Two transformants were isolated as heterozygous diploids. A third diploid was isolated as a stable mitotic segregant from one of the mitotically unstable transformants.

Arginine↗
PubMed1985

Early events in insect neurogenesis. I. Development and segmental differences in the pattern of neuronal precursor cells.

The earliest events in the development of the central nervous system in insect embryos involve the differentiation of a stereotyped pattern of individually identified neuronal precursor cells, called neuroblasts (NBs), each of which generates a stereotyped family of neuronal progeny. After gastrulation, the midventral region of the ectoderm becomes a neuroepithelium; it is from this sheet of seemingly uniform ectodermal cells that certain cells enlarge to become NBs while the rest of the cells either die or acquire other nonneuronal phenotypes. Here we focus on three aspects of neurogenesis. First, we examine the morphological changes associated with the differentiation of neuronal precursor cells and nonneuronal support cells. The differentiation of each cell type is reflected by its morphology; moreover, the identity of individual cells of certain cell types (e.g., a particular NB) is reflected by their position within the neuroepithelium. Second, we show that there is a characteristic temporal sequence of differentiation in the stereotyped pattern of NBs. Third, we show that this stereotyped pattern of NBs varies in a segment-specific way by the addition or deletion of particular neuronal precursor cells. These studies on the events of early neurogenesis set the stage for the experimental manipulations described in the following paper (C. Q. Doe and C.S. Goodman, 1985, Dev. Biol. 110, 206-219).

Abdomen↗