Sequence of the Escherichia coli O26 O antigen gene cluster and identification of O26 specific genes

Escherichia coli associated with outbreaks of gastroenteritis and hemolytic uremic syndrome include clones with O antigens O157 and O111. However, O26 has emerged as an O antigen present in pathogenic strains, particularly those implicated in cases of infantile gastroenteritis worldwide. The O26 O antigen gene cluster was sequenced. It was found to contain the genes expected for biosynthesis of nucleotide sugars L-rhamnose, N-acetyl-L-fucosamine and N-acetyl-glucosamine, as well genes for O unit flippase, O antigen polymerase and potential transferase genes. By polymerase chain reaction testing against representative strains for the 166 Escherichia coli O serogroups and some randomly selected Gram-negative bacteria, we identified three O antigen genes that are highly specific to O26. This work provides the basis for a sensitive test for the rapid detection of pathogenic clones with the O26 antigen, which has implications for public health, especially in the control of food-borne outbreaks.     

The 26S proteasome Rpn10 gene encoding splicing isoforms: evolutional conservation of the genomic organization in vertebrates

Recognition of polyubiquitinated substrates by the 26S proteasome is a key step in the selective degradation of various cellular proteins. The Rpn10 subunit of the 26S proteasome can bind polyubiquitin conjugates in vitro. We have previously reported the unique diversity of Rpn10, which differs from other multiple proteasome subunits, and that the mouse Rpn10 mRNA family is generated from a single gene by developmentally regulated alternative splicing. To determine whether such alternative splicing mechanisms occur in other species, we searched for Rpn10 isoforms in databases and in our original PCR products. Here we report the genomic organization of the Rpn10 gene in lower vertebrates and provide evidence for the competent generation of distinct forms of Rpn10 by alternative splicing through evolution.     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

Plant 21D7 protein, a nuclear antigen associated with cell division, is a component of the 26S proteasome.

Previously, we cloned a carrot (Daucus carota L.) cDNA encoding a 45-kD protein, 21D7, located in the nuclei of proliferating cells. The 21D7 protein is similar to the partial sequence of a regulatory subunit of the bovine 26S proteasome, p58 (G. DeMartino, C.R. Moomaw, O.P. Zagnitko, R.J. Proske, M. Chu-Ping, S.J. Afendis, J.C. Swaffield, C.A. Slaughter [1994] J Biol Chem 269: 20878-20884) and to the deduced sequence encoded by the Saccharomyces cerevisiae gene SUN2 (M. Kawamura, K. Kominami, J. Takeuchi, A. Toh-e [1996] Mol Gen Genet 251: [146-152]). In our work, the expression of plant 21D7 cDNA rescued the yeast sun2 mutant. Fractionation of carrot and spinach (Spinacia oleracea L.) crude extracts showed that the 21D7 protein sedimented with the active 26S proteasomes. The cessation of cell proliferation in carrot suspensions at the stationary phase caused 26S proteasome dissociation and, correspondingly, the 21D7 protein sedimented together with the free regulatory complexes of the 26s proteasomes. Large-scale purification of carrot 26s proteasomes resulted in co-isolation of the 21D7 protein. Polyacrylamide gel electrophoresis under nondenaturing conditions showed that the 21D7 protein had the same mobility as the 26S proteasome and that proteasome dissociation changed the mobility of the 21D7 protein accordingly. We conclude that the 21D7 protein is a subunit of the plant 26S proteasome and that it probably belongs to the proteasome regulatory complex.     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

Mapping of five new putative anion transporter genes in human and characterization of SLC26A6, a candidate gene for pancreatic anion exchanger

A second distinct family of anion transporters, in addition to the classical SLC4 (or AE) family, has recently been delineated. Members of the SLC26 family are structurally well conserved and can mediate the electroneutral exchange of Cl(-) for HCO(-)(3) across the plasma membrane of mammalian cells like members of the SLC4 family. Three human transporter proteins have been functionally characterized: SLC26A2 (DTDST), SLC26A3 (CLD or DRA), and SLC26A4 (PDS) can transport with different specificities the chloride, iodine, bicarbonate, oxalate, and hydroxyl anions, whereas SLC26A5 (prestin) was suggested to act as the motor protein of the cochlear outer hair cell. We report the expansion of the SLC26 family with five new members in chromosomes 3, 6, 8, 12, and 17 and mapping of SLC26A1 to 4p16.3. We have characterized one of them, SLC26A6, in more detail. It maps to chromosome 3p21.3, encodes a predicted 738-amino-acid transmembrane protein, and is most abundantly expressed in the kidney and pancreas. Pancreatic ductal cell lines Capan-1 and Capan-2 express SLC26A6, and immunohistochemistry localizes SLC26A6 protein to the apical surface of pancreatic ductal cells, suggesting it as a candidate for a luminal anion exchanger. The functional characterization of the novel members of this tissue-specific gene family may provide new insights into anion transport physiology in different parts of the body.     

The biosynthetic gene cluster for the 26-membered ring polyene macrolide pimaricin. A new polyketide synthase organization encoded by two subclusters separated by functionalization genes

The biosynthetic gene cluster for the 26-membered ring of the polyene macrolide pimaricin extends for about 110 kilobase pairs of contiguous DNA in the genome of Streptomyces natalensis. Two sets of polyketide synthase (PKS) genes are separated by a group of small polyketide-functionalizing genes. Two of the polyketide synthase genes, pimS0 and pimS1, have been fully sequenced and disrupted proving the involvement of each of these genes in pimaricin biosynthesis. The pimS0 gene encodes a relatively small acetate-activating PKS (approximately 193 kDa) that appears to work as a loading protein which "presents" the starter unit to the second PKS subunit. The pimS1 gene encodes a giant multienzyme (approximately 710 kDa) harboring 15 activities responsible for the first four cycles of chain elongation in pimaricin biosynthesis, resulting in formation of the polyene chromophore.     

Haplotypic variation of the transporter associated with antigen processing (TAP) genes and their extension of HLA class II region haplotypes

Stable cell surface presentation of HLA class I molecules requires active transport of antigenic peptides across the endoplasmic reticulum by products of two genes, TAP1 and TAP2, which map in the major histocompatibility complex class II region. Alleles of each gene are derived from a combination of variable sitesaat each locus. In this study, TAP1 and TAP2 alleles were identified in homozygous typing cell (HTC) lines, allowing resolution of specific haplotypes in conjunction with the highly polymorphic HLA class II region haplotypes. Three alleles at each TAP locus were found from which eight haplotypes could be assigned. Determination of TAP1 and TAP2 alleles in cell lines homozygous at DR, DQ, and DP created eight additional haplotypes beyond the number observed with these class II genes alone. Complete analysis of DR, DQ, TAP, and DP genotypes in 66 HTCs resulted in the following groups: 1) 46 homozygotes; 2) nine homozygous at DR, DQ, and TAP, but heterozygous at DP; 3) four homozygous at DR, DQ, and DP, but heterozygous at one or both TAP genes; 4) four homozygous at DR and DQ, but heterozygous at TAP and DP; and 5) three complex genotypes heterozygous at DP, TAP, and at least one of DQA1, DQB1, or DRB1 loci. TAP1 and TAP2 genes map in an area of frequent recombination. TAP alleles were determined in five DQB1, DPB1 recombinant individuals, three of which were informative. Recombination was found between DQB1 and the TAP loci in two individuals and between TAP and DPB1 in the other individual.     

Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato

Summary We have cloned and sequenced all five members of the gene family for the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato, Lycopersicon esculentum cv. VFNT LA 1221 cherry line. Two of the five genes, designated Rbcs-1 and Rbcs-2, are present as single genes at individual loci. Three genes, designated Rbcs-3A, Rbcs-3B and Rbcs-3C, are organized in a tandem array within 10 kb at a third independent locus. The Rbcs-2 gene contains three introns; all the other members of the tomato gene family contain two introns. The coding sequence of Rbcs-1 differs by 14.0% from that of Rbcs-2 and by 13.3% from that of Rbcs-3 genes. Rbcs-2 shows 10.4% divergence from Rbcs-3. The exon and intron sequences of Rbcs-3A are identical to those of Rbcs-3C, and differ by 1.9% from those of Rbcs-3B. Nucleotide sequence analysis suggests that the five rbcS genes encode four different precursors, and three different mature polypeptides. S₁ nuclease mapping of the 5 end of rbcS mRNAs revealed that the mRNA leader sequences vary in length from 8 to 75 nucleotides. Northern analysis using gene-specific oligonucleotide probes from the 3 non-coding region of each gene reveals a four to five-fold difference among the five genes in maximal steady-state mRNA levels in leaves.     

Susceptibility to relapsing-progressive multiple sclerosis is associated with inheritance of genes linked to the variable region of the TcR beta locus: use of affected family-based controls.

We tested the hypothesis that susceptibility to relapsing-progressive (RP) (but not to relapsing-remitting [RR]) multiple sclerosis (MS) is associated with a gene linked to the TcR beta-chain variable region delimited by the Vbeta8-BamHI and Vbeta11-BamHI RFLP alleles in DRw15+ MS patients, using a contingency-table test of patient data and affected family-based controls. Control alleles and haplotypes were composed of parental marker alleles and haplotypes not transmitted to the affected child, in 90 simplex and 31 multiplex families from British Columbia. A total of 6,164 alleles at 11 loci were segregated through families of probands with RP MS or RR MS. The Vbeta8-Vbeta11 subhaplotype frequencies in the DRw15+ RP MS (but not RR MS) patients differed from control frequencies, because of an increase of the 2-1 subhaplotype (P=.02). Vbeta8-BamHI and Vbeta11-BamHI allele frequencies (P=.05 and .009, respectively) in the DRw15+ RP MS (but not RR MS) patients differed from control frequencies. The Vbeta1-Vbeta8 subhaplotype frequencies in the DRw15- RP MS (but not RR MS) patients differed from control frequencies (P=.03), with a significantly increased frequency of the 1-1 subhaplotype (P=.01; RR=7.1) in RP MS versus RR MS patients. Susceptibility to RP MS is associated both with a recessive inheritance of a gene linked to the 3' (Vbeta11) end of the 2-1 subhaplotype defined by the Vbeta8-BamHI and Vbeta11-BamHI alleles in DRw15+ patients and with a gene, located on the 1-1 subhaplotype, defined by the Vbeta1-TaqI and Vbeta8-MspI alleles of the TcR beta-chain complex in DRw15- patients.     

Genetic analysis of essential genes in the ftsE region of the Escherichia coli genetic map and identification of a new cell division gene, ftsS

Summary Several conditional lethal mutants of Escherichia coli have been analysed genetically using generalized transduction and lambda transducing vectors. Three temperature-sensitive ftsE mutants were found as was a cold-sensitive ftsE mutant. A new gene was foud which mapped close to ftsE, namely ftsS. Both cell division genes map close to the gene which controls the heat-shock regulon (htpR).     

Nucleotide sequence analysis of five putative Streptomyces griseus genes, one of which complements an early function in daunorubicin biosynthesis that is linked to a putative gene cluster involved in TDP-daunosamine formation

Sequence analysis of the lkmB region of the daunorubicin biosynthetic gene cluster of Streptomyces griseus JA3933 revealed two contiguous open reading frames (ORF) in the same orientation, and three ORFs in the opposite orientation together extending over a 4.6 kb region adjacent to a homologue of the S. peucetius dnrJ gene. ORF1 complemented in trans the lkmB mutation, which seems to affect an early step in daunorubicin biosynthesis. Its deduced product showed no similarity to any known enzyme in the databases. The mutation in ORF1 was localised to a C-T transition at position 1172, leading to the change from a glycine to aspartic acid in the deduced protein. The lack of any homology to known polyketide synthesis enzymes indicates a regulatory role for the product of ORF1, despite the ability of lkmB mutants to further metabolise aklanonic acid. The genes of the oppositely oriented cluster seem to be involved in sugar metabolism. The putative ORF3 protein revealed strong homology to eukaryotic acyl CoA dehydrogenases and might encode an enzyme for the oxidoreduction preceding the introduction of the amino group into daunosamine, and the ORF4 protein is homologous to several epimerases, central enzymes in the formation of the l,-2,3,6-trideoxy-3-aminohexoses from TDP-d-glucose. ORF5 seems also to be related to enzymes metabolising nucleotide-activated hexoses.     

Mutational analysis of the Bordetella pertussis fim/fha gene cluster: identification of a gene with sequence similarities to haemolysin accessory genes involved in export of FHA

The chromosome of Bordetella pertussis harbours a region of 27 contiguous kb, which contains the bvg, fha and flm genes, involved in the co-ordinate regulation of virulence genes, FHA production and fimbriae production, respectively. The linkage of FHA and fimbrial genes has resulted in some confusion concerning the existence and location of genes required for the production of FHA and the function of the fimbrial genes fimB-D, which were proposed to be involved in both FHA and fimbriae biosynthesis. Through the use of non-polar mutations in each of these genes, we found that fimB-D are required for the production of both serotype 2 and 3 fimbriae, but not for FHA biosynthesis. Furthermore, a large open reading frame, designated fhaC, was identified downstream of fimD. It was shown that fhaC is essential for FHA production but not for fimbriae biogenesis. We propose that insertion mutations in fimB-D affect FHA production because of polar effects on fhaC expression. An insertion in the region downstream of fhaC had only a slight effect on FHA and fimbriae production. The fhaC gene product shows homology with ShIB and HpmB, two outer membrane proteins involved in export and activation of the haemolysins, ShIA and HpmA, of Serratia marcescens and Proteus mirabilis, respectively. Homology is also observed between the N-termini of FHA, ShIA and HpmA. Export of the haemolysins requires the Af-termini of these molecules, and when this region was removed from FHA by an in-frame deletion, FHA biosynthesis was abolished. These results suggest that the N-terminus of FHA interacts with FhaC, and that as a result FHA is transported across the outer membrane.