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PubMed1995

Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA.

Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates. We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da. Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction. Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Amino Acid Sequence↗
PubMed1991

Unusual codon bias occurring within insertion sequences in Escherichia coli.

The large open reading frames of insertion sequences from Escherichia coli were examined for their spatial pattern of codon usage bias and distribution of rarely used codons. There is a bias in codon usage that is generally lower toward the terminal ends of the coding regions, which is reflected in the occurrence of an excess of nonpreferred codons in the 3' portions of the coding regions as compared with the 5' portions. In contrast, typical chromosomal genes have a lower codon usage bias toward the 5' ends of the coding regions. These results imply that the selective forces reflected in codon usage bias may differ according to position within the coding sequence. In addition, these constraints apparently differ in important ways between genes contained in insertion sequences and those in the chromosome.

Chromosomes, Bacterial↗
PubMed1999

Regulation of Escherichia coli cell division genes ftsA and ftsZ by the two-component system rcsC-rcsB.

Genes rcsC and rcsB form a two-component system in which rcsC encodes the sensor element and rcsB the regulator. In Escherichia coli, the system positively regulates the expression of the capsule operon, cps, and of the cell division gene ftsZ. We report the identification of the promoter and of the sequences required for rcsB-dependent stimulation of ftsZ expression. The promoter, ftsA1p, located in the ftsQ coding sequence, co-regulates ftsA and ftsZ. The sequences required for rcsB activity are immediately adjacent to this promoter.

Bacterial Proteins↗
PubMed1989

Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon.

A second nitrogen fixation (nif) operon in the cyanobacterium (blue-green alga) Anabaena (Nostoc) sp. strain PCC 7120 has been identified and sequenced. It is located just upstream of the nifHDK operon and consists of four genes in the order nifB, fdxN, nifS, and nifU. The three nif genes were identified on the basis of their similarity with the corresponding genes from other diazotrophs. The fourth gene, fdxN, codes for a bacterial type ferredoxin (Mulligan, M. E., Buikema, W. J., and Haselkorn, R. (1988) J. Bacteriol. 167, 4406-4410). The four genes are probably transcribed as a single operon, but are expressed at a lower level than the nifHDK operon, and only after a developmentally induced DNA rearrangement occurs that excises a 55-kilobase pair element from within the fdxN gene (Golden, J. W., Mulligan, M. E., and Haselkorn, R. (1987) Nature 327, 526-529; Golden, J. W., Carrasco, C. D., Mulligan, M. E., Schneider, G. J., and Haselkorn, R. (1988) J. Bacteriol. 170, 5034-5041). The promoter for the nifB operon was located by primer extension. Comparison of the nifB 5'-flanking sequence with the nifH 5'-flanking sequence did not reveal any consensus base pairs that would define a nif promoter for Anabaena. The operon contains two instances of 7-base pair directly repeated sequences: seven copies of the repeated sequence are found between the nifB and fdxN genes and six copies are found between the nifS and nifU genes. The function of these repeats is unknown.

Amino Acid Sequence↗
PubMed2002

The bacterial histone-like protein HU specifically recognizes similar structures in all nucleic acids. DNA, RNA, and their hybrids.

HU, a major component of the bacterial nucleoid, shares properties with histones, high mobility group proteins (HMGs), and other eukaryotic proteins. HU, which participates in many major pathways of the bacterial cell, binds without sequence specificity to duplex DNA but recognizes with high affinity DNA repair intermediates. Here we demonstrate that HU binds to double-stranded DNA, double-stranded RNA, and linear DNA-RNA duplexes with a similar low affinity. In contrast to this nonspecific binding to total cellular RNA and to supercoiled DNA, HU specifically recognizes defined structures common to both DNA and RNA. In particular HU binds specifically to nicked or gapped DNA-RNA hybrids and to composite RNA molecules such as DsrA, a small non-coding RNA. HU, which modulates DNA architecture, may play additional key functions in the bacterial machinery via its RNA binding capacity. The simple, straightforward structure of its binding domain with two highly flexible beta-ribbon arms and an alpha-helical platform is an alternative model for the elaborate binding domains of the eukaryotic proteins that display dual DNA- and RNA-specific binding capacities.

Bacterial Proteins↗
PubMed1992

Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5.

The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver.

Amino Acid Sequence↗
PubMed2002

Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.).

Three genes from Ralstonia eutropha necessary for poly(3-hydroxybutyrate) (PHB) synthesis were introduced into the hairy roots of sugar beet. Transformation of a vector construct harbouring the PHB genes, each fused to the coding region of the pea ribulose-bisphosphate carboxylase plastid targeting sequence, resulted in 20 transgenic hairy-root clones, producing up to 55 mg high molecular PHB/g dry weight, as identified by gas chromatography, gel permeation chromatography and HPLC. Accumulation of PHB polymer in sugar beet root leucoplasts was confirmed by transmission electron microscopy. Thus, for the first time, plastidic PHB production was demonstrated for roots of a carbohydrate-storing crop plant.

Beta vulgaris↗
PubMed2006

ISfinder: the reference centre for bacterial insertion sequences.

ISfinder (www-is.biotoul.fr) is a dedicated database for bacterial insertion sequences (ISs). It has superseded the Stanford reference center. One of its functions is to assign IS names and to provide a focal point for a coherent nomenclature. It is also the repository for ISs. Each new IS is indexed together with information such as its DNA sequence and open reading frames or potential coding sequences, the sequence of the ends of the element and target sites, its origin and distribution together with a bibliography where available. Another objective is to continuously monitor ISs to provide updated comprehensive groupings or families and to provide some insight into their phylogenies. The site also contains extensive background information on ISs and transposons in general. Online tools are gradually being added. At present an online Blast facility against the entire bank is available. But additional features will include alignment capability, PsiBLAST and HMM profiles. ISfinder also includes a section on bacterial genomes and is involved in annotating the IS content of these genomes. Finally, this database is currently recommended by several microbiology journals for registration of new IS elements before their publication.

DNA Transposable Elements↗
PubMed1984

Selective amplification in methotrexate-resistant mouse cells of an artificial dihydrofolate reductase transcription unit making use of cryptic splicing and polyadenylation sites.

We have constructed a recombinant ( pMOP ) which is based on the bacterial plasmid pML2 and contains bovine papilloma virus type 1 (BPV 1) sequences linked to an artificial mouse dihydrofolate reductase (DHFR) transcription unit. This unit consists of the SV40 early gene promoter, a complete DHFR coding sequence and splice and polyadenylation sites from a rabbit beta-globin gene. Intact pMOP or a fragment thereof devoid of pML2 sequences will transform mouse cells to methotrexate resistance. The lines obtained contain approximately 200 copies of the DHFR transcription unit. In no case, however, did we find evidence of extrachromosomal maintenance of BPV 1 or DHFR sequences. One line, when selected for resistance to elevated levels of methotrexate, contained amplified copies of a 'novel' DHFR transcription unit resulting from fusion of two normal units. This line contained the DHFR RNA species present in the parent line plus some additional species. The structure of these various RNA species was determined and evidence found for the use of cryptic splice and polyadenylation sites.

Animals↗
PubMed1997

Mouse connexin40: gene structure and promoter analysis.

A family of related connexin genes encodes the subunit gap junction proteins that form intercellular channels in different tissues. Connexin40 (Cx40) is one of these proteins, and it exhibits limited expression only in a few cells of the cardiovascular system. To begin to analyze Cx40 expression, we isolated a 3.3-kb rat Cx40 cDNA by hybridization screening of a bacteriophage library prepared from BWEM cells and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library. Restriction mapping, sequencing, and comparison to the rat cDNA showed that the mouse Cx40 gene contained a short first exon, an 11.4-kb intron, and a second exon containing the complete coding region and 3'-UTR. Exon I contained only 1 base that differed between rat and mouse. Primer extension experiments yielded a single band and confirmed the position of the transcriptional start site. We obtained 1.2 kb of sequence 5' of the transcriptional start site and 400 bp 3' of exon I. Exon I was closely preceded by a consensus TATA box. The flanking sequences contained a number of potential transcription factor binding sites (including AP-1, AP-2, SP1, TRE, and p53). To identify transcriptional regulatory elements in the Cx40 promoter region, a series of DNA deletion fragments flanking exon I was prepared, subcloned adjacent to a luciferase reporter gene, and used for transient transfections of BWEM, SHM, and N2A cells. The resulting luciferase activity determinations suggested that an area of 300 bp 5' of the transcription start site acted as a basal promoter for Cx40 and that there was a strong negative regulatory element in the region from +100 to +297.

Animals↗
PubMed2000

Cloning and characterization of a putative human glycerol 3-phosphate permease gene (SLC37A1 or G3PP) on 21q22.3: mutation analysis in two candidate phenotypes, DFNB10 and a glycerol kinase deficiency.

Using multiple exons trapped from human chromosome 21 (HC21)-specific cosmids with homology to a putative Arabidopsis thaliana glycerol 3-phosphate permease, we have determined the full-length cDNA sequence of a novel HC21 gene encoding a putative sugar-phosphate transporter (HGMW-approved symbol SLC37A1, aka G3PP). The predicted protein has 12 putative transmembrane domains and is also highly homologous to bacterial glpT proteins. The transcript was precisely mapped to 21q22.3 between D21S49 and D21S113. Comparison of the SLC37A1 cDNA to genomic sequence revealed that the gene encompasses 82 kb, and it is split into 19 coding exons and 7 untranslated exons, which are alternatively spliced in a complex and tissue-specific manner. Glycerol 3-phosphate (G3P) is produced by glycerol kinase (GK) and is found in several biochemical pathways in different cellular compartments, such as the glycerol phosphate shuttle and glycerophospholipid synthesis. Thus SLC37A1 mutations may cause a phenotype similar to GK deficiency. Mutational analyses of SLC37A1 in seven patients with no mutations in the GK gene and low GK activity revealed only nonpathogenetic sequence variants, excluding SLC37A1 as the gene for the phenotype in these patients. SLC37A1 maps in the refined critical region of the autosomal recessive deafness locus, DFNB10, on 21q22.3. Mutation analyses also excluded SLC37A1 as the gene for DFNB10.

Amino Acid Sequence↗
PubMed1991

Structure and properties of luciferase from Photobacterium phosphoreum.

The nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits, respectively, of luciferase from Photobacterium phosphoreum have been determined. The predicted amino acid sequences of the alpha and beta subunits were shown to be significantly different from other bacterial luciferases with 62 to 88% identity with the alpha subunits and 47 to 71% identity with the beta subunits of other species. Expression of the different luciferases appear to correlate with the number of modulator codons. Kinetic properties of P. phosphoreum luciferase were shown to reflect the bacterium's natural cold temperature habitat.

Amino Acid Sequence↗
PubMed1992

Regulation of the Escherichia coli lac operon expressed in human cells.

We have investigated the use of various Epstein-Barr virus (EBV)-based vectors bearing the two components of the Escherichia coli lac operator-repressor (lacO, lacI) complex. Our aim was to develop a model system of gene expression by looking at the transcription of the bacterial beta-galactosidase coding gene (lacZ) in 293 human embryonic kidney cells. Several vectors have been built carrying different promoters upstream of the lacI and lacZ genes and in which natural or synthetic operator sequences were inserted in the 5' part of the lacZ gene. In transient expression assays we achieved efficient lacZ gene repression which could be released by the specific inducer isopropyl beta-D-thiogalactoside (IPTG). A stable transformed cell line carrying two EBV-derived plasmids with the building blocks of the lac operator/repressor system was established. This cell line allowed us to achieve a wide range of lacZ gene regulation. In this cell line IPTG alone could remove the repression to trigger a 5-fold increase of lacZ expression. Heavy metal ions, which induced the mouse metallothionein I promoter located upstream of the lacZ gene, added together with IPTG gave rise to a 40-fold induction of lacZ expression.

Base Sequence↗
PubMed2000

GnRH-Bik/Bax/Bak chimeric proteins target and kill adenocarcinoma cells; the general use of pro-apoptotic proteins of the Bcl-2 family as novel killing components of targeting chimeric proteins.

In recent years chimeric proteins carrying bacterial toxins as their killing moiety, have been developed to selectively recognize and kill cell populations expressing speciific receptors. The involvement of Gonadotropin releasing hormone (GnRH) has been demonstrated in several adenocarcinomas and a GnRH-bacterial toxin chimeric protein (GnRH-PE66) was thus developed and found to specifically target and kill adenocarcinoma cells both in vitro and in vivo. Because of the immunogenicity and the non-specific toxicity of the bacterial toxins, we have developed new chimeric proteins, introducing apoptosis inducing proteins of the Bcl-2 family as novel killing components. Sequences encoding the human Bik, Bak or Bax proteins were fused to the GnRH coding sequence at the DNA level and were expressed in E. coli. GnRH-Bik, GnRH-Bak and GnRH-Bax new chimeric proteins efficiently and specifically inhibited the cell growth of adenocarcinoma cell lines and eventually led to cell death. All three Bcl2-proteins-based chimeric proteins seem to induce apoptosis within the target cells, without any additional cell death stimulus. Apoptosis-inducing-proteins of the Bcl-2 family targeted by the GnRH are novel potential therapeutic reagents for adenocarcinoma treatment in humans. This novel approach could be widely applied, using any molecule that binds a specific cell type, fused to an apoptosis-inducing protein.

Adenocarcinoma↗
PubMed1989

Phase variation in Bordetella pertussis by frameshift mutation in a gene for a novel two-component system.

Bordetella pertussis, the aetiological agent of whooping cough, coordinately regulates the expression of many virulence-associated determinants, including filamentous haemagglutinin, pertussis toxin, adenylyl cyclase toxin, dermonecrotic toxin and haemolysin. The coordinate regulation is apparent in the repression of synthesis of these determinants in response to environmental stimuli; a phenomenon known as antigenic or phenotypic modulation. B. pertussis also varies between metastable genetic states, or phases. There is a virulent phase in which virulence-associated determinants are synthesized, and an avirulent phase in which they are not. Previous studies have shown that a genetic locus, vir, is required for expression from many virulence-associated loci, and that replacing the cloned vir locus in trans can restore the virulent phase phenotype to spontaneously occurring avirulent phase strains. Here, we show that phase variation in one series of strains is due to a frameshift mutation within an open reading frame that is predicted to code for a Vir protein product. The deduced protein sequence is similar to both components of the 'two-component' regulatory system which control gene expression in response to environmental stimuli in a range of bacterial species.

Base Sequence↗