Positive regulator for the expression of Mba protein of the virulence plasmid, pKDSC50, of Salmonella choleraesuis.

A positive regulator was identified within a 2.3 kb fragment of the 6.4 kb mouse bacteremia region (mba region) of the virulence pKDSC50 plasmid of Salmonella choleraesuis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that Escherichia coli K-12 carrying the recombinant plasmids of the 2.3 kb fragment produced Mba1 protein with a molecular mass of 32 kDa. The recombinant plasmids carrying a 4.1 kb fragment, the other part of 6.4 kb region, produced Mba2 (32 kDa), Mba3 (70 kDa) and Mba4 (29 kDa) proteins. All three proteins were expressed by using the lacZ promoter under isopropyl thiogalactoside induction. In contrast to this, Mba3 protein was overexpressed independently of the lacZ promoter when the 2.3 kb fragment coexisted either in cis or trans. These results suggest that Mba1 is a trans-acting positive regulator for the expression of the Mba3 protein of mba region of pKDSC50.     

Organization of the 5.8S, 16–18S,and 23–28S ribosomal RNA genes of Cephalosporium acremonium

Summary A 9.2 kb Pst1 restriction fragment, repeated tandemly head-to-tail in the genome, contains the 5.8S, 16–18S, and 23–28S ribosomal RNA (rRNA) genes of Cephalosporium acremonium, a filamentous fungus used at the industrial scale for production of cephalosporin antibiotics. These rRNA genes were located in Pst1 digests of C. acremonium genomic DNA using a hybridization probe that contained the 5.8S, 18S, and 25S rRNA genes from the yeast Saccharomyces cerevisiae. This probe was also used in screening a recombinant lambda library to identify phage carrying rRNA genes of C. acremonium. Yeast rRNA genes contained separately on individual ³²P-labeled plasmids were used to demonstrate that a cloned 7.2 kb C. acremonium sequence, represented in the repeated 9.2 kb Pst1 fragment, contained DNA from the C. acremonium 5.8S, 16–18S, and 23–28S rRNA genes. These genes were ordered with the 5.8S gene located between the 16–18S and 23–28S rRNA genes. The order of the 16–18S, 5.8S, and 23–28S rRNA genes observed in C. acremonium is the same as that observed in rRNA repeats of many other lower eucaryotes, e.g. S. cerevisiae, Aspergillus nidulans, and Neurospora crassa.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker

Summary Rhizopus niveus has been transformed to blasticidin S resistance by vectors containing the bacterial blasticidin S resistance gene under the control of a Rhizopus promoter. Southern analysis of the total DNA from transformants indicated that the introduced DNA was rearranged, and that one of the transformants harbored extrachromosomal plasmids with rearranged DNA. Using this transformation system, the introduction of pUBSR101, a plasmid carrying the Escherichia coli lacZ gene fused to the promoter and the N-terminal region of the R. niveus aspartic proteinase-II (RNAP-II) gene, resulted in an increase of -galactosidase activity in the cell extract, indicating expression of the lacZ fusion gene in R. niveus. This is the first report of a transformation system for filamentous fungi using the blasticidin S resistance gene as a dominant selectable marker.     

Molecular characterization of a trans-acting, positive effector (ipaR) of invasion plasmid antigen synthesis in Shigella flexneri serotype 5

A trans-acting, positive effector of invasion plasmid antigen (Ipa) synthesis has been identified and mapped on the pWR100 invasion plasmid of Shigella flexneri serotype 5 (strain M90T-W). Recombinant plasmids carrying this regulatory gene, designated ipaR, were found to restore full virulence to a non-invasive ipaR::Tn5 insertion mutant [M90T-W(pHS1042)] that had lost the ability to synthesize four Ipa antigens (IpaA, 70 kDa; IpaB, 62 kDa; IpaC, 42 kDa; and IpaD, 37 kDa). Genetic mapping of the ipaR gene positioned the locus on a 2.6 kb PstI-AccI fragment contained within a larger 8.0 kb EcoRI molecule that also encoded IpaD, IpaA, and two small proteins (27 kDa and 28 kDa). The trans regulatory effect of the ipaR product on ipaB, ipaC, ipaD, and ipaA expression was demonstrated by transforming compatible ipaBC, ipaDA, ipaR and ipaDAR plasmid recombinants, in various combinations, into M90T-A3, an isogenic invasion plasmid mutant of M90T-W that contained a deletion of the pWR100 ipaBCDA and ipaR loci; such transformants produced wild type levels of the IpaB, IpaC, IpaD and IpaA antigens only in the presence of IpaR+ plasmids. DNA sequence analysis of the ipaR region established that the intiation codon for ipaR is 459 bp from the 3'-end of the ipaA gene and that ipaR encodes a 309 amino acid residue protein. An interesting feature of the IpaR polypeptide was its strong sequence homology with the bacteriophage P1 partition protein ParB, consisting of a 42.8% amino acid identity over a 278 residue section of the aligned proteins.     

Cloning of a Candida utilis gene which complements leu2 mutation in Saccharomyces cerevisiae

Summary DNA fragments containing the LEU2 gene of Candida utilis have been isolated, utilizing the genome library (constructed in YRp12) of this organism. Two recombinant plasmids pZR84 and pZR32, containing the cloned LEU2 gene, were 4.24 kb and 10.4 kb, respectively, and were shown to complement leu2 mutation in Saccharomyces cerevisiae and leuB mutation in Escherichia coli. The cloned fragment in pZR84 contained one restriction site each for EcoRI and PvuII, and two for HindIII, but none for SalI, BamHI or Pstl. This cloned fragment hybridized with the total DNA from C. utilis and from Leu⁺ transformants of S. cerevisiae, but not with that from untransformed S. cerevisiae. Subcloning analyses showed that a 2.34 kb BamHI HindIII fragment of the cloned C. utilis sequence contains the region essential for the expression of the LEU2 gene.Journal Article No. 11669 from the Michigan Agricultural Experiment Station     

Isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae by functional complementation in yeast

Summary This paper describes the isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae. Two pools of plasmids consisting of BamHI and Sa1GI yeast DNA inserts into the bifunctional yeast — Escherichia coli vector pLC544 (Kingsman et al. 1979) were constructed in E. coli and used for the isolation of the two genes by selection for functional complementation of trp2 and trp3 mutations, respectively, in yeast.The TRP2 gene was isolated on a 6.2 kb BamHl and a 5.8 kb Sa1GI yeast DNA fragment which shared an identical 4.5 kb BamHI-SaIGI fragment. The TRP3 gene was located on a 5.2 kb BamHl fragment.By physical, genetic and physiological experiments it could be shown that the cloned yeast DNA fragments contained the whole structural sequences as well as the regulatory regions of the TRP2 and the TRP3 genes.     

Multiple-copy integration in the yeast Yarrowia lipolytica

Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25–60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.     

Cloning of maltase regulatory genes in Saccharomyces cerevisiae

Summary By hybridization with a putative MAL2p regulatory sequence we have identified a 19 kb long BamH1 DNA fragment to contain the MALp sequence in a MAL4 strain. A mixture of recombinant plasmids was prepared by ligation of purified 19 kb BamH1 fragments partially digested with Sau3A into the multicopy vector YEp1357. The source of DNA was a strain carrying the MAL4 locus. Yeast maltose non-fermenting strains were transformed with the plasmid mixture. A recombinant plasmid, pRM-4, containing the MAL4p regulatory gene was isolated that complements the maltose-negative phenotype. The plasmid was shown to confer the ability to synthesize maltase to recipient strains grown under inducing as well as under repressing conditions.The MAL4p regulatory sequence cloned was used as a probe in hybridization experiments to study the degrees of homology between the different MAL regulatory genes. The results showed that the sequence from MAL4 strains is strongly homologous to that of MAL3 strains whereas it shows significant differences to the ones of MAL1 and MAL2 strains.Southern analysis of the segregants of crosses between maltose-positive strains and ma10 strains allowed us to localize the maltase regulatory sequence of each MAL locus within a characteristic BamH1 fragment of genomic DNA hybridizing to the isolated sequence.     

Protein profiles of Legionella pneumophila Philadelphia-1 grown in macrophages and characterization of a gene encoding a novel 24 kDa Legionella protein

Legionella pneumophila Philadelphia-1 strain was grown in cultured macrophages of guinea pigs, hamsters, and A/J mice and the bacteria were purified by Percoll density gradient centrifugation without any detergent. Patterns of the bacterial proteins were compared by SDS-PAGE with those of organisms cultured in vitro. A 24 kDa protein was a major protein of intracellularly grown bacteria: its expression was about four times as much as a 24 kDa protein of agar-grown bacteria. At least three novel proteins (100, 65, and 16 kDa) that were not found on agar-grown bacteria were also observed. In this paper, we focused on the 24 kDa major Legionella protein expressed within macrophages. Western blot and N-terminal amino acid analysis revealed that this protein is a novel protein different from Legionella proteins previously reported, including a 24 kDa macrophage infectivity potentiator protein (Mip). On the basis of amino acid sequence (MQRIKKI and IANAQGK), two kinds of oligonucleotides were synthesized and radiolabeled. Using these oligonucleotides as DNA probes, a 7.2 kb EcoRI-digested DNA fragment encoding the 24 kDa protein was cloned into λZAP II phage vector in Escherichia coli XL1-Blue. A 0.9 kb DNA fragment from the 7.2 kb fragment was further subcloned into pUC118 in E. coli CSR603 for maxicell analysis or XL1-Blue for DNA sequencing. Maxicells which carry recombinant plasmids consisting of the 0.9 kb DNA fragment and vector plasmid pUC118 expressed the 24 kDa protein. When the DNA fragment encoding the protein was sequenced, an open reading frame of 555 base pairs was identified. The inferred polypeptide had a molecular weight of 20 kDa and an estimated isoelectric point of 8.14. Both the nucleotide sequence and the deduced amino acid sequence were distinct from those of bacterial proteins reported previously, suggesting that the protein is a novel Legionella protein.     

Prokaryotic promoters in the chloroplast DNA replication origin of Chlamydomonas reinhardtii

Summary In Chlamydomonas reinhardtii, one displacement loop region which initiates the replication of chloroplast DNA was located on a 1.05 kb restriction fragment. This fragment was cloned and sequenced. In this report, the galK expression plasmid, pK01 was used to screen for the presence of any prokaryotic promoter within the cloned fragment. The insertion of 2 AluI fragments yielded galK⁺ colonies. Sequence analyses of these Alul inserts revealed prokaryotic promoter consensus regions. Cloning into pKOTWI and subsequent DNA sequencing were used to determine the promoter-active orientation of each insert. Two back-to-back prokaryotic promoters were mapped on a 79 by Alul fragment located within the displacement loop region.     

The characterization of lectin-binding proteins of Chlamydia trachomatis as glycoproteins

The 18 kDa and 32 kDa lectin binding proteins of Chlamydia trachomatis were characterized as glycoproteins by treatments with glycosidases. The proteins of the serovar L₂ whole cell lysate were separated by SDS-PAGE and transferred to nitrocellulose paper. After treatment with an enzyme, the proteins were reacted with a biotinylated lectin. Each of the endoglycosidases tested affected the binding of the lectin to the protein. PNGase F inhibited the binding of Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin I (UEAI) to both the 18 kDa and 32 kDa proteins. Endoglycosidase F and H inhibited the binding of these lectins to the 32 kDa protein completely and to the 18 kDa protein partially. In the exoglycosidase treatments, alpha- -fucosidase prevented binding of only UEAI to the two proteins while beta-galactosidase inhibited the binding of SBA. Mannosidase abolished the binding of all the lectins tested. Neuraminidase had no effect. The proteins isolated by electroelution from the excised gels after SDS-PAGE were digested with an endoglycosidase. PNGase F-treated proteins showed a lower molecular weight mobility in which the lectin binding ability was destroyed. Endo-alpha-N-acetylgalactosaminidase had no effect. The polysaccharide stain of isolated proteins with p-phenylenediamine showed a positive reaction. Radiolabeling with [³H]glucosamine did not reveal the 18 kDa and 32 kDa proteins in autoradiography but [³H]galactose did.     

Localization of three chloroplast ribosomal protein genes at the left junction of the large single copy region and the inverted repeat of Spirodela oligorhiza chloroplast DNA

Summary In order to determine the localization of ribosomal protein genes on the chloroplast genome of Spirodela, we have followed two different approaches: First, antisera were prepared against purified 30S, 50S and 70S chloroplast ribosomal proteins from Spinacia. These antisera react with about two third of the chloroplast ribosomal proteins as shown by protein blot and immunoprecipitation experiments. Recombinant plasmids carrying the Spirodela BamHI-G or PstI-I cpDNA fragment both direct the synthesis of a 15 kD chloroplast ribosomal protein in a DNA dependent E. coli cell free system. This was confirmed by molecular weight determination, immunoprecipitation and competition immunoprecipitation experiments. Second, heterologous hybridization with the rps19 gene probe from Nicotiana revealed the localization of this gene on the chloroplast DNA of Spirodela within the BamHI-G fragment at the left junction of the large single copy region and the inverted repeat. Furthermore we show that the recombinant plasmid carrying Nicotiana rps19 also directs the synthesis of another chloroplast ribosomal protein in an E. coli cell free system. The identity of this protein is discussed.Abbreviations Bis-Tris 2,2(Bis(hydroxymethyl)2,2,2 nitrilotriethanol - (k)bp (kilo)base pairs - BPB bromphenol blue - cp chloroplast - IR inverted repeat - kD kilo dalton - LSCR large single copy region - Mw molecular weight - r-protein ribosomal protein - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodiumdodecyl sulphate     

Recombination within the inverted repeat sequences of the Chlamydomonas reinhardii chloroplast genome produces two orientation isomers

Summary Two orientations of the Chlamydomonas reinhardii chloroplast (ct) genome are shown to be produced by recombination within the inverted repeat (IR) sequences that separate the two single copy (SC) regions. SC region 1 is bounded on its two ends by EeoRI restriction endonuclease fragments of 3.2 and 4.7 kilobase pairs (kb) (Rochaix 1978). The 3.2 kb EeoRI fragment overlaps a 51.3 kb BglII fragment spanning one of the 19.7 kb IR sequences, and the 4.7 kb EcoRI fragment overlaps a 42.1 kb BglII fragment spanning the other 19.7 kb IR sequence. We have shown by hybridization analysis that the 3.2 kb fragment also overlaps a BgIII fragment with a predicted size of 52.3 kb, and that the 4.7 kb fragment also overlaps a BglII fragment of a predicted size of 41.1 kb. The second set of BglII fragments are isomers produced by recombination localized to the IR region. The two isomers are present in approximately equimolar ratio. Knowledge of the isomeric composition of the C. reinhardii ctDNA is essential for establishing a correlation between genetic and physical maps of the ct genome.     

Molecular cloning of a ribosomal protein gene from the fission yeast Schizosaccharomyces pombe

Summary Using the structural gene for the ribosomal protein L3 from Saccharomyces cerevisiae as a probe, we isolated a homologous fragment from genomic DNA of Schizosaccharomyces pombe. Analysis of the plasmid carrying this fragment by hybridization selection and 2D-electrophoresis revealed a 31 kDa ribosomal protein. Transformation of the vector pDB248x containing this fragment into Schizosaccharomyces pombe leads to an increased level of mRNA suggesting that we have cloned the entire and actively transcribed gene.     

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.     

Identification of an upstream activation site in the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae

Summary The upstream region of the Saccharomyces cerevisiae pyruvate decarboxylase structural gene, PDC1, has been isolated and fused to the indicator gene Escherichia coli lacZ. 1.2 kb of the upstream region has been sequenced. The PDC1-lacZ fusion has been integrated at the ura3-52 locus in the yeast genome, and has a basal level of expression on ethanol. On glucose media this level is increased 30–50 fold. An upstream activation site, UAS_pdc, between 793 and 535 by upstream from the ATG of PDC1, which mediates the response to glucose has been identified by deletion analysis. The UAS_pdc contains a consensus RPG box, originally identified in ribosomal protein genes (Leer et al. 1985). The function of UAS_pdc is independent of distance from the ATG. There is also an upstream repressing sequence located between 535 and 385 by upstream from the translational start of PDC1.     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.