The Transposon-Like Structure of IS26-Tetracycllne,and Kanamycin Resistance Determinant Derived from Transferable R Plasmid of Fish Pathogen,Pasteurella piscicida

Tetracycline (pp-tet), and kanamycin (pp-kan) resistance genes were cloned from a transferable R plasmid of fish pathogen Pasteurella piscicida, and complete nucleotide sequences were determined. The pp-tet was a class D Tet determinant constructed with the tetA resistance gene of 1,182 bp encoding a protein with a deduced molecular mass of 41 kDa and the tetR repressor gene of 654 bp encoding a product of 24 kDa. The pp-tet was highly homologous to the tet(D) of plasmid RA1 isolated from Aeromonas hydrophila with two nucleotide differences in the tetR, and of plasmid pIP173 from Salmonella ordonez with two nucleotide differences in the tetA. The pp-kan contained 813 bp encoding a 31 kDa protein of 271 amino acids, and was classified into type aph-Ic. It was identical to the aphA7 in the IAB operon of pBWH77, in which was originally found an isolate of Klebsiella pneumoniae, in its nucleotide sequences and hybrid promoter construction. The genes were connected by an insertion sequence IS 26 of 820 bp, and were flanked by repeated copies in direct orientation at the 3′ flanking region of the pp-tetA and in inverted orientation at the 3′ flanking region of the pp-kan. The genetic elements are organized like a complex transposon by close linkage of the IS 26 and the pp-tet and -kan.     

Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.     

Comparative analysis of 18 sex pheromone plasmids fromEnterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3-terminus of the surface exclusion gene,sep1, of sex pheromone plasmid pPD1 inEnterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.     

Isolation and genetic structure of IS112, an insertion sequence responsible for the inactivation of the SalI restriction-modification system of Streptomyces albus G.

Summary IS112 is a transposable element identified in Streptomyces albus G by its frequent mutagenic insertion into the genes for the SalI restriction-modification system. IS112 is present in several copies in the genome of S. albus G. Homologous sequences were detected in other Streptomyces strains. Sequence analysis revealed that IS112 has a length of 883 by with a GC content of 67.4%. The copy that was isolated contained imperfect inverted repeats (16/20 match) at its ends and was flanked by a 2 by duplication at the target site, which was located within the gene (salIR) for the Sall endonuclease. A long open reading frame (ORF) encoding a putative polypeptide of 256-253 amino acids spans almost the entire sequence. Significant homology was detected between this polypeptide and that corresponding to ORFB of IS493, an insertion sequence recently isolated from Streptomyces lividans 66.

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IS231 and otherBacillus thuringiensis transposable elements: A review

Bacillus thuringiensis is an entomopathogenic bacterium whose toxicity is due to the presence in the sporangia of -endotoxin crystals active against agricultural pests and vectors of human and animal diseases. Most of the genes coding for these toxin proteins are plasmid-borne and are generally structurally associated with insertion sequences (IS231, IS232, IS240, ISBT1 and ISBT2) and transposons (Tn4430 and Tn5401). Several of these mobile elements have been shown to be active and are believed to participate in the crystal gene mobility, thereby contributing to the variation of bacterial toxicity. Structural analysis of the iso-IS231 elements indicates that they are related to IS1151 fromClostridium perfringens and distantly related to IS4 and IS186 fromEscherichia coli. Like the other IS4 family members, they contain a conserved transposase-integrase motif found in other IS families and retroviruses. Moreover, functional data gathered from IS231 A inEscherichia coli indicate a non-replicative mode of transposition, with a marked preference for specific targets. Similar results were also obtained inBacillus subtilis andB. thuringiensis, and a working model for DNA-protein interactions at the target site is proposed.     

Gene duplication and gene conversion in class II MHC genes of New Zealand robins (Petroicidae)

In contrast to mammals, the evolution of MHC genes in birds appears to be characterized by high rates of gene duplication and concerted evolution. To further our understanding of the evolution of passerine MHC genes, we have isolated class II B sequences from two species of New Zealand robins, the South Island robin (Petroica australis australis), and the endangered Chatham Island black robin (Petroica traversi). Using an RT-PCR based approach we isolated four transcribed class II B MHC sequences from the black robin, and eight sequences from the South Island robin. RFLP analysis indicated that all class II B loci were contained within a single linkage group. Analysis of 3-untranslated region sequences enabled putative orthologous loci to be identified in the two species, and indicated that multiple rounds of gene duplication have occurred within the MHC of New Zealand robins. The orthologous relationships are not retained within the coding region of the gene, instead the sequences group within species. A number of putative gene conversion events were identified across the length of our sequences that may account for this. Exon 2 sequences are highly diverse and appear to have diverged under balancing selection. It is also possible that gene conversion involving short stretches of sequence within exon 2 adds to this diversity. Our study is the first report of putative orthologous MHC loci in passerines, and provides further evidence for the importance of gene duplication and gene conversion in the evolution of the passerine MHC.Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers AY258333–AY258335, AY428561–AY428570, and AY530534–AY530535     

Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium synechococcus sp. PCC 7002

The unicellular photoheterotrophic cyanobacterium Synechococcus sp. PCC 7002 was shown to encode two genes for the Photosystem II reaction center core protein D2 and one gene for the reaction center chlorophyhll-binding protein CP43. These three genes were cloned and their DNA sequences determined along with their flanking DNA sequences. Northern hybridization experiments show that both genes which encode D2, psbD1 and psbD2, are expressed at roughly equivalent levels. For each of the two psbD genes, there are 18 nucleotide differences among the 1059 nucleotides which are translated. The DNA sequences surrounding the coding sequences are nearly 70% divergent. Despite the DNA sequence differences in the genes, the proteins encoded by the two genes are predicted to be identical. The proteins encoded by psbD1 and psbD2 are 92% homologous to other sequenced cyanobacterial psbD genes and 86% homologous to sequenced chloroplast-encoded psbD genes.The single gene for CP43, psbC, overlaps the 3 end of psbD1 and is co-transcribed with it. Results from previous sequencing of psbC genes encoded by chloroplasts suggest that the 5 end of the psbC gene overlaps the 3 end of the coding sequence of psbD by 50 nucleotides. In Synechococcus sp. PCC 7002, the methionine codon previously proposed to be the start codon for psbC is replaced by an ACG (threonine) codon. We propose an alternative start for the psbC gene at a GTG codon 36 nucleotides downstream from the threonine codon. This GTG codon is preceded by a consensus E. coli-like ribosome binding sequence. Both the GTG start codon and its preceding ribosome binding sequence are conserved in all psbC genes sequenced from cyanobacteria and chloroplasts. This suggests that all psbC genes start at this alternative GTG codon. Based on this alternative start codon, the gene product is 85% identical to other cyanobacterial psbC gene products and 77% identical to eucaryotic chloroplast-encoded psbC gene products.     

Organization of the genes encoding chalcone synthase in Pisum sativum

To analyze the regulation of defense-related genes by signal molecules produced by phytopathogens, we isolated genes that encode chalcone synthase (CHS) in Pisum sativum. We have obtained seven independent genomic clones that contain at least seven classes of CHS genes, identified by the hybridization analysis to CHS cDNA and by the restriction mapping analysis. Two of the genomic clones (clone 5 and 6) each contain two CHS genes in a tandem repeat. The nucleotide sequence analysis of CHS genomic clone 5 revealed that PsCHS1 and PsCHS2 were corresponding genes of the CHS cDNA clones, pCC6 and pCC2, respectively, as reported earlier. Both genes are interrupted by a single intron of 88 nucleotides with identical sequences, although exonic sequences and 5-flanking sequences are divergent. Nucleotide sequences of the introns in five other classes of CHS genes showed that three classes had an intron of 87 nt with a striking homology to each other, but that the intron of the other two classes of CHS genes showed heterogeneity both in size and nucleotide sequence. 5-upstream regions of PsCHS1 and PsCHS2 did not show sequence homology except the 31 bp identical sequence that contains the CCTACC motif resembling the box-1 sequence. Both PsCHS1 and PsCHS2 genes are shown to be induced by fungal elicitor by a primer extension analysis and a transient transformation analysis using pea protoplasts prepared from suspension cultured-cells.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

Accurate mapping of the Escherichia coli pepD gene by sequence analysis of its 5′ flanking region

Summary A cloned DNA fragment, carrying the gene for peptidase D (pepD) of Escherichia coli, was partially sequenced. By purification of peptidase D and sequence determination of an amino-terminal oligopeptide the reading frame of the pepD gene, starting with a GTG initiator codon, was unambiguously identified.An overlap of the established nucleotide sequence with the previously sequenced 5 flanking region of the gpt gene allowed the exact distance between pepD and gpt to be calculated. The two genes are pointing towards each other and are separated by 260 bp. A search for open reading frames (ORFs) and the analysis of possible codon usage in the intercistronic region indicate the absence of an additional gene (lpcA) between pepD and gpt.     

Structural and functional analysis of the rcsA gene from Erwinia stewartii

Summary ResA is a positive regulator of genes for extracellular polysaccharide biosynthesis in the Enterobacteriaceae. The nucleotide sequence of the rcsA gene from Erwinia stewartii was determined and compared to rcsA sequences from E. amylovora, Escherichia coli, and Klebsiella pneumoniae. Three highly conserved regions of the gene were identified. The C-terminal end of the open reading frame (ORF) shared significant amino acid homology to the LuxR class of bacterial activator proteins. Insertion and deletion mutagenesis of the 5 non-coding region indicated that maximal expression of rcsA was dependent upon cis-acting regulatory sequences located more than 300 by upstream of the translational start site.     

Structure,regulation and evolution of the crystal-Stellate system of Drosophila

The crystal–Stellate system is one of the most known example of interaction between heterochromatin and euchromatin: a heterochromatic locus on the Y chromosome (crystal) 'represses a euchromatic locus (Stellate) on the X chromosome in Drosophila melanogaster. The molecular mechanism regulating this interaction is not completely understood. It is becoming clear that an RNA interference (RNAi) mechanism could be responsible for the silencing carried out by crystal on the Stellate sequences. Here, a detailed structural analysis of all the sequences involved in the system is reported, demonstrating a their 'puzzling structure. In addition three autosomal mutations: sting, scratch and sirio are described that interfere with the system. All of them are male sterile mutations and exhibit crystals made by the STELLATE protein in their primary spermatocytes. They are requested during oogenesis and early in embryogenesis as well. Hypothesis on the involvement of these genes in activating the Stellate sequences are discussed.     

Molecular cloning and nucleotide sequence of the psaA and psaB genes of the cyanobacterium Synechococcus sp. PCC 7002

The psaA and psaB genes, which encode the P700 chlorophyll a apoproteins of the Photosystem I complex, have been cloned from the unicellular, transformable cyanobacterium Synechococcus sp. PCC 7002. The nucleotide sequence of these genes and of their flanking sequences have been determined by the chain termination method. As found in the chloroplast genomes of higher plants, the psaA gene lies 5 to the psaB gene; however, the cyanobacterial genes are separated by a greater distance (173 vs. 25–26 bp). The psaA gene is predicted to encode a polypeptide of 739 amino acid residues (81.7 kDa), and the psaB gene is predicted to encode a polypeptide of 733 residues (81.4 kDa). The cyanobacterial psa gene products are 76% to 81% identical to their higher plant homologues; moreover, because of conservative amino acid replacements, the cyanobacterial sequences are more than 95% homologous to those determined for higher plants. These results provide the basis for a genetic analysis of Photosystem I, and are discussed in relationship to structural and functional aspects of the Photosystem I complexes of both cyanobacteria and higher plants.     

IS1-mediated mobility of the aerobactin system of pColV-K30 in Escherichia coli

Summary Genes determining the high affinity iron transport system mediated by the siderophore aerobactin are flanked in the enterobacterial plasmid pColV-K30 by inverted repeats of IS1 sequences, suggesting that the aerobactin genes are part of a transposon. To study this possibility, the entire region between the two IS1 sequences was cloned as an 18 kb HindIII-BamHI restriction fragment in pUC8 giving plasmid pMO1. A number of derivatives of pMO1, in which aerobactin genes were tagged with a kanamycin resistance gene, were prepared in order to assess the ability of both IS1s to promote the formation of cointegrates with pCJ105, an F derivative devoid of insertion sequences. Mating-out assays indicated that both flanking IS1s were active in cointegrate formation at detectable frequencies. In some cases, the cointegrates could be resolved, the final result being a transposition-like event for the entire aerobactin system.     

Organization,inheritance and expression of acetohydroxyacid synthase genes in the cotton allotetraploid Gossypium hirsutum

The acetohydroxyacid synthase (AHAS) gene family of the cotton AD allotetraploid Gossypium hirsutum has been cloned and characterized. We have identified six different AHAS genes from an analysis of genomic clones and Southern blots of genomic DNA. Four of the six genes are organized as tandem pairs, in which the genes are separated by only 2–3 kb. Conservation of restriction fragment length polymorphisms between G. hirsutum and A-genome and D-genome-containing diploid cottons was sufficient to assign the single genes in clones A5 and A19 to the A and D subgenomes, respectively. Each diploid genome has one tandem pair, but in these cases we could not make specific subgenomic assignments. DNA and deduced amino acid sequences were determined for the A5 and A19 genes, and an AHAS cDNA clone isolated from a leaflibrary. The sequence of the A19 gene matches that of the cDNA clone, while the A5 gene is 97.8% similar. The four genes comprising the tandem pairs are much less similar to the cDNA clone. The deduced amino acid sequences of the mature polypeptides encoded by the A5 and A19 genes are collinear with the housekeeping forms of AHAS from Arabidopsis thaliana, Nicotiana tabacum and Brassica napus. The constitutive expression of A5 and A19 was confirmed with RNase protection assays and northern blots. We conclude that these genes encode the main house-keeping froms of AHAS in G. hirsutum. Among the four AHAS genes comprising the two tandem pairs, at least two are functional. These genes exhibit either low-level constitutive expression (one or both of the downstream genes of each pair), or highly specific expression in reproductive tissue (one or both of the upstream genes of each pair). The AHAS gene family of G. hirsutum is more complex than that of other plants so far examined.     

Different mechanisms generating sequence variability are revealed in distinct regions of the hydroxyproline-rich glycoprotein gene from maize and related specie*

Summary The sequences of the genes coding for a hydroxyproline-rich glycoprotein from two varieties of maize (Zea mays, Ac1503 and W22), a teosinte (Zea diploperennis) and sorghum (Sorghum vulgare) have been obtained and compared. Distinct patterns of variability have been observed along their sequences. The 500 by region immediately upstream of the TATA box is highly conserved in theZea species and contains stretches of sequences also found in the sorghum gene. Further upstream, significant rearrangements are observed, even between the two maize varieties. These observations allow definition of a 5 region, which is common to the four genes and is probably essential for their expression. The 3 end shows variability, mostly due to small duplications and single nucleotide substitutions. There is an intron present in this region showing a high degree of sequence conservation among the four genes analyzed. The coding region is the most divergent, but variability arises from duplications of fragments coding for similar protein blocks and from single nucleotide substitutions. These results indicate that a number of distinct mechanisms (probably point mutation, transposon insertion and excision, homologous recombination and unequal crossing-over) are active in the production of sequence variability in maize and related species. They are revealed in different parts of the gene, probably as the result of the different types of functional constraints acting on them, and of the specific nature of the sequence in each region.The sequences reported in this paper have been deposited in the EMBL/GenBank Database (Bolt, Beranek, and Newman Laboratories, Cambridge, Mass., and EMBL, Heidelberg), accession nos. M36635 (maize Ac1503), X63134 (maize W22), X64173 (teosinte) and X56010 (sorghum)     

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

Summary The Bacillus subtilis cdd gene encoding cytidine/2-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis 43 and 37 RNA polymerase holoenzymes during growth and stationary phase.Abbreviations SDS sodium dodecyl sulphate - Ap ampicillin resistance - Tet^r tetracycline resistance - Km^r kanamycin resistance     

Sequence of the iaa and ipt region of different Agrobacterium tumefaciens biotype III octopine strains: reconstruction of octopine Ti plasmid evolution

The TA regions of biotype III octopine/cucumopine (OC) Ti plasmids are closely related to the TL region of the biotype I octopine Ti plasmids pTiAch5 and pTi15955. Sequence analysis shows that the limited and wide host range biotype III OC TA regions are derived from a common ancestor structure which lacked the 6a gene found in the biotype I octopine TL region. The TA region of the wide host range OC Ti plasmids has conserved most of the original TL-like structure. In most wide host range OC isolates the TA-iaaH gene is inactivated by the insertion of an IS866 element. However, the TA region of the wide host range isolate Hm1 carries an intact TA-iaaH gene. This gene encodes a biologically active product, as shown by root induction tests and indole-3-acetic acid measurements.The limited host range OC Ti plasmids pTiAB3 and pTiAg57 have shorter TA regions which are derived from a wide host range TA region. The AB3 type arose by an IS868-mediated, internal TA region deletion which removed the iaa genes and part of the ipt gene and left a copy of IS868 at the position of the deleted fragment. The pTiAB3 iaa/ipt deletion was followed by insertion of a second IS element, IS869, immediately 3 of the ipt gene. pTiAg57 underwent the same iaa-ipt deletion as pTiAB3, but lacks the IS868 and IS869 elements.Analysis of the various TA region structures provides a detailed insight into the evolution of the biotype III OC strains.     

Sequence variation, differential expression and chromosomal location of rice chitinase genes

Rice chitinases are encoded by a small multigene family. To clarify the overall organization of rice chitinase genes, we have isolated and characterized the genes Cht-1, Cht-2 and Cht-3. Although all the three genes encode class I chitinase, the nucleotide sequences of the coding regions of Cht-1 and Cht-3 are very similar (90%), while that of Cht-2 is clearly more divergent (78%). Only Cht-2 has a 130 by intron and encodes a C-terminal peptide sequence similar to that known to function as a vacuolar targeting signal. In 5 flanking regions of Cht-1 and Cht-3, but not of Cht-2, conserved sequences (GGCCGGCYGCCCYAG) were found. Related sequences were found also in the 5 flanking regions of another chitinase gene and a -glucanase gene which has also been reported to be stress-induced in rice. RNA blot hybridization analysis demonstrated that the stress-induced expression patterns of the Cht-1 and Cht-3 genes are similar, but quite different from that of Cht-2. However, all three genes are active in unstressed roots. By restriction fragment length polymorphism (RFLP) linkage analysis, Cht-1 and Cht-3 were mapped onto chromosome 6 and shown to be closely linked (0.8 cM). Cht-2 was mapped onto chromosome 5. All these features suggest that the expression patterns of rice class I chitinase genes may be correlated with their levels of sequence divergence and their chromosomal location.     

Genetic organization ofAcetobacter for acetic acid fermentation

Plasmid vectors for the acetic acid-producing strains ofAcetobacter andGluconobacter were constructed from their cryptic plasmids and the efficient transformation conditions were established. The systems allowed to reveal the genetic background of the strains used in the acetic acid fermentation. Genes encoding indispensable components in the acetic acid fermentation, such as alcohol dehydrogenase, aldehyde dehydrogenase and terminal oxidase, were cloned and characterized. Spontaneous mutations at high frequencies in the acetic acid bacteria to cause the deficiency in ethanol oxidation were analyzed. A new insertion sequence element, IS1380, was identified as a major factor of the genetic instability, which causes insertional inactivation of the gene encoding cytochromec, an essential component of the functional alcohol dehydrogenase complex. Several genes including the citrate synthase gene ofA. aceti were identified to confer acetic acid resistance, and the histidinolphosphate aminotransferase gene was cloned as a multicopy suppressor of an ethanol sensitive mutant. Improvement of the acetic acid productivity of anA. aceti strain was achieved through amplification of the aldehyde dehydrogenase gene with a multicopy vector. In addition, spheroplast fusion of theAcetobacter strains was developed and applied to improve their properties. ADH membrane-bound alcohol dehydrogenase - ALDH membrane-bound aldehyde dehydrogenase - IS insertion sequence - NTG N-methyl-N-nitro-N-nitrosoguanidine - PQQ pyrroloquinoline quinone