Coordinate expression of Escherichia coli dnaA and dnaN genes

Summary The defects of temperature-sensitive dnaA and dnaN mutants of Escherichia coli are complemented by a recombinant lambda phage, which carries the bacterial DNA segment composed of two EcoRI segments of 1.0 and 3.3 kilobases. Derivatives of the phage, which have an insertion segment of Tn3 in the dnaA gene, are much less active in expressing the dnaN gene function than the parent phage. The dnaN gene activity was determined as the efficiency of superinfecting phage to suppress loss of the viability of lysogenic dnaN59 cells at the nonpermissive temperature. Deletions that include the end of the dnaA gene distal to the dnaN gene also reduce the expression of the dnaN gene fuction. Deletion and insertion in the dnaN gene do not affect the expression of the dnaA gene function. The expression of the dnaN gene function by the dnaA ^- dnaN ⁺ phages remains weak upon simultaneous infection with dnaA ⁺ dnaN ^- phages. Thus the insertion and deletion in the dnaA gene influence in cis the expression of the dnaN gene. We propose that the dnaA and dnaN genes constitute an operon, where the former is upstream to the latter.     

The 5''-untranslated leader sequence of potato virus X RNA enhances the expression of a heterologous gene in vivo.

Summary We have cloned and sequenced the wild-type CDC26 gene and a mutant allele, cdc26-1, of Saccharomyces cerevisiae. Nucleotide sequence analysis revealed that the gene we cloned was the same as SCD26, a dosage-dependent suppressor of cdc26. However, the cloned gene is in fact the CDC26 gene, because a nucleotide substitution in cdc26-1 was found to be a nonsense mutation in this sequence. Disruption of this gene conferred thermosensitive cell growth and the disrupted cdc26 gene could not complement the cdc26-1 mutant allele. Thus, the CDC26 gene is required for cell growth only at high temperature.     

Identification of the dnaA and dnaN gene products of Escherichia coli

Summary A specialized transducing lambda phage carrying the dnaN and dnaA genes of Escherichia coli specifies two proteins of about 41 and 48 kilodaltons (kd). The temperature-sensitive mutations, dnaN59 and dnaA167, were found to result in altered isoelectric points of the 41 and 48 kd proteins, respectively. Thus the dnaN gene product was identified as a weakly acidic 41 kd protein, and the dnaA gene product as a weakly basic 48 kd protein. The synthesis of the dnaN gene product is greatly reduced by insertion of a transposon Tn3 in the dnaA gene and by deletion in the gene at the distal end to the dnaN gene. Temperature-sensitive dnaA mutations, on the other hand, do not affect the synthesis of the dnaN gene product. These results indicate that the synthesis of the dnaN gene product is dependent on the structural integrity of the dnaA gene.Abbreviations kd kilodaltons - SDS sodium dodecyl sulfate     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Genomic organization of the HSET locus and the possible association of HLA-linked genes with immotile cilia syndrome (ICS)

 The kinesin-related protein (HSET) gene belongs to the kinesin superfamily, the members of which are involved in cellular transport processes. The HSET gene product was previously characterized by partial cDNA sequencing. The gene is located on the short arm of human Chromosome 6 (6p21.3), at the centromeric end of the major histocompatibility complex. Here, we report the genomic structure of the complete HSET gene together with its flanking loci. Sequence analysis of the 40 kilobase (kb) cosmid clone containing the HSET gene also revealed the presence of several new genes not related to the kinesin superfamily. These include a 60S ribosomal protein L35A-like pseudogene (rPL35A-like) on the telomeric side and a polycomb-like gene (PHF1), a copper tolerance-like gene (CUTA1) and the 5' part of the synaptic ras-GTPase-activating protein (SynGAP) gene centromeric of HSET. In addition, a complete 60S ribosomal protein L12-like (rPL12L) gene in intron 3 of the HSET gene was identified which appears to have an open reading frame. The possible involvement of the HSET gene and a β-tubulin gene (TUBB) in the pathogenesis of immotile cilia syndrome (ICS) was studied by screening two unrelated ICS families with microtubular defects and suspected HLA linkage for mutations within the HSET gene and the TUBB gene. Four single base substitutions were detected in the HSET gene, and none in the TUBB gene. On the basis of these data, a role of the HSET and TUBB products in the pathogenesis of ICS in the two families is unlikely. Received: 22 October / Revised: 15 February 1999     

Comparison of lantibiotic gene clusters and encoded proteins

Lantibiotics form a group of modified peptides with unique structures, containing post-translationally modified amino acids such as dehydrated and lanthionine residues. In the gram-positive bacteria that secrete these lantibiotics, the gene clusters flanking the structural genes for various linear (type A) lantibiotics have recently been characterized. The best studied representatives are those of nisin (nis), subtilin (spa), epidermin (epi), Pep5 (pep), cytolysin (cyl), lactocin S (las) and lacticin 481 (lct). Comparison of the lantibiotic gene clusters shows that they contain conserved genes that probably encode similar functions.The nis, spa, epi and pep clusters contain lanB and lanC genes that are presumed to code for two types of enzymes that have been implicated in the modification reactions characteristic of all lantibiotics, i.e. dehydration and thio-ether ring formation. The cyl, las and lct gene clusters have no homologue of the lanB gene, but they do contain a much larger lanM gene that is the lanC gene homologue. Most lantibiotic gene clusters contain a lanP gene encoding a serine protease that is presumably involved in the proteolytic processing of the prelantibiotics. All clusters contain a lanT gene encoding and ABC transporter likely to be involved in the export of (precursors of) the lantibiotics. The lanE, lanF and lanG genes in the nis, spa and epi clusters encode another transport system that is possibly involved in self-protection. In the nisin and subtilin gene clusters two tandem genes, lanR and lanK, have been located that code for a two-component regulatory system.Finally, non-homologous genes are found in some lantibiotic gene clusters. The nisl and spal genes encode lipoproteins that are involved in immunity, the pepI gene encodes a membrane-located immunity protein, and epiD encodes an enzyme involved in a post-translational modification found only in the C-terminus of epidermin. Several genes of unknown function are also found in the las gene cluster.A database has been assembled for all putative gene products of type A lantibiotic gene clusters. Database searches, multiple sequence alignment and secondary structure prediction have been used to identify conserved sequence segments in the LanB, LanC, LanE, LanF, LanG, LanK, LanM, LanP, LanR and LanT gene products that may be essential for structure and function. This database allows for a rapid screening of newly determined sequences in lantibiotic gene clusters.     

Sequence and diversity of rabbit T-cell receptor gamma chain genes

The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes.The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases and have been assigned the accession numbers D38134-D38144 and D42090     

An essential gene of Escherichia coli that has sequence similarity to a chloroplast gene of unknown function

Summary The dedB gene of Escherichia coli has sequence similarity to the zfpA gene of the chloroplast chromosome. The functions of dedB and zfpA are unknown. We constructed derivatives of temperature-sensitive polA strains into whose chromosomes a plasmid containing the disrupted dedB gene was integrated by homologous recombination. These strains contained normal and disrupted dedB genes in their chromosomes. We then selected plasmid-segregated strains and found no cells containing the disrupted dedB gene, indicating that disruption of the dedB gene was lethal in polA strains of E. coli.     

The arsD gene encodes a second trans-acting regulatory protein of the plasmid-encoded arsenical resistance operon

The plasmid-encoded arsenical resistance (ars) operon produces resistance to trivalent and pentavalent salts of arsenic and antimony. The first gene in the operon, arsR, was previously shown to encode a repressor protein. A newly identified gene, arsD, is shown here to encode a regulatory protein, the ArsD protein. The gene was identified by construction of an in-frame fusion between the C-terminally truncated arsD gene and the coding region for the mature form of β-lactamase (blaM). The native arsD gene product was overexpressed and radioactively labelled as a 13kDa polypeptide. A frameshift mutation within the arsD gene resulted in elevated levels of expression of downstream ars genes. Co-expression of a wild-type arsD gene in trans with the operon containing the mutated arsD gene reduced expression of the downstream genes to wild-type levels. The presence of the arsD gene had no effect on the basal level of operon expression set by the arsR gene product, and the repression produced by the arsD gene product was not affected by inducers of the operon. The results indicate that the ArsD protein is an inducer-independent trans-acting regulatory protein.     

Elimination of marker genes and targeted integration via FLP/<Emphasis Type="BoldItalic">FRT</Emphasis> recombination system from yeast in hybrid aspen (<Emphasis Type="BoldItalic">Populus tremula</Emphasis> L. × <Emphasis Type="Italic">P. tremuloides</Emphasis> Michx.)

Marker gene elimination was investigated in hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) using the FLP/FRT recombination system. The construct contained the FLP recombinase under control of a heat inducible promoter, the antibiotic resistance gene nptII driven by the CaMV 35S promoter, and a promoterless uidA gene. The construct was integrated into poplar via Agrobacterium-mediated transformation. The active FLP recombinase excised the nptII marker gene and combined the promoterless uidA gene with the CaMV 35S promoter to form an active uidA gene. For targeted transgene integration, two constructs were used. The first one carried FLP under control of the heat-inducible Gmhsp17.5-E promoter from soybean as well as an active nptII gene flanked by two FRT sites; the second contained the promoterless bar selection marker gene also flanked by two FRT sites. Following transformation and induction of FLP, the enzyme mediated a site-specific recombination at the FRT sites of both constructs. This recombination leads to an excision of the FLP and nptII gene from the first as well as an excision of the promoterless bar gene from the second construct. The promoterless bar gene reintegrated exactly at the former position of the FLP and nptII genes in the first construct to form an active bar gene. The FLP/FRT recombination system from yeast forms a promising basis for the production of antibiotic-free transgenic plants and a useful tool for directed integration of transgenes into plant genomes.     

Identification and Ds‐tagged isolation of a new gene at the Cf‐4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5

Leaf mould disease in tomato is caused by the biotrophic fungus Cladosporium fulvum. An Ac/Ds targeted transposon tagging strategy was used to isolate the gene conferring resistance to race 5 of C. fulvum, a strain expressing the avirulence gene Avr4. An infection assay of 2-week-old seedlings yielded five susceptible mutants, of which two had a Ds element integrated in the same gene at different positions. This gene, member of a gene family, showed high sequence homology to the C. fulvum resistance genes Cf-9 and Cf-2. The gene is predicted to encode an extracellular transmembrane protein containing a divided domain of 25 leucine-rich repeats. Three mutants exhibited a genomic deletion covering most of the Lycopersicon hirsutum introgressed segment, including the Cf-4 locus. Southern blot analysis revealed that this deletion includes the tagged gene and five homologous sequences. To test whether the tagged gene confers resistance to C. fulvum via Avr4 recognition, the Avr4 gene was expressed in planta. Surprisingly, expression of the Avr4 gene still triggered a specific necrotic response in the transposon-tagged plants, indicating that the tagged resistance gene is not, or is not the only gene, involved in Avr4 recognition. Mutants harbouring the genomic deletion did not show this Avr4-specific response. The deleted segment apparently contains, in addition to the tagged gene, one or more other genes, which play a role in the Avr4 responses. The tagged gene is present at the Cf-4 locus, but it does not necessarily recognize Avr4 and is therefore designated Cf-4A.     

Functional complementation between the two homologous genes, ops and tps, during differentiation of Myxococcus xanthus

Summary Protein S is a development-specific protein of Myxococcus xanthus encoded by the tps gene. It has been shown that there are two extensively homologous genes (ops and tps) tandemly repeated in the same direction with a 1.4 kb spacer fragment between them (Inouye et al. 1983). Seven deletion mutants were constructed by removing the ops gene, the tps gene, segments of the spacer sequence or combinations of these regions. The deleted regions were replaced with DNA fragments carrying the Tn5 gene for kanamycin resistance.The effects of deleting different regions on morphological changes and on patterns of protein synthesis during fruiting body formation were examined. The process of fruiting body formation was severely delayed when both the ops and the tps genes were deleted. However, this delay could be suppressed by either the ops gene or the tps gene, individually, although in the latter case, a slight delay was still observed. These results indicate that the ops gene is expressed during fruiting body formation and plays a role in the normal program of M. xanthus differentiation. Furthermore, the role of the ops gene can be complemented by the tps gene. The deletion of the ops and/or tps genes had no effect on glycerol-spore formation.     

Complete Mitochondrial DNA Sequences of the Decapod Crustaceans Pseudocarcinus gigas (Menippidae) and Macrobrachium rosenbergii (Palaemonidae)

The complete mitochondrial DNA sequence was determined for the Australian giant crab Pseudocarcinns gigas (Crustacea: Decapoda: Menippidae) and the giant freshwater shrimp Macrobrachium rosenbergii (Crustacea: Decapoda: Palaemonidae). The Pse gigas and Mrosenbergii mitochondrial genomes are circular molecules, 15,515 and 15,772 bp in length, respectively, and have the same gene composition as found in other metazoans. The gene arrangement of M. rosenbergii corresponds with that of the presumed ancestral arthropod gene order, represented by Limulus polyphemus, except for the position of the tRNA^Leu(UUR) gene. The Pse. gigas gene arrangement corresponds exactly with that reported for another brachyuran, Portunus trituberculatus, and differs from the M. rosenbergii gene order by only the position of the tRNA^His gene. Given the relative positions of intergenic nonoding nucleotides, the “duplication/random loss” model appears to be the most plausible mechanism for the translocation of this gene. These data represent the first caridean and only the second brachyuran complete mtDNA sequences, and a source of information that will facilitate surveys of intraspecific variation within these commercially important decapod species.     

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.     

Evolution of mercuric reductase (<Emphasis Type="Italic">merA</Emphasis>) gene: A case of horizontal gene transfer

In the present study the role of horizontal gene transfer events in providing the mercury resistance is depicted. merA gene is key gene in mer operon and has been used for this swtudy. Phylogenetic analysis of aligned merA gene sequences shows broad similarities to the established 16S rRNA gene phylogeny. But there is no separation of bacterial merA gene from archael merA gene which suggests that merA gene in both these groups share considerable sequence homology. However, inconsistencies between merA gene and 16S rRNA gene phylogenetic trees are apparent for some taxa. These discrepancies in the phylogenetic trees for merA gene and 16S rRNA gene have lead to the suggestion that horizontal gene transfer (HGT) is a major contributor for its evolution. The close association among members of different groups in merA gene tree, as supported by high bootstrap values, deviations in GC content and codon usage pattern indicate the possibility that horizontal gene transfer events might have taken place during the evolution of this gene.     

Mapping of the multiple regulatory sites for putP and putA expression in the putC region of Escherichia coli

Summary The effects of regulatory proteins on the expression of putP and putA were studied using put-lacZ fusion genes. The expression of the putP-lacZ gene was activated by the glnG gene product and the catabolite gene activator protein (CAP). The putA gene product inhibited activation of putP-lacZ gene expression by CAP or the glnG gene product and its inhibition was greater in the absence of proline. The expression of the putA-lacZ gene was activated by CAP and repressed by the glnG gene product. The putA gene product acted as a repressor in the absence of proline, but not in its presence. Studies using put-lacZ fusion genes with upstream deletions showed that the region required for the activation of putP by CAP was within 234 bp upstream of the translational initiation site and that that for the activation of putP was within 107 bp upstream of the translational initiation site of the putA gene. This supported the suggested locations of CAP binding sites. The region required for induction of putP and putA expression by proline was located at the Hpal site 182 bp upstream of the translational starting site of putA, suggesting that a sequence of dyad symmetry located 1 bp to the left of the HpaI site is a candidate for the binding site of the putA gene product.Abbreviations AC L-azetidine-2-carboxylic acid - Ap ampicillin - CAP catabolite gene activator protein - NR_I nitrogen regulator I - RF DNA DNA replicative form - Str streptomycin - Tc tetracycline - TTC 2,3,5-triphenyl tetrazolium chloride - UV ultraviolet