Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.     

Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes.

Biochemical analysis of the enzymatic activity catalyzing the conversion of chorismate to isochorismate in the enterobactin biosynthetic pathway attributed the reaction to the isochorismate synthetase enzyme, designated EntC. However, the lack of mutations defining this activity has hampered the precise identification of the entC structural gene. In this study, we engineered a stable insertion mutation into the chromosomal region between the enterobactin genes fepB and entE. This mutation disrupted the structural gene for a previously identified 44-kilodalton protein and eliminated production of 2,3-dihydroxybenzoic acid, the catechol precursor of enterobactin. The complete nucleotide sequence of this gene was determined and compared with the sequences of other genes encoding chorismate-utilizing proteins. The similarities observed in these comparisons not only indicated that the locus is entC but also supported the premise that these enzymes constitute a family of related proteins sharing a common evolutionary origin. In addition, in this and the accompanying paper (M. S. Nahlik, T. J. Brickman, B. A. Ozenberger, and M. A. McIntosh, J. Bacteriol. 171:784-790, 1989), evidence is presented indicating that the entA product is potentially a secondary factor in the chorismate-to-isochorismate conversion and that the prototypic entC lesion (entC401) resides in the structural gene for the EntA protein. Finally, polarity effects from the insertion mutation in entC on downstream biosynthetic genes indicated that this locus is the promoter-proximal cistron in an ent operon comprising at least five genes. Appropriate regulatory signals upstream of entC suggest that this operon is regulated by iron through interaction with the Fur repressor protein.     

Nucleotide sequence of an actin-encoding gene from Hydra attenuata: structural characteristics and evolutionary implications

We have determined the complete nucleotide sequence of an actin-encoding gene from Hydra attenuata as well as partial sequences of cDNA clones from two additional actin-encoding genes. The gene from the genomic clone contains a single intron, and has promoter and polyadenylation signals similar to those found in other species. The hydra genome has a very A + T-rich base composition (71%). This is reflected in the codon usage of the actin-encoding genes, which is strongly biased towards codons having A or T in the third position. The hydra actin-encoding gene family consists of three or more transcribed genes, two of which are very closely related to each other and probably arose by a recent gene duplication. Hydra actin, like other invertebrate actins, is more similar to the non-muscle isotypes of vertebrates than to the vertebrate muscle actins. Hydra actin is more similar to animal actins than to those of plants or fungi, which is consistent with the view that all metazoans arose from a single protist ancestor.     

Nucleotide sequence of the ARGRII regulatory gene and amino acid sequence homologies between ARGRII PPRI and GAL4 regulatory proteins

We report here the DNA sequence of the ARGRII gene, one of the three regulatory genes involved in controlling the anabolism and catabolism of arginine in yeast. This gene encodes a protein of 880 amino acids with a deduced molecular mass of about 100 kDa. The ARGRII protein shows significant homology with two other regulatory proteins of yeast, PPRI and GAL4.     

Nucleotide sequence of pig plasma gelsolin. Comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats

Pig plasma gelsolin (Mr = 81595; 739 residues) contains 704 identical residues out of a maximum 730 when compared to the cytoplasmic form of human gelsolin. The cDNA sequence also codes for a peptide of 33 residues N-terminal to the nine-residue plasma extension sequence previously reported: these 33 residues are highly homologous to the human signal peptide and plasma extension. Comparison of the gelsolin sequences with chicken brush border villin, severin from Dictyostelium discoideum and fragmin from Physarum polycephalum shows a strong evolutionary relationship between all these proteins. There are six large repeating segments in gelsolin and villin, and three similar segments in severin and fragmin. Although these multiple repeats cannot be related to any known function of these actin-severing proteins, this superfamily of proteins appears to have evolved from an ancestral sequence of 120 to 130 amino acid residues.     

Nucleotide sequence of Fujinami sarcoma virus: evolutionary relationship of its transforming gene with transforming genes of other sarcoma viruses

We determined the entire nucleotide sequence of the molecularly cloned DNA of Fujinami sarcoma virus (FSV). The sequence of 1182 amino acids was deduced for the FSV transforming protein P130, the product of the FSV gag-fps fused gene. The P130 sequence was highly homologous to the amino acid sequence obtained for the gag-fes protein of feline sarcoma virus, supporting the view that fps and fes were derived from a cognate cellular gene in avian and mammalian species. In addition, FSV P130 and p60^src of Rous sarcoma virus were 40% homologous in the region of the carboxyterminal 280 amino acids, which includes the phosphoacceptor tyrosine residue. These results strongly suggest that the 3′ region of fps/fes and src originated from a common progenitor sequence. A portion (the U3 region) of the long terminal repeat of FSV DNA appears to be unusual among avian retroviruses in its close similarity in sequence and overall organization to the same region of the endogenous viral ev1 DNA.     

Nucleotide sequence of the kanamycin resistance determinant of the pneumococcal transposon Tn1545: evolutionary relationships and transcriptional analysis of aphA-3 genes

The nucleotide sequence of the kanamycin resistance determinant aphA-3 encoded by transposon Tn1545 from Streptococcus pneumoniae was determined and compared to those of plasmids pJH1 and pIP1433 from Streptococcus faecalis and Campylobacter coli, respectively. The three sequences were found to be identical and differed by two substitutions and the deletion of a codon from that of plasmid pSH2 from Staphylococcus aureus. Comparison of the 5' noncoding sequences indicated that the regions containing the aphA-3 gene in pJH1 and in Tn1545 evolved independently by deletion from a sequence similar to that found in pIP1433. In the latter plasmid, aphA-3 is transcribed from a promoter, P1, which is flanked by two 12-base pair direct repeats. The rearrangement observed in pJH1 removed one of these recombinogenic sites and altered the -10 and 3' flanking sequences of P1. The promoter thus generated. P1', allows expression of similar level of kanamycin resistance as P1. However, fusion experiments carried out with a promotorless chloramphenicol acetyltransferase gene indicated that the canonical promoter P1 is significantly less efficient than P1'. From analysis of the thermodynamic properties of these promoters, we conclude that this difference in strength reflects the melting properties of the -10 sequences. The transition from pIP1433 to pJH1 may correspond to the progression of a molecule structurally unstable to a more stable one combined with the need to maintain an efficient promoter upstream of the aphA-3 gene. The deletion event in Tn1545, which occurred between the two 12-base pair directly repeated sequences, removed P1 in its entirety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase

The nucleotide sequence of a transposon Tn7 DNA fragment encoding a 3"(9)-O-nucleotidyltransferase, an aminoglycoside-modifying enzyme, which mediates bacterial resistance to spectinomycin and streptomycin, was determined. The aadA structural gene was 786 bases long and predicted a polypeptide of 262 amino acids with a calculated molecular weight of 29,207. Comparison of the DNA sequences of Tn7 and plasmid R538-1 indicated that their aadA genes were nearly identical. Comparison of the polypeptides predicted by the aadA genes of Tn7 and Tn554 indicated that the genes were related.     

Identification of an Acinetobacter baumannii gene region with sequence and organizational similarity to Tn2670.

A chloramphenicol resistance gene was cloned from chromosomal DNA prepared from a clinical Acinetobacter baumannii isolate. Sequence analysis of this gene (cat) and the flanking DNA regions shows that this gene is linked to Tn21 and to IS1 in a manner similar to that found in Tn2670.     

Acinetobacter baumannii biofilms: Variations among strains and correlations with other cell properties

Acinetobacter baumannii is an opportunistic pathogen that causes serious infections in humans by colonizing and persisting on surfaces normally found in hospital settings. The capacity of this pathogen to persist in these settings could be due to its ability to form biofilms on inanimate surfaces. This report shows that although the ATCC 19606(T) type strain and 8 different clinical isolates form biofilms, there are significant variations in the cell density and microscopic structures of these cell aggregates, with 3 of the isolates forming pellicles floating on the surface of stagnant broth cultures. PCR indicated that, like ATCC 19606(T), all 8 clinical isolates harbor all the genetic components of the CsuA/BABCDE chaperone-usher pili assembly system, which is needed for biofilm formation on plastic. Pili detection in cells of all strains examined supports the presence and function of a pilus assembly system. However, only one of them produced the putative ATCC 19606(T) CsuA/B pilin subunit protein. Hydrophobicity tests and motility assays also showed significant variations among all tested strains and did not result in direct correlations between the biofilm phenotype and cell properties that could affect biofilm formation on abiotic surfaces. This lack of correlation among these 3 phenotypes may reflect some of the variations already reported with this pathogen, which may pose a challenge in the treatment of the infections this pathogen causes in humans using biofilm formation on abiotic surfaces as a target.     

Nucleotide sequence of the chicken cardiac alpha actin gene: absence of strong homologies in the promoter and 3'-untranslated regions with the skeletal alpha actin sequence

The entire nucleotide sequence of the chicken cardiac alpha-actin (CC alpha A) gene has been determined. This is the first complete sequence of a cardiac actin gene that includes the promoter region, cap site, all the introns, and the polyadenylation site. The gene contains six introns, five of which interrupt the coding region at amino acids (aa) 41, 150, 204, 267, and 327. The first intron is in the 5'-noncoding region and is 438 bp in length. The CC alpha A gene encodes an mRNA of approx. 1400 bp with 5'- and 3'-untranslated region of 59 and 184 nucleotides (nt), respectively. Like the chicken skeletal alpha-actin gene, the CC alpha A gene has the codon for the aa cysteine between the initiator ATG and the codon for the N-terminal aspartic acid residue of the mature protein. There are no strong homologies (less than 13 consecutive nt) in the promoter or 3'-untranslated regions between the CC alpha A and chicken skeletal alpha-actin genes even though both are expressed in skeletal muscle during development. However, the 3'-untranslated region of the CC alpha A gene demonstrates significant sequence homology (76% over a 200-nt region) with the same region in the partial sequence of the human cardiac gene. The conservation of these sequence homologies between identical isoforms rather than the different alpha actin genes suggests these conserved regions may have a role in regulation rather than tissue-specific expression, as previously proposed.     

Physical organisation of simple sequence repeats (SSRs) in Triticeae: structural, functional and evolutionary implications

A significant fraction of the nuclear DNA of all eukaryotes is occupied by simple sequence repeats (SSRs) or microsatellites. This type of sequence has sparked great interest as a means of studying genetic variation, linkage mapping, gene tagging and evolution. Although SSRs at different positions in a gene help determine the regulation of expression and the function of the protein produced, little attention has been paid to the chromosomal organisation and distribution of these sequences, even in model species. This review discusses the main achievements in the characterisation of long-range SSR organisation in the chromosomes of Triticum aestivum L., Secale cereale L., and Hordeum vulgare L. (all members of Triticeae). We have detected SSRs using an improved FISH technique based on the random primer labelling of synthetic oligonucleotides (15-24 bases) in multi-colour experiments. Detailed information on the presence and distribution of AC, AG and all the possible classes of trinucleotide repeats has been acquired. These data have revealed the motif-dependent and non-random chromosome distributions of SSRs in the different genomes, and allowed the correlation of particular SSRs with chromosome areas characterised by specific features (e.g., heterochromatin, euchromatin and centromeres) in all three species. The present review provides a detailed comparative study of the distribution of these SSRs in each of the seven chromosomes of the genomes A, B and D of wheat, H of barley and R of rye. The importance of SSRs in plant breeding and their possible role in chromosome structure, function and evolution is discussed.     

Nucleotide sequence of the structural gene for class I pilin from Neisseria meningitidis: homologies with the pilE locus of Neisseria gonorrhoeae

The nucleotide sequence has been determined for the expressed pilin (pilE) locus of Neisseria meningitidis strain C311 which produces class I pili that are antigenically and structurally similar to those of gonococci. The deduced amino acid sequence of the N. meningitidis pilE translation product contains a 7 amino acid N-terminal pre-pilin leader sequence which is identical to that found in gonococcal pilin and which is characteristic of N-methylphenylalanine pili in general. The succeeding N-terminal 53 amino acids are identical to those found in the equivalent position in antigenically variant gonococcal pilins and confirm direct peptide sequencing of the amino-terminus of at least one type of meningococcal pilin. Other regions that are conserved in variant pilin polypeptides from Neisseria gonorrhoeae are conserved at the amino acid level in the class I meningococcal pilin but the coding DNA contains numerous base substitutions when compared with the equivalent gonococcal pil sequence. Sequences extending downstream for about 140 bp on the 3' side of the coding region for both pilin genes are only about 85% homologous.