Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans.

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homology (74%) and Clostridium pasteurianum (nifH 1) showed the least homology (54%). In a comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH 1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.     

Genetic organization of the KpnI restriction--modification system.

The KpnI restriction-modification (KpnI RM) system was previously cloned and expressed in E. coli. The nucleotide sequences of the KpnI endonuclease (R.KpnI) and methylase (M. KpnI) genes have now been determined. The sequence of the amino acid residues predicted from the endonuclease gene DNA sequence and the sequence of the first 12 NH2-terminal amino acids determined from the purified endonuclease protein were identical. The kpnIR gene specifies a protein of 218 amino acids (MW: 25,115), while the kpnIM gene codes for a protein of 417 amino acids (MW: 47,582). The two genes transcribe divergently with a intergeneic region of 167 nucleotides containing the putative promoter regions for both genes. No protein sequence similarity was detected between R.KpnI and M.KpnI. Comparison of the amino acid sequence of M.KpnI with sequences of various methylases revealed a significant homology to N6-adenine methylases, a partial homology to N4-cytosine methylases, and no homology to C5-methylases.     

ScrFI restriction-modification system of Lactococcus lactis subsp. cremoris UC503: cloning and characterization of two ScrFI methylase genes.

Two genes from the total genomic DNA of dairy starter culture Lactococcus lactis subsp. cremoris UC503, encoding ScrFI modification enzymes, have been cloned and expressed in Escherichia coli. No homology between the two methylase genes was detected, and inverse polymerase chain reaction of flanking chromosomal DNA indicated that both were linked on the Lactococcus genome. Neither clone encoded the cognate endonuclease. The DNA sequence of one of the methylase genes (encoded by pCI931M) was determined and consisted of an open reading frame 1,170 bp long, which could encode a protein of 389 amino acids (M(r), 44.5). The amino acid sequence contained the highly characteristic motifs of an m5C methylase. Extensive regions of homology were observed with the methylases of NlaX, EcoRII, and Dcm.     

DNA sequence conservation at the gene level in a conserved chromosomal segment in twoPseudomonas species

A comparative analysis has been made of the DNA sequences of the isofunctional genes encodingN-acetylglutamate synthase of the arginine biosynthetic pathway of the bacterial speciesPseudomonas aeruginosa andPseudomonas putida. Overall homologies of 81% and 84% at the nucleotide and deduced amino acid sequence levels, respectively, were observed. This high homology was also reflected in the strikingly similar hydropathy profiles of the encoded proteins; patterns of codon usage, including rare codon usage; and amino acid composition of the proteins. This high level of homology at the DNA sequence level is consistent with the location of these genes in the genetically conserved chromosomal region (called auxotrophic-rich region) of the respectivePseudomonas species. Despite chromosomal rearrangements identified in this region the conservation observed at the chromosomal level between thesePseudomonas species is also maintained at the level of the DNA sequence, and in the deduced amino acid sequence, of the genes reported here and of six other pairs of genes of the tryptophan biosynthetic pathway, reported by others, which are also located within this chromosomal region.     

Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli.

Immunochemical cross-reaction between the endo-exonuclease of Neurospora crassa, an enzyme previously implicated in recombination and recombinational DNA repair, and the recC-encoded polypeptide of Escherichia coli has been detected by immunoblotting extracts of strains of E. coli having a deletion that includes the recBCD genes but carrying multicopy plasmids bearing all three of the recBCD genes or only one or two of these genes. It was predicted that homology would also be found at the amino acid sequence level between the recC polypeptide and both nuclear and mitochondrial endo-exonucleases of Saccharomyces cerevisiae, which cross-react with antibodies raised to the N. crassa endo-exonuclease. Since the gene for the S. cerevisiae mitochondrial enzyme, NUC1, has been cloned and sequenced and the predicted amino acid sequence is known, this sequence was aligned with the predicted amino acid sequence of the recC polypeptide. Extensive homology was found by aligning 306 of the 329 amino acids of the yeast mitochondrial nuclease sequence with the carboxy-terminal one-quarter of the amino acid sequence of the recC polypeptide.     

How reliable is amino acid sequence homology in predicting similarity of structure and function of c-myc and Ad12 E1A oncogenic proteins?

Summary Adenovirus E1A and c-myc genes are known to be capable of transforming primary rat cells when they occur in combination with either polyoma middle-T or T24 Harvey-ras 1 genes. There was a low level of amino acid sequence homology between the nuclear adenovirus-12 (Ad12) E1A protein product (289 amino acids) and the c-myc protein based on optimal alignment and percentage identity. In contrast to others [Ralston R, Bishop JM (1983) Nature 306:803–806], we concluded that this low level of amino acid sequence homology was not significant, since rabies glycoprotein (RGP), which has no transforming function and localizes to the cell surface, had a similar low level of amino acid sequence homology to the c-myc protein. Furthermore, dot-matrix analysis, when used to test the overall level of amino acid sequence homology, showed no significant homology between c-myc and Ad12 E1A, E1B, or RGP. Thus, low levels of amino acid sequence homology between two proteins may not be sufficient to predict structural and functional similarities between them reliably, even if the two proteins appear to share a common function.     

Isolation and structure of a rat cytochrome c gene

We screened a Charon 4A-rat genomic library using the cloned iso-1 cytochrome c gene from Saccharomyces cerevisiae as a specific hybridization probe. Eight different recombinant phages homologous to a coding region subfragment of the yeast gene were isolated. Nucleotide sequence analysis of a 0.96-kilobase portion of one of these established the existence of a gene coding for a cytochrome c identical in amino acid sequence with that of mouse. The rat polypeptide chain sequence had not previously been determined. In contrast to the yeast iso-1 and iso-2 cytochrome c genes, neither of which have introns, the rat gene contains a single 105-base pair intervening sequence interrupting glycine codon 56. The overall nucleotide sequence homology between cytochrome c genes of yeast and rat is about 62%, with areas of greater homology coinciding with four regions of functionally constrained amino acid sequences. Two of these regions displayed 85-90% DNA sequence homology, including the longest consecutive homologous stretch of 14 nucleotides, corresponding to amino acids 47-52 of the rat protein. Somewhat less homology was observed in the DNA-specifying amino acids 70-80, which are invariant residues in most known cytochrome c molecules. Thermal dissociation of the yeast probe from the homologous rat DNA was at about 58 degrees C in 0.39 M Na+. These results establish that cytochrome c genes may be isolated by interspecies hybridization between widely divergent organisms.     

The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.     

Homology between mammalian DNA polymerase beta and terminal deoxynucleotidyltransferase

Nucleotide sequence analysis of the cDNA and the genomic clones for rat DNA polymerase beta revealed the existence of a 1,005-base pair open reading frame capable of encoding a Mr = 38,269 polypeptide of 335 amino acid residues. The region of 174 amino acid residues between the 42nd and 215th residues of the DNA polymerase beta polypeptide has extensive amino acid sequence homology with the region between the 195th and 366th residues of human terminal deoxynucleotidyltransferase. The two enzymes share extensive homology not only in primary structures but also in the computer-derived higher structures in these particular regions. The genes for DNA polymerase beta and terminal deoxynucleotidyltransferase are proposed to be derived from a common ancestral DNA polymerase gene.     

Cloning and analysis of the L-lactate utilization genes from Streptococcus iniae.

The presence of lactate oxidase was examined in eight Streptococcus species and some related species of bacteria. A clone (pGR002) was isolated from a genomic library of Streptococcus iniae generated in Escherichia coli, containing a DNA fragment spanning two genes designated lctO and lctP. We show that these genes are likely to be involved in the L-lactic acid aerobic metabolism of this organism. This DNA fragment has been sequenced and characterized. A comparison of the deduced amino acid sequence of LctP protein demonstrated that the protein had significant homology with the L-lactate permeases of other bacteria. The amino acid sequence of the LctO protein of S. iniae also showed a strong homology to L-lactate oxidase from Aerococcus viridans and some NAD-independent lactate dehydrogenases, all belonging to the family of flavin mononucleotide-dependent alpha-hydroxyacid-oxidizing enzymes. Biochemical assays of the gene products confirm the identity of the genes from the isolated DNA fragment and reveal a possible role for the lactate oxidase from S. iniae. This lactate oxidase is discussed in relation to the growth of the organism in response to carbon source availability.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

Evolutionary and structural relationships among the group-specific component, albumin and alpha-fetoprotein.

The group-specific component (Gc) is a plasma protein that binds vitamin D. Recent characterization of human Gc cDNA demonstrated homology with serum albumin and alpha-fetoprotein. This study compares the sequences of the three proteins and demonstrates a strong evolutionary relationship. Albumin, alpha-fetoprotein and Gc evolved from an ancestral gene containing an intragenic triplication. Comparison of the amino acid sequences and patterns of double disulfide bonds suggests that the Gc gene may have diverged from an ancestral gene earlier in evolution than the genes encoding albumin and alpha-fetoprotein. Analysis of the amino acid and nucleotide sequences of the three internal domains of Gc revealed 19-23% amino acid sequence identity and the localization of three homology blocks with 40-44% nucleotide sequence identity. The deduced amino sequence of Gc furnished data for comparing its molecular configuration based on the predicted secondary structure with those predicted for human albumin and alpha-fetoprotein. Utilization of Gc cDNA has also led to the identification of its genomic DNA and detection of a human DNA polymorphism.     

A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene

The DNA sequence of the ompC gene which encodes one of the outer membrane porins has been determined. The gene appears to encode a secretory precursor of OmpC protein consisting of a total of 367 amino acid residues with a signal peptide of 21 amino acid residues at its NH2-terminal end. The 5' end noncoding region including the promoter of the ompC gene is extremely [A-T]-rich, and the codon usage in the ompC gene is unusual as are those in genes for other abundant outer membrane proteins. The promoter sequence of the ompC gene was compared with that of the ompF gene, both of which are controlled by the osmoregulatory operon, ompB. The deduced amino acid sequence of the OmpC protein showed extensive homology with that of the other porins (OmpF and PhoE proteins). The homology in the primary amino acid sequences, as well as the coding DNA sequences among the porins, indicates that the structural genes for the three porins evolved from a common ancestral gene. Comparison of the amino acid sequences among the OmpC, OmpF, and PhoE porins will be discussed with regard to structure and function.     

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.     

Complete nucleotide sequence of a Selenomonas ruminantium plasmid and definition of a region necessary for its replication in Escherichia coli.

A plasmid from Selenomonas ruminantium subspecies lactilytica has been subcloned in Escherichia coli K-12 and completely sequenced. Three open reading frames (ORFs) of 909, 801, and 549 bp were identified and the complete sequence was analyzed by comparison with DNA and protein databases. No significant deoxynucleotide or amino acid sequence homology with other published genes or proteins was detected. The plasmid was shown to replicate independently in E. coli K-12 by a DNA polymerase I-dependent mechanism and deletion analysis defined the DNA sequence responsible for this phenotype.     

Cloning, nucleotide sequence, and expression of the HincII restriction-modification system.

Two genes, coding for the HincII from Haemophilus influenzae Rc restriction-modification system, were cloned and expressed in Escherichia coli RR1. Their DNA sequences were determined. The HincII methylase (M.HincII) gene was 1,506 base pairs (bp) long, corresponding to a protein of 502 amino acid residues (Mr = 55,330). The HincII endonuclease (R.HincII) gene was 774 bp long, corresponding to a protein of 258 amino acid residues (Mr = 28,490). The amino acid residues predicted from the R.HincII and the N-terminal amino acid sequence of the enzyme found by analysis were identical. These methylase and endonuclease genes overlapped by 1 bp on the H. influenzae Rc chromosomal DNA. The clone, named E. coli RR1-Hinc, overproduced R.HincII. The R.HincII activity of this clone was 1,000-fold that from H. influenzae Rc. The amino acid sequence of M.HincII was compared with the sequences of four other adenine-specific type II methylases. Important homology was found between tne M.HincII and these other methylases.     

Chlamydia psittaci ewe abortion agent: complete nucleotide sequence of the major outer membrane protein gene

The complete nucleotide sequence encoding the major outer membrane protein (MOMP) of Chlamydia psittaci strain A22/M, responsible for enzootic abortion of ewes (EAE), has been determined. An 800bp Eco RI/ Xba I fragment containing a portion of the MOMP coding sequence from C. trachomatis serovar L1 was used to probe a λL47.1 genomic library constructed from DNA obtained from C. psittaci EAE A22/M. The recombinant L47.1/EA1 was selected and contained the entire C. psittaci MOMP gene within a 7.5 kb Bam HI fragment. The DNA sequence revealed an open reading frame encoding 402 amino acids, including a 22 amino acid signal peptide, which exhibited 17/22 conservation with the signal peptide of C. trachomatis MOMP. The calculated molecular mass of the C. psittaci MOMP was 43 kDa. A comparison of the MOMP genes of C. psittaci and C. trachomatis revealed only 34% nucleotide sequence homology, but 65% amino acid homology.     

The nucleotide sequence of the LPD1 gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae: comparison between eukaryotic and prokaryotic sequences for related enzymes and identification of potential upstream control sites

The complete nucleotide sequence of the LPD1 gene, which encodes the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Saccharomyces cerevisiae, has been established. The flanking region 5' to the LPD1 gene contains DNA sequences which show homology to known control sites found upstream of other yeast genes. The primary structure of the protein, determined from the DNA sequence, shows strong homology to a group of flavoproteins including Escherichia coli lipoamide dehydrogenase and pig heart lipoamide dehydrogenase. The amino acid sequence also reveals the presence of a potential targeting sequence at its N-terminus which may facilitate transport to and entry into mitochondria.     

Genetic analysis of acetan biosynthesis in Acetobacter xylinum: DNA sequence analysis of the aceM gene encoding an UDP-glucose dehydrogenase

Analysis of the nucleotide sequence of a 1592 bp region of Acetobacter xylinum genomic DNA involved in acetan biosynthesis revealed the presence of an open-reading frame (aceM) encoding a protein of 449 amino acids with a molecular weight of 48.5 kDa. The deduced amino acid sequence of aceM displayed high homology to the protein sequences of genes encoding UDP-glucose dehydrogenase (UGDH) activities from other organisms. AceM is likely to encode the UGDH involved in the biosynthesis of UDP-glucuronic acid required for acetanbiosynthesis. This revised version was published online in November 2006 with corrections to the Cover Date.