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PubMed1999

Sequence and expression analysis of a novel Xenopus laevis cDNA that encodes a protein similar to bacterial and chloroplast ribosomal protein L24.

We report here the cloning and the characterization of a Xenopus laevis cDNA that encodes a basic protein of 276 amino acids with a central core region, which shows a substantial degree of homology to bacterial and chloroplast ribosomal protein L24, and additional diverged N- and C-terminal polypeptide extensions. The N-terminal extension displays similarities to the mitochondrial targetting sequence, thereby suggesting that the cDNA probably codes for a mitochondrial ribosomal protein. Although the gene was expressed ubiquitously, at fairly constant levels, during embryogenesis, the abundance of the transcripts in the different tissues varies with the mRNA levels in the kidney, adipose tissue, muscle and liver being greater than that present in the brain, heart, ovary and lung.

Amino Acid Sequence↗

PubMed1994

Regulation of interleukin-8 expression in porcine alveolar macrophages by bacterial lipopolysaccharide.

Interleukin (IL)-8 is a macrophage-derived neutrophil chemotactic factor that plays an important role in the recruitment of neutrophils to inflammatory loci. Hence, expression of IL-8 by alveolar macrophages may be a significant factor in host defense in the lung and in the pathogenesis of pneumonia in swine. To initiate molecular studies of IL-8 regulation in pigs, we cloned IL-8 cDNA and examined the regulation of its mRNA in alveolar macrophages. The porcine IL-8 cDNA consists of 1491 base pairs including a coding region of 309 base pairs. The deduced amino acid sequence was 75 and 81% similar to human and rabbit IL-8, respectively. Resting macrophages contained low levels of IL-8 mRNA, which increased markedly after exposure to bacterial lipopolysaccharide (LPS). LPS induction of IL-8 was direct, not mediated through elevation of tumor necrosis factor or interleukin-1. The effect of LPS on IL-8 expression was dose dependent, and induction was observed at a concentration of 10 pg/ml. IL-8 mRNA expression was detectable within 0.5 h after stimulation with LPS, peaked at 3-6 h at about 30-fold higher levels than in resting cells, and was maintained for 24 h. Secreted IL-8, measured by neutrophil chemotaxis, was induced within 4 h by LPS, and accumulated in the media throughout the 24-h period. The mechanism of induction of IL-8 mRNA appeared to involve transcription and RNA processing. Nuclear run-on analysis showed that the IL-8 gene was actively transcribed in noninduced cells; upon stimulation with LPS, the rate of IL-8 transcription was increased about 4-fold. A single mature mRNA species was detected by primer extension analysis. The half-life of IL-8 mRNA transcripts in aveolar macrophages was approximately 2 h and did not change after LPS stimulation. The ability of LPS to induce IL-8 expression was suppressed by recombinant human IL-4 and dexamethasone in a concentration-dependent manner. These observations indicate that the expression of IL-8 is an early event in the sequelae to bacterial infection in the lung.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and nucleotide sequence of the 90k serine protease gene, hspK, from Bacillus subtilis (natto) No. 16.

We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues. The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases. The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.

Amino Acid Sequence↗

PubMed2003

Targeting RNA: new opportunities to address drugless targets.

Historically, pharmaceutical industries have focussed on the discovery of compounds that target the protein products of genes. The intermediary product between gene and protein, consisting of RNA, has remained largely unexplored. Several drugs targeting the rRNA of bacteria have been, however, in clinical use for over half a century. One of these drug classes, the aminoglycoside antibiotics, also targets human rRNA, and have been developed as therapeutics for genetic disorders. Targeting at the RNA level is an economical approach to address non-drugable proteins and targets that have failed to give leads by hits in HTS, as it can build on biological knowledge gathered over years. RNA also offers entirely new opportunities for drug development, such as targeting of non-coding RNA sequences.

Aminoglycosides↗

PubMed1978

Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein.

Two mutant strains of Escherichia coli have been isolated in which the cellular location of an outer membrane protein, the phage lambda receptor (the lamB gene product), is altered. These mutations were initially selected in a strain containing a lamB-lacZ fusion. In the parent strain the protein coded for by the hybrid gene is located, at least in part, in the outer membrane. In the mutants it is located in the cytoplasm. The mutations responsible for the alteration of cellular location lie very early in the lamB gene, in a region corresponding to the NH2-terminus of the lambda receptor protein. One of these mutations is a small deletion internal to the lamB gene. When this mutation is present in an otherwise wild-type lamB gene, the protein produced is of lower molecular weight than normal receptor. The other mutation behaves as a point mutation; when it is present in an otherwise normal lamB gene, reversion can be demonstrated. The molecular weight of this mutant protein, which is located in the cytoplasm, is larger than that of the wild-type gene product by approximately 2000. It is suggested that these two mutations are in the portion of the lamB gene coding for a signal sequence and thereby block export of the protein.

Bacterial Proteins↗

PubMed1993

Structure and metabolic control of the Yarrowia lipolytica peroxisomal 3-oxoacyl-CoA-thiolase gene.

Using a Yarrowia lipolytica genomic library, several overlapping clones of the peroxisomal 3-oxoacyl-CoA-thiolase gene, POT1, were isolated. The library was prepared in the bacterial expression vector lambda gt11, thus allowing an immunological screening of recombinant bacteriophages with specific antibodies raised against purified peroxisomal thiolase. The isolated POT1 clones hybridized to a 1.4 kb RNA species, which was induced approximately 30-fold when oleate was the carbon source. A 3634-bp segment of the cloned DNA was sequenced. This segment contained, on both strands, three major overlapping open-reading frames of 678, 1122 and 1242 bp. Northern-hybridization analysis showed that only the largest of these reading frames was transcribed. It encodes a protein of 414 amino acids and molecular mass 43.059 kDa. Its deduced amino acid sequence has 30-60% identity and 50-70% sequence similarity when compared to other known thiolases. According to both the amount (68-71%) and location of conserved amino acids, the encoded protein belongs to the peroxisomal rather than the mitochondrial or cytoplasmic class of thiolases. Compared to bacterial and yeast cytosolic thiolases, the POT1 gene product contains a N-terminal extension of 25 amino acids which clearly differs from typical mitochondrial import signals. One of the isolated clones contained, in addition to the POT1 coding sequence, 784 bp of the corresponding 5' flanking region. Nevertheless, it was efficiently expressed in Escherichia coli suggesting the correct recognition of this fungal promoter by the prokaryotic transcriptional and translational machinery. The Y. lipolytica genomic POT1 gene was disrupted by replacing 120 bp of its coding sequence with 2.7 kbp of DNA including the Y. lipolytica LEU2 gene. The resulting delta pot1::LEU2 cells were free of immunologically cross-reacting thiolase. Western-blot analysis showed that the product of the non-disrupted gene had a molecular mass of approximately 42 kDa. This corresponds well to the molecular mass of purified Y. lipolytica peroxisomal thiolase. Disruption of POT1 abolished the ability of Y. lipolytica cells to grow on solid media with oleate as a carbon source. This inability to grow in the presence of oleate suggests both the catabolic function of POT1 and the absence of additional catabolic thiolases in Y. lipolytica. However, the delta pot1::LEU2 cells were unaffected in their ability to elongate externally added tridecanoic acid to its higher-chain-length homologues. Hence, another, POT1-independent and biosynthetic 3-oxoacyl-CoA thiolase must be responsible for this reaction in Y. lipolytica.

Acetyl-CoA C-Acyltransferase↗

PubMed1985

Thermal regulation of beta-galactosidase synthesis using anti-sense RNA directed against the coding portion of the mRNA.

The in vivo production of RNA that is complementary to the mRNA of a particular target gene (anti-sense RNA) appears to be an effective tool for the regulation of genes in Escherichia coli (Coleman, J., Green, P.J., and Inouye, M. (1984) Cell 37, 429-436). These investigators demonstrated that short anti-sense transcripts which are complementary to the ribosome binding site of the target mRNA are overwhelmingly the most effective in blocking protein synthesis. We have constructed plasmids which produce thermally regulated anti-sense transcripts of three regions of the E. coli lac Z gene coding sequence, and have examined the relative effects of these constructs on the synthesis of the lac Z gene product, beta-galactosidase. We conclude that there is a strong correlation between the length of RNA complementarity and the suppression of beta-galactosidase synthesis. Furthermore, a significant inhibition of translation can be obtained when the anti-sense transcript lacks complementarity to the 5' noncoding region of the mRNA, provided that the extent of complementarity with the coding sequence is considerable.

DNA Restriction Enzymes↗

PubMed1988

DNA sequence of a gene cluster coding for subunits of the F0 membrane sector of ATP synthase in Rhodospirillum rubrum. Support for modular evolution of the F1 and F0 sectors.

A region was cloned from the genome of the purple non-sulphur photobacterium Rhodospirillum rubrum that contains genes coding for the membrane protein subunits of the F0 sector of ATP synthase. The clone was identified by hybridization with a synthetic oligonucleotide designed on the basis of the known protein sequence of the dicyclohexylcarbodi-imide-reactive proteolipid, or subunit c. The complete nucleotide sequence of 4240 bp of this region was determined. It is separate from an operon described previously that encodes the five subunits of the extrinsic membrane sector of the enzyme, F1-ATPase. It contains a cluster of structural genes encoding homologues of all three membrane subunits a, b and c of the Escherichia coli ATP synthase. The order of the genes in Rsp. rubrum is a-c-b'-b where b and b' are homologues. A similar gene arrangement for F0 subunits has been found in two cyanobacteria, Synechococcus 6301 and Synechococcus 6716. This suggests that the ATP synthase complexes of all these photosynthetic bacteria contain nine different polypeptides rather than eight found in the E. coli enzyme; the chloroplast ATP synthase complex is probably similar to the photosynthetic bacterial enzymes in this respect. The Rsp. rubrum b subunit is modified after translation. As shown by N-terminal sequencing of the protein, the first seven amino acid residues are removed before or during assembly of the ATP synthase complex. The subunit-a gene is preceded by a gene coding for a small hydrophobic protein, as has been observed previously in the atp operons in E. coli, bacterium PS3 and cyanobacteria. A number of features suggest that the Rsp. rubrum cluster of F0 genes is an operon. On its 5' side are found sequences resembling the -10 (Pribnow) and -35 boxes of E. coli promoters, and the gene cluster is followed by a sequence potentially able to form a stable stem-loop structure, suggesting that it acts as a rho-independent transcription terminator. These features and the small intergenic non-coding sequences suggest that the genes are cotranscribed, and so the name atp2 is proposed for this second operon coding for ATP synthase subunits in Rsp. rubrum. The finding that genes for the F0 and F1 sectors of the enzyme are in separate clusters supports the view that these represent evolutionary modules.

Base Sequence↗

PubMed2003

Combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method.

Recombinogenic engineering or recombineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. We report here a general method for using recombineering to combine overlapping bacterial artificial chromosomes (BACs) to build larger, unified BACs. In order to test the feasibility of using recombineering to combine two large DNA fragments (>20 kb), we constructed a unified BAC containing the full-length tyrosinase-related protein-1 (Tyrp-1) gene from two library-derived BACs, one containing the 5' regulatory elements and the other containing the 3' coding exons. This was achieved using a two-step homologous recombination method enabled by the bacteriophage lambda Red proteins. In the first step, retrieval, a large DNA fragment (approximately 22 kb) was retrieved from one of the original BACs. In the second step, recombination, the retrieved DNA fragment was inserted into the second original BAC to form the unified BAC containing all the desired Tyrp-1 sequence. To further demonstrate the general applicability of our approach, an additional DNA fragment (approximately 20 kb) was inserted into the unified BAC downstream of the coding region. This method should prove very useful for enabling BAC manipulation in a variety of scenarios.

Chromosomes, Artificial, Bacterial↗

PubMed1993

A bipartite DNA-binding domain in yeast Reb1p.

The REB1 gene encodes a DNA-binding protein (Reb1p) that is essential for growth of the yeast Saccharomyces cerevisiae. Reb1p binds to sites within transcriptional control regions of genes transcribed by either RNA polymerase I or RNA polymerase II. The sequence of REB1 predicts a protein of 809 amino acids. To define the DNA-binding domain of Reb1p, a series of 5' and 3' deletions within the coding region was constructed in a bacterial expression vector. Analysis of the truncated Reb1p proteins revealed that nearly 400 amino acids of the C-terminal portion of the protein are required for maximal DNA-binding activity. To further define the important structural features of Reb1p, the REB1 homolog from a related yeast, Kluyveromyces lactis, was cloned by genetic complementation. The K. lactis REB1 gene supports active growth of an S. cerevisiae strain whose REB1 gene has been deleted. The Reb1p proteins of the two organisms generate almost identical footprints on DNA, yet the K. lactis REB1 gene encodes a polypeptide of only 595 amino acids. Comparison of the two Reb1p sequences revealed that within the region necessary for the binding of Reb1p to DNA were two long regions of nearly perfect identity, separated in the S. cerevisiae Reb1p by nearly 150 amino acids but in the K. lactis Reb1p by only 40 amino acids. The first includes a 105-amino-acid region related to the DNA-binding domain of the myb oncoprotein; the second bears a faint resemblance to myb. The hypothesis that the DNA-binding domain of Reb1p is formed from these two conserved regions was confirmed by deletion of as many as 90 amino acids between them, with little effect on the DNA-binding ability of the resultant protein. We suggest that the DNA-binding domain of Reb1p is made up of two myb-like regions that, unlike myb itself, are separated by as many as 150 amino acids. Since Reb1p protects only 15 to 20 nucleotides in a chemical or enzymatic footprint assay, the protein must fold such that the two components of the binding site are adjacent.

Amino Acid Sequence↗

PubMed1987

DISTAN--a program which detects significant distances between short oligonucleotides.

We present an algorithm to detect distances between oligonucleotides in large collections of nucleic acids sequences. The ratios of actual frequencies of occurrence of short oligonucleotides at a given distance to the corresponding expected frequencies were analyzed in four categories of DNA sequences leukaryotic exons, bacterial genes, introns and non-Alu repeated DNAs). Three base periodic occurrences (independent of the reading frame) of all combinations of mononucleotides and repeats of all dinucleotides was characteristic for protein coding regions. This was also the case with the majority of trinucleotides (including translational stop signals) in these regions. Mirror-symmetric trinucleotides (except GCG and CGC) displayed a strong tendency to be two base periodically repeated in introns. Some two and three base periodic motifs were also observed in repeated DNAs. The possible biological implications of outstanding three base periodicities in bacterial genes and eukaryotic exons are discussed.

Algorithms↗

PubMed1987

Molecular cloning, characterization, and complete nucleotide sequence of the gene for pneumolysin, the sulfhydryl-activated toxin of Streptococcus pneumoniae.

A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.

Amino Acid Sequence↗

PubMed2002

A single Ala139-to-Glu substitution in the Renibacterium salmoninarum virulence-associated protein p57 results in antigenic variation and is associated with enhanced p57 binding to chinook salmon leukocytes.

The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2. The 5' and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1 and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala(139)-to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala139-to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57.

Actinomycetales↗

PubMed1990

Isolation, characterization and expression of the complementary DNA for human tumor necrosis factor (TNF-alpha).

A cDNA for human TNF-alpha (615bp) was isolated by means of polymerase chain reaction (PCR) using first strand cDNA from PMA-induced HL-60 cells as template. The result from sequencing the 615 bp cDNA fragment indicated that it corresponded to the entire sequence of mature human TNF coding region. Direct expression of mature human TNF was achieved using a plasmid pHT-1 constructed by ligation of the cDNA and a synthetic DNA. The IPTG-induced bacterial product (hTNF) showed cytotoxicity to mouse L-929 cells. The TNF activity was further identified by neutralization of a specific monoclonal antibody against human TNF-alpha. Approximately 80,000 units of activity were detected per ml of culture at A600 = 2.

Animals↗

PubMed1999

FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G + C content.

FramePlot is a web-based tool for predicting protein-coding regions in bacterial DNA with a high G + C content, such as Streptomyces. The graphical output provides for easy distinction of protein-coding regions from non-coding regions. The plot is a clickable map. Clicking on an ORF provides not only the nucleotide sequence but also its deduced amino acid sequence. These sequences can then be compared to the NCBI sequence database over the Internet. The program is freely available for academic purposes at http://www.nih.go.jp/jun/cgi-bin/frameplot.pl.

Amino Acid Sequence↗

PubMed1986

Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli.

We isolated a collection of mutants defective in the export of alkaline phosphatase to the periplasm. Two classes of mutants were obtained: one class with lesions unlinked to the phoA gene and a second class harboring linked mutations. Among the former class, one mutant is cold sensitive for growth and may be defective in a component of the Escherichia coli secretory apparatus. Included in the latter class are 47 mutants which are characterized in detail in this report. To facilitate DNA sequence analysis of these mutants, we devised a convenient method that relies on homologous recombination in vivo to transfer phoA mutations from the bacterial chromosome directly onto the genome of a single-stranded M13 phage vector. DNA sequence analysis revealed that our collection of mutants comprises six unique mutations, all of which reside in the phoA signal sequence coding region and lend further support to the notion that the length of the hydrophobic core of the signal sequence is crucial for its function in protein export. Kinetic studies showed that in these mutants, the small fraction of alkaline phosphatase which succeeds in reaching a periplasmic location, despite a defective signal sequence, is translocated across the membrane in a slow, posttranslational fashion.

Alkaline Phosphatase↗

PubMed2002

Simultaneous horizontal gene transfer of a gene coding for ribosomal protein l27 and operational genes in Arthrobacter sp.

Phylogenetic analysis of bacterial L27 ribosomal proteins showed that, against taxonomy, the L27 protein from the Actinobacteria Arthrobacter sp. clusters with protein sequences from the Bacillus group. The L27 gene clusters in the Arthrobacter sp. genome with six genes responsible for creatinine and sarcosine degradation. Phylogenetic analyses of orthologue proteins encoded by three of these genes also showed a phylogenetic relationship with Bacillus species. Comparisons between the synonymous codon usage of the Arthrobacter sp. genes and those from complete genomes showed that Arthrobacter genes encoding the L27 ribosomal protein and the proteins responsible for the degradation of creatinine and sarcosine have a codon usage that is more similar to that of Bacillus species than that of Arthrobacter. We suggest that the Arthrobacter sp. genes encoding the L27 ribosomal protein and the proteins responsible for the degradation of creatinine and sarcosine were acquired simultaneously through horizontal gene transfer from an unknown Bacillus species.

Amino Acid Sequence↗

PubMed1994

Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting.

Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mumol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mumol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP- and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.

Amino Acid Sequence↗