The sequence of the 6S RNA gene of Pseudomonas aeruginosa.

From the gram-negative eubacterium Pseudomonas aeruginosa we have isolated a stable 6S RNA, approximately 180 nucleotides in length. The RNA was partially sequenced and identified by comparison with the known Escherichia coli 6S RNA sequence. Southern hybridizations revealed a single copy gene coding for the 6S RNA. DNA from other prokaryotes, i.e. E. coli, Thermus thermophilus, Bacillus subtilis, Bacillus stearothermophilus and Halobacterium maris mortui, did not give detectable hybridization signals. The 6S RNA gene was cloned in E. coli and its complete primary structure was determined. Although the 6S RNA sequences from P. aeruginosa and E. coli share only a 60.4% homology, we are able to propose a common secondary structural model.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.     

Molecular cloning of pertussis toxin genes.

We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.     

Nucleotide sequence and stability of the RNA component of RNase P from a temperature-sensitive mutant of E. coli.

The gene coding for the RNA component of RNase P was cloned from a temperature-sensitive mutant of Escherichia coli defective in RNase P activity (ts709) and its parental wild-type strain (4273), and the complete nucleotide sequences of the gene and its flanking regions were determined. The 5'- and 3'-terminal sequences of the RNA component were determined and mapped on the DNA sequence. The mutant gene has GC-to-AT substitutions at positions corresponding to 89 and 365 nucleotides downstream from the 5' terminus of the RNA sequence. Comparing to the wild-type RNA, the mutant RNA is less stable and rapidly degraded in vivo and in vitro.     

Molecular cloning and nucleotide sequence of the gene for the ribosomal protein S11 from the archaebacterium Halobacterium marismortui

The gene encoding ribosomal protein S11 (Escherichia coli S15 homologue) from Halobacterium marismortui was cloned employing two synthetic oligonucleotide mixtures, 23 and 32 bases in length, as hybridization probes. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (1300 base pairs) were then determined by the dideoxy chain termination method. Comparison of the nucleotide sequence of the H. marismortui S11 gene with that of the E. coli S15 gene (rpsO) showed that the 3'-end of the S11 gene can be aligned with the entire E. coli S15 gene, sharing 44% identical nucleotides. It has been found that the S11 gene has a higher G + C content (G + C = 65%) than that of the E. coli S15 gene (G + C = 53%). This increase in G + C content specifically shows up as a preference for G + C in the 3rd position of the codon. Upstream of the S11 gene, an archaebacterial promoter sequence (GGACTTTCA) and a putative ribosomal binding site (GCGGT) have been found, 88 and 15 (or 24) base pairs from the initiation codon of the gene. In addition, an open reading frame could be identified immediately after the stop codon for the S11 gene. Northern blotting analysis using the S11 coding region as probe has shown that the S11 gene is located on a 2.4-kilobase mRNA, suggesting that it is cotranscribed with other downstream gene(s).     

Genetic selection and DNA sequences of 4.5S RNA homologs.

A general strategy for cloning the functional homologs of an Escherichia coli gene was used to clone homologs of 4.5S RNA from other bacteria. The genes encoding these homologs were selected by their ability to complement a deletion of the gene for 4.5S RNA. DNA sequences of the regions encoding the homologs were determined. Since this approach does not require that the homologous genes hybridize with probes generated from the E. coli sequence, the sequences of the homologs were not all similar to the sequence of the E. coli gene. Despite the dissimilarity of the primary sequences of some of the homologs, all could be folded to obtain a similar structure.     

Complete DNA sequence coding for the large ribosomal RNA of yeast mitochondria.

The mitochondrial gene coding for the large ribosomal RNA (21S) has been isolated from a rho- clone of Saccharomyces cerevisiae. A DNA segment of about 5500 base pairs has been sequenced which included the totality of the sequence coding for the mature ribosomal RNA and the intron. The mature RNA sequence corresponds to a length of 3273 nucleotides. Despite the very low guanine-cytosine content (20.5%), many stretches of sequence are homologous to the corresponding Escherichia coli 23S ribosomal RNA. The sequence can be folded into a secondary structure according to the general models for prokaryotic and eukaryotic large ribosomal RNAs. Like the E.coli gene, the mitochondrial gene contains the sequences that look like the eukaryotic 5.8S and the chloroplastic 4.5S ribosomal RNAs. The 5' and 3' end regions show a complementarity over fourteen nucleotides.     

High level expression of porcine growth hormone in Escherichia coli from an expression vector containing bacteriophage lambda PL and N gene untranslated region

An Escherichia coli expression vector, pG408N containing a PL promoter and the upstream untranslated region of the N gene of bacteriophage lambda has been constructed. We have designed a PvuII site immediately behind the untranslated region. A DNA fragment starting with an initiation codon ATG could be inserted into this site for expression. This vector also contains 7 additional cloning sites downstream from the PvuII site. A gene could be cloned into one of these sites and the 5' sequence of this gene could be modified with synthetic oligonucleotides and ligated to the PvuII for the purpose of increasing gene expression. We have also cloned the lambda cl gene into a p15A plasmid. Cotransformation of this plasmid with the expression vector allows the cloning vector pG408N to be used in any E. coli strain. Using this system, we were able to express porcine growth hormone to approximately 35% of total proteins in E. coli DH5 alpha.     

Molecular cloning and sequencing of the beta-isopropylmalate dehydrogenase gene from the cyanobacterium Spirulina platensis.

The gene for beta-isopropylmalate dehydrogenase (EC 1.1.1.85) of Spirulina platensis (leuB) was cloned from a lambda EMBL3 genomic library by heterologous hybridization using the Nostoc UCD 7801 leuB gene as a probe. The sequence of the entire leuB coding region was determined as well as 645 bp of 5' flanking region and 956 bp of 3' flanking region. DNA sequencing revealed an open reading frame of 1065 nucleotides capable of encoding a polypeptide of 355 amino acids. Homologies between the amino acid sequence deduced from the nucleotide sequence of the S. platensis leuB gene and the amino acid sequences published for corresponding proteins either from bacteria or yeasts are 45% or more. Northern hybridization analysis indicated that the S. platensis leuB gene is transcribed as a single monocistronic RNA of approximately 1200 bases.     

Total DNA synthesis and cloning in Escherichia coli of a gene coding for the human growth hormone releasing factor

A DNA containing a sequence coding for the human growth hormone releasing factor (hGRF) has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 140 base-pair fragment containing initiation and termination signals for translation and appropriate protruding ends for cloning into a newly constructed plasmid vector (pULB1219). Eleven oligodeoxyribonucleotides, from 14 to 31 bases in length, sharing pairwise stretches of complementary regions of at least 13 bases were prepared by phosphotriester solid-phase synthesis. The DNA sequence was designed to take into account the optimal use of E. coli codons. Oligomers were annealed in one step and assembled by ligation. The DNA fragment of the expected size (140 bp) was recovered and cloned into the pULB1219 vector. The expected sequence was confirmed by DNA sequencing.     

Organization and nucleotide sequences of the genes encoding the biotin carboxyl carrier protein and biotin carboxylase protein of Pseudomonas aeruginosa acetyl coenzyme A carboxylase.

The genetic organization of the Pseudomonas aeruginosa acetyl coenzyme A carboxylase (ACC) was investigated by cloning and characterizing a P. aeruginosa DNA fragment that complements an Escherichia coli strain with a conditional lethal mutation affecting the biotin carboxyl carrier protein (BCCP) subunit of ACC. DNA sequencing and RNA blot hybridization studies indicated that the P. aeruginosa accB (fabE) homolog, which encodes BCCP, is part of a 2-gene operon that includes accC (fabG), the structural gene for the biotin carboxylase subunit of ACC. P. aeruginosa homologs of the E. coli accA and accD, encoding the alpha and beta subunits of the ACC carboxyltransferase, were identified by hybridization of P. aeruginosa genomic DNA with the E. coli accA and accD. Data are presented which suggest that P. aeruginosa accA and accD homologs are not located either immediately upstream or downstream of the P. aeruginosa accBC operon. In contrast to E. coli, where BCCP is the only biotinylated protein, P. aeruginosa was found to contain at least three biotinylated proteins.