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Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However,...
The genomic and complementary DNA sequences were determined for the unique actin-encoding gene inChlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5 -untranslated region and eight introns in the coding...
A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site...
In Escherichia coli, the biosynthesis of the electron carrier menaquinone (vitamin K 2 ) involves at least seven identified enzymatic activities, five of which are encoded in the men cluster. One of these, the conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid, requires the formation of o-succinylbenzoyl-CoA (OSB-CoA) as an intermediate. Formation of the intermediate...
The PHO81 gene product functions as an inhibitor of the cyclin-cyclin-dependent kinase pair, Pho80-Pho85, and is required for derepression of acid phosphatase-encoding gene (PHO5) expression. PHO81 is the only known regulator of this system whose transcriptional expression is regulated by the level of inorganic phosphate. This effect is mediated by the gene products of the PHO4 and PHO2 (BAS2, GRF10)...
The production of phenazine (Ph) antibiotics in Pseudomonas aureofaciens (Pau) 30-84 is positively regulated by PhzR, a protein belonging to the LuxR family of transcriptional activators. We have now identified phzI, a second gene required for Ph production. The product of phzI is a member of the LuxI family of N-acyl-homoserine lactone ( (N-acyl-HSL) synthases. Inactivation of phzI results in the...
An 18.5-kb DNA fragment was cloned from Sphingomonas yanoikuyae (Sy) B1 (previously Beijerinckia B1). Analysis of a 4.3-kb sequence revealed an isocitrate dehydrogenase (Idh)-encoding gene (idhA), an unidentified open reading frame (ORF) and a partial glucosamine synthetase-encoding ORF (glmS). As in a number of bacteria, Tn7 insertion was found specifically at a site past the stop codon of glmS...
The display of antibody (Ab) fragments (Fab) on the surface of filamentous bacteriophage (phage) and selection of phage that interact with a particular antigen (Ag) has enabled the isolation of Fab that bind nucleic acids. Nucleic acid (NA) binding Ab occur in vivo in connective tissue disease patients and certain inbred strains of mice and are thought to be pathogenic. Although there is ample data...
The complete sequence of the Fe 2+ -containing superoxide dismutase (Fe-SOD)-encoding gene of Chlamydomonas reinhardtii was determined from genomic DNA by use of the direct (PCR) and inverse PCR (IPCR). It was confirmed by reverse transcription-PCR. Primers employed in the PCR represented oligodeoxyribonucleotides corresponding to conserved amino acid (aa) residues of Fe-SOD. From...
A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec). Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptides of 89 and 88 amino acids (aa) (10 044 and 9764 Da, respectively). Ec cells harboring...
Major alterations of fungal gene expression are induced by the development of ectomycorrhiza, a symbiosis between tree roots and filamentous fungi. Several cDNAs corresponding to highly expressed transcripts of the BasidiomycetePisolithus tinctorius (Pt) were isolated from symbiotic tissues. Two of these abundant transcripts (hydPt-1 andhydPt-2 ) encoded polypeptides belonging to the hydrophobin...
A tightly regulated gene-expression system was developed using SP6 RNA polymerase (RpoSP6). The RpoSP6-encoding gene (rpoSP6) was inserted into a mini-F plasmid (mini-F) and expression was controlled by the lactose promoter (P lac ) and operator (O lac ) on the plasmid. Therefore, a controlled expression system for the target genes can easily be constructed in...
A cDNA clone encoding grapevine (Vitis vinifera L. cv Sultanina) NAD(H)-glutamate dehydrogenase (GDH) was isolated from a cDNA expression library by immunoscreening with a polyclonal antibody raised against grapevine GDH. Nucleotide sequence analysis revealed an open reading frame (ORF) encoding a precursor protein of 411 amino acids (aa) with a calculated molecular mass of 44.517 kDa. The deduced...
We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169° on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol...
Two motility genes (fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced. FliH and FliI showed extensive homology to the proteins involved in the export of flagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway...
The gene-sized DNA molecule in the macronucleus (mac) of Oxytricha nova (On) encoding heat-shock protein 70 (Hsp70) was cloned and sequenced. It contains 2654 bp, including telomeres. It consists of a 394-bp A + T-rich 5 leader, a 1956-bp open reading frame (ORF) encoding a putative polypeptide of 651 amino acids (aa), and a 240-bp A + T-rich 3 trailer. The 5 leader contains two copies of...
A type-II topoisomerase (Topo-IV) encoded by the parC and parE genes in Escherichia coli and Salmonella typhimurium is thought to be involved in cell septation and in the decatenation of newly replicated chromosomes. We have identifiedparC and parE homologs in the pleomorphic, wall-less organism Mycoplasma genitalium. Since the mechanics of cell septation in conventional eubacterial species is believed...
Degenerate oligodeoxyribonucleotide primers, designed on the basis of conserved regions of the chitin synthase gene family, were used to amplify a fragment of the Ampelomyces quisqualis (Aq) chsA gene. Subsequently, the PCR product was used as a probe in order to identify and isolate genomic clones harboring the entire chsA gene. Aq chsA is 2786-nt long, has one intron and encodes a 910-amino-acid...
Genes (EFA) encoding the translation elongation factor EF-1α (EFA) or the prokaryotic homolog EFTu frequently occur in multiple copies in the same organism. This has been intepreted either in terms of a potential of differential gene expression during different phases of development, or as gene dosage adaptation to the need of high-level production of the gene products. Since ciliates can differentially...
Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor fromBorrelia burfdorferi strain B31 was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa). Comparison with Escherichia coli σ 70 and Bacillus subtilis σ 43 showed a high degree of similarity...
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