Primary structure of cold-adapted alkaline phosphatase from a Vibrio sp. as deduced from the nucleotide gene sequence.

Alkaline phosphatases (AP) are widely distributed in nature, and generally have a dimeric structure. However, there are indications that either monomeric or multimeric bacterial forms may exist. This paper describes the gene sequence of a psychrophilic marine Vibrio AP, previously shown to be particularly heat labile. The kinetic properties were also indicative of cold adaptation. The amino acid sequence of the Vibrio G15-21 AP reveals that the residues involved in the catalytic mechanism, including those ligating the metal ions, have precedence in other characterized APs. Compared with Escherichia coli AP, the two zinc binding sites are identical, whereas the metal binding site, normally occupied by magnesium, is not. Asp-153 and Lys-328 of E. coli AP are His-153 and Trp-328 in Vibrio AP. Two additional stretches of amino acids not present in E. coli AP are found inserted close to the active site of the Vibrio AP. The smaller insert could be accommodated within a dimeric structure, assuming a tertiary structure similar to E. coli AP. In contrast the longer insert would most likely protrude into the interface area, thus preventing dimer formation. This is the first primary structure of a putative monomeric AP, with indications as to the basis for a monomeric existence. Proximity of the large insert loop to the active site may indicate a surrogate role for the second monomer, and may also shape the catalytic as well as stability characteristics of this enzyme.

DNA sequences of yeast H3 and H4 histone genes from two non-allelic gene sets encode identical H3 and H4 proteins.

The complete DNA sequences of two loci encoding H3 and H4 histones in Saccharomyces cerevisiae have been determined. Each locus contains one H3 and one H4 gene. The genes at each locus are divergently transcribed and the coding sequences are separated by 646 base-pairs at one locus and 676 base-pairs at the other. The H3 genes code for identical histone H3 proteins and the H4 genes code for identical histone H4 proteins. The yeast proteins differ from histones H3 and H4 of calf by 15 and 8 amino acid substitutions, respectively, and these differences are largely confined to the carboxy-terminal halves of the proteins. The genes demonstrate a bias in synonymous codon usage similar to that noted for other yeast genes. This bias is confined to the coding sequences of the genes and is specific for the reading frame encoding the proteins. The coding sequence of each gene is flanked on both sides by DNA with an A + T content of 70 to 80%. Possible regulatory sequences are located relative to the 5' and 3'-termini of the histone H3 and H4 RNA transcripts.

Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases.

In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm). We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig. The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79,487 Da. Comparison with published sequences of bacterial Lig revealed significant homology between the NAD(+)-utilizing Lig, and alignment of their aa sequences revealed several blocks of conserved residues. Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures. Under our assay conditions the Rm Lig was active at 5-75 degrees C with apparent optimal activity above 55 degrees C. The Ts enzyme showed activity at 15-75 degrees C with optimal activity above 65 degrees C. The half-life of the Lig at 91 degrees C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig.

Immune response to recombinant visna virus Gag and Env precursor proteins synthesized in insect cells.

Two different recombinant visna virus (VV) gag-baculoviruses were constructed for the expression of precursor VV Gag in insect cells. Both recombinant Gag viruses expressed proteins migrating on SDS PAGE at the predicted rate for VV Gag precursor, Pr50gag. However, differences were seen in the morphology of the virus-like particles produced. Monoclonal antibody directed against the VV Gag capsid protein (p25) and sera from sheep infected with ovine lentiviruses reacted to both 50-kDa proteins. A recombinant VV env-baculovirus was constructed, substituting sequences encoding the signal peptide of VV Env with the murine IFN-gamma analogue. Sera from ovine lentivirus infected sheep reacted in immunoblots with two proteins of approximately 100 and 200 kDa found in the plasma membrane of insect cells infected with env-recombinant virus. Sheep immunized with either the recombinant Gag or the Env proteins developed high antibody titers to VV in ELISA. The serum of sheep and ascitic fluid of mice immunized with the recombinant Gag reacted with native Pr50gag and the processed Gag proteins in immunoblots, whereas serum of the recombinant Env immunized sheep reacted with VV gp135 and a putative oligomer of gp135. The immunized sheep responded specifically to visna virus by lymphocyte proliferation in vitro.

Expression and purification of Clostridium perfringens beta-toxin glutathione S-transferase fusion protein.

The beta-toxin gene from Clostridium perfringens type C was cloned and expressed as a glutathione S-transferase fusion protein in Escherichia coli. The DNA sequence was determined and compared to the type B sequence. Two nucleotide differences were found in the protein coding sequence, resulting in one amino acid difference between the two proteins. The purified beta-toxin fusion protein is not toxic in mice, but rabbit antiserum raised against it neutralises the toxic effect of C. perfringens type C culture filtrate in mice.

Site-directed mutagenesis of Clostridium perfringens beta-toxin: expression of wild-type and mutant toxins in Bacillus subtilis.

Recombinant beta-toxin has been expressed and secreted from Bacillus subtilis. Biological activity was tested in vivo and in vitro. The lethal dose in mice was determined. Hemolysis of rabbit and sheep erythrocytes was tested but no effect was observed. Seven mutant proteins were produced. Targets for mutagenesis were mostly selected on the basis of the similarity between beta-toxin and alpha-toxin from Staphylococcus aureus, a pore-forming toxin. Mutations of two amino acids affected the lethal dose in mice. Both residues have counterparts in the membrane binding region of alpha-toxin. Alteration of the single cysteine residue did not affect protein function, contrary to previous suggestions.

Human and ovine lentiviral infections compared.

Maedi-visna virus (MVV) of sheep was the first lentivirus to be isolated. The genomic organization of MVV is very similar to that of human immunodeficiency virus (HIV) with several genes regulating the expression of the viral genome. Viral replication is severely restricted in the host and some cells apparently contain the genetic information in a DNA provirus form with little or no expression of viral antigens. This seems to be a major factor in causing the "slowness" of lentiviral infections and the persistence of the virus in the host since the immune system may not recognize the provirus-containing cells. The target cells for HIV and MVV are similar although T4 lymphocytes are not specifically destroyed in maedi-visna. There are also certain similarities in the pathological changes in both diseases, both in the central nervous system, the lungs and the lymphatic system. Although the severe final immunodeficiency state characteristic of AIDS has not been observed in maedi-visna, the basic biological features of the MVV and its interaction with host cells are so similar to HIV infection, that we consider ovine maedi-visna useful animal model for the human lentivirus infections.

Isolation and characterization of lambda transducing phages for the E. coli genes ksgA and pdxA.

Lambda phages carrying the Escherichia coli genes ksgA and pdxA were isolated from secondary site lysogens in araB. 1) The phage genomes were characterized by genetic complementation tests, restriction endonuclease digestion and electron microscopy. 2) A 6.3 kilobasepair (kb) EcoRI restriction fragment carrying both ksgA and pdxA was cloned in a lambda vector; this fragment has proven useful in further characterization of the ksgA gene (Andrésson and Davies, 1980a, b). The ksgA and pdxA genes are about 14 and 12-13 kb, respectively, counterclockwise of the arabinose operon and 1.5 and 2.5-3.5 kb clockwise of folA.

Genetic organization and restriction enzyme cleavage map of the ksgA-pdxA region of the Escherichia coli chromosome.

Small multicopy plasmids carrying the Escherichia coli genes ksgA and pdxA were constructed by ligation in vitro of an EcoRI restriction fragment from lambda ksg10 (Andrésson and Davies, 1980a) into the EcoRI sites of the ColE1 plasmids RSF2124 and pVH51. Cleavage maps of the plasmids were determined for 21 different restriction enzymes. The ksgA gene was located in a 750 basepairs (bp) region 1,450 bp clockwise of the EcoRI site in folA: pdxA is in a 2,040 bp region immediately clockwise of ksgA.

Some properties of the ribosomal RNA methyltransferase encoded by ksgA and the polarity of ksgA transcription.

The assay for the ksgA-encoded S-adenosylmethionine--6-N',N'-adenosyl (rRNA) dimethyltransferase has been improved; the gel-filtration molecular weight of partially purified enzyme under two different sets of conditions was found to be 55,000 or 26,000 daltons. We have determined methyltransferase activities in strains where ksgA was brought under the control of the mitomycin C-inducible promoter of the colicin E1 gene. Our studies show that ksgA is transcribed counterclockwise on the Escherichia coli chromosome.

The sequence of the single 16S rRNA gene of the thermophilic eubacterium Rhodothermus marinus reveals a distant relationship to the group containing Flexibacter, Bacteroides, and Cytophaga species.

Rhodothermus marinus, a gram-negative heterotrophic marine thermophile, has been the subject of several recent studies. Isolation, sequencing, and analyses of a 16S rRNA gene have shown that R. marinus diverges sharply from major bacterial phyla and is most closely allied to the Flexibacter-Cytophaga-Bacteroides group. Further analyses revealed that the R. marinus chromosome contains a single rRNA operon with a 16S-23S intergenic region coding for tRNA(Ile) and tRNA(Ala).

Clostridium perfringens beta-toxin forms multimeric transmembrane pores in human endothelial cells.

Beta-toxin is one of the lethal toxins of Clostridium perfringens. It shares sequence homology with the pore-forming alpha-toxin of Staphylococcus aureus and structural homology has been indicated by mutagenesis studies. Human endothelial cells are sensitive to the toxic effect of alpha-toxin and in order to investigate the function of beta-toxin we have looked at the effect of the protein on human umbilical vein endothelial cells. We show that like alpha-toxin beta-toxin induces release of arachidonic acid in a dose dependent manner. In addition we show that both toxins cause leakage of inositol from the cells, consistent with the formation of transmembrane pores. The effect of toxin mutants on endothelial cells correlates with the lethal dose of each mutant in mice. Furthermore, we demonstrate the formation of heat stable toxin multimers in the cell membrane. Multimer formation was not observed on other cell types tested. We conclude that beta-toxin is a cell specific pore-forming toxin, structurally and functionally related to alpha-toxin of Staphylococcus aureus.

Deletions of ribosomal protein genes in Escherichia coli merodiploids heterozygous for resistance to streptomycin and spectinomycin.

In a merodiploid strain of Escherichia coli heterozygous for the ribosomal protein genes spc and str, deletions were observed preventing the expression of either gene but permitting the expression of the other. This suggests that the spc and str genes are in separate transcriptional units.

Cloning and heterologous expression of Solorina crocea pyrG.

A pyrG gene, encoding orotidine 5'-monophosphate decarboxylase, was cloned from a phage library derived from the lichen Solorina crocea. Phylogenetic analysis and a survey of geographically well-separated specimens were used to verify that the gene represented the fungal component of the lichen. Both coding and upstream sequences of S. crocea pyrG exhibited features typical of fungal genes. A 132-bp intron interrupting the coding region between nucleotides 157 and 288 was confirmed by RT-PCR and sequencing. Transformation of Aspergillus nidulans with S. crocea pyrG, controlled by either its native promoter or the A. nidulans trpC promoter, resulted in uridine-independent strains that exhibited appreciable growth only at 24 degrees C. Southern analysis indicated multiple integrations of S. crocea pyrG. These results demonstrate that heterologous expression may be used to investigate genes from lichens.

Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene.

We describe the cloning and the DNA sequence of an amber suppressor allele of the Escherichia coli leuX (supP) gene. The suppressor allele codes for a tRNA with anticodon CUA, presumably derived by a single base change from a CAA anticodon. The mature coding sequence of the leuX gene is preceded by a putative Pribnow box sequence (TATAAT) and followed by a termination signal. The sequence of the leuX-coded tRNA is compared with the sequences of the four remaining tRNALeu isoacceptors of E. coli and with two tRNALeu species from bacteriophage T4 and T5. The conserved nucleotides in these seven tRNAs recognized by E. coli leucyl-tRNA synthetase are located mainly in the aminoacyl stem and in the D-stem/loop region.

Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcription-PCR of 16S rRNA sequences.

An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue.

supN ochre suppressor gene in Escherichia coli codes for tRNALys.

We describe the cloning and nucleotide sequence of a new tRNALys gene, lysV, in Escherichia coli. An ochre suppressor allele of this gene, supN, codes for a tRNALys with anticodon UUA, presumably derived by a single base change from a wild-type UUU anticodon. The sequence of the supN tRNALys is identical to the sequence of ochre suppressor tRNAs encoded by mutant alleles at the lysT locus. This locus, which contains the two previously known tRNALys genes of E. coli, is located far from the lysV locus on the chromosome.