Sequence variation in the mitochondrial DNA control region of wild African cheetahs (Acinonyx jubatus).

Five hundred and twenty-five bp of mitochondrial control region were sequenced and analysed for 20 Acinonyx jubatus and one Felis catus. These sequences were compared with published sequences from another domestic cat, 20 ocelots (Leopardus pardalus) and 11 margays (Leopardus weidii). The intraspecific population divergence in cheetahs was found to be less than in the other cats. However variation was present and distinct groups of cheetahs were discernible. The 80 bp RS2 repetitive sequence motif previously described in other felids was found in four copies in cheetah. The repeat units probably have the ability to form secondary structure and may have some function in the regulation of control region replication. The two central repeat units in cheetah show homogenization that may have arisen by convergent evolution.

Interaction of the Pseudomonas cepacia DSM3959 lipase with its chaperone, LimA.

The lipA gene of Pseudomonas cepacia DSM3959 requires a downstream gene, limA, in oder to express lipase activity. The product of the lim gene, LimA, is a molecular chaperone required during the folding of lipase in oder for the lipase to adopt an active conformation. The lipase and LimA proteins have been shown to form a complex precipitable with either an anti-lipase or anti-LimA antibody. LimA has been shown to form a 1:1 complex with with prelipase and lipase isolated from "natural" P. cepacia system. The mature lipase (lacking its signal peptide) has been expressed in the presence and absence of LimA in Escherichia coli. LimA can activate mature lipase during a urea denaturation-renaturation experiment, indicating that the signal peptide is not required for the lipase to be activated by LimA. The effects of various reagents on the renaturation of lipase from 8 M urea have been examined. We propose a mechanism for the function of the LimA chaperone during the production of active extracellular lipase.

Chaperone-mediated activation in vivo of a Pseudomonas cepacia lipase.

An extracellular Pseudomonas cepacia lipase, LipA, is inactive when expressed in the absence of the product of the limA gene. Evidence has been presented that LimA is a molecular chaperone. The lipA and limA genes have been cloned in separate and independently inducible expression systems in Escherichia coli. These systems were used to test the molecular chaperone hypothesis by investigating whether LimA could activate presynthesized prelipase and whether presynthesized LimA could activate newly synthesized prelipase. The results show that LimA cannot activate presynthesized prelipase and that presynthesized LimA can activate only a limited number of de novo synthesized prelipase molecules. Co-immunoprecipitation of prelipase/lipase with LimA generated a 1:1 complex of prelipase/lipase and LimA. The results suggest that a 1:1 complex of LipA and LimA is required for prelipase processing and secretion of active lipase.

Overproduction, isolation, and DNA-binding characteristics of Xre, the repressor protein from the Bacillus subtilis defective prophage PBSX.

PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168. Lysogeny is maintained by the PBSX-encoded repressor, Xre. The Xre protein was overproduced in Escherichia coli and isolated by affinity chromatography. Gel retardation and DNase I footprinting studies indicated that Xre binds to four sites close to its own gene. These sites overlap putative promoters for xre and a divergent transcriptional unit, containing the middle genes.

Genetic control of bacterial suicide: regulation of the induction of PBSX in Bacillus subtilis.

PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168. Bacteria carrying the PBSX genome are induced by DNA-damaging agents to lyse and produce PBSX particles. The particles cannot propagate the PBSX genome. The particles produced by this suicidal response kill strains nonlysogenic for PBSX. A 5.2-kb region which controls the induction of PBSX has been sequenced. The genes identified include the previously identified repressor gene xre and a positive control factor gene, pcf. Pcf is similar to known sigma factors and acts at the late promoter PL, which has been located distal to pcf. The first two genes expressed from the late promoter show homology to genes encoding the subunits of phage terminases.

Molecular genetic analysis of the pullulanase B gene of Bacillus acidopullulyticus.

A fragment from Bacillus acidopullulyticus strain 294-16 encoding a pullulanase activity has been cloned into Bacillus subtilis. The nucleotide sequence of the 3972 base pairs (bp) fragment has been determined and shown to include only one complete open reading frame (ORF) of 863 codons. The deduced amino acid sequence of this ORF, denoted pulB, shows homology to a number of amylolytic enzymes. Primary and secondary structure analysis indicates that the central region of the protein forms the catalytic domain in a characteristic (beta/alpha)8 barrel. Three carboxylic acid residues essential for catalysis were identified. Regions within the catalytic domain proposed to be involved in substrate binding have been identified by homology.

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids.

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F1 hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F1 hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.

Activation of a bacterial lipase by its chaperone.

The gene lipA of Pseudomonas cepacia DSM 3959 encodes a prelipase from which a signal peptide is cleaved during secretion, producing a mature extracellular lipase. Expression of lipase in several heterologous hosts depends on the presence of another gene, limA, in cis or in trans. Lipase protein has been overproduced in Escherichia coli in the presence and absence of the lipase modulator gene limA. Therefore, limA is not required for the transcription of lipA or for the translation of the lipA mRNA. However, no lipase activity is observed in the absence of limA. limA has been overexpressed and encodes a 33-kDa protein, Lim. If lipase protein is denatured in 8 M urea and the urea is removed by dialysis, lipase activity is quantitatively recovered provided Lim protein is present during renaturation. Lip and Lim proteins form a complex precipitable either by an anti-lipase or anti-Lim antibody. The Lim protein has therefore the properties of a chaperone.

Optimization of the synthesis of porcine somatotropin in Escherichia coli.

We report on the influence of choice of promoter and RNA polymerase, 5'-untranslated regions and ribosome binding sites, codon usage, leader peptide coding sequences and poly A tail in the 3'-untranslated region on the synthesis of porcine somatotropin (PST) in Escherichia coli. A total of 12 different constructs were tested in this study for the production of porcine somatotropin (PST) in E. coli. Several factors have significant effects on PST synthesis. In the presence of a strong promoter and a strong ribosome binding site, the next most important factor seems to be the combination of sequences at the 5'-end of the mRNA including both the 5'-untranslated region and the start of the coding sequence. Codon usage in the 5'-coding sequence per se is not important in determining the level of PST synthesis where high level expression is achieved from a strong ribosome binding site. However, where low level synthesis of recombinant PST (rPST) is achieved, codon usage in the 5'-coding sequence is important in determining the level of PST synthesis. Leader sequences dramatically reduce the level of PST synthesis. The presence of a poly A tail in the 3'-untranslated region has no significant effect on PST synthesis.

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm.

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.

Molecular analysis of an IS200 insertion in the gpt gene of Salmonella typhimurium LT2.

A strain of Salmonella typimurium LT2 has been isolated which carries an insertion of approximately 700 bp in the gpt gene. The insertion in the gpt gene was shown to be the Salmonella-specific element IS200. The mutation in strain CR1 arose without selection during storage and is only the second phenotypically identified mutation caused by the insertion of IS200.

Characterisation of a repressor gene (xre) and a temperature-sensitive allele from the Bacillus subtilis prophage, PBSX.

The defective prophage of Bacillus subtilis 168, PBSX, is a chromosomally based element which encodes a non-infectious phage-like particle with bactericidal activity. PBSX is induced by agents which elicit the SOS response. In a PBSX thermoinducible strain which carries the xhi1479 mutation, PBSX is induced by raising the growth temperature from 37 degrees C to 48 degrees C. A 1.2-kb fragment has been cloned which complements the xhi1479 mutation. The nucleotide sequence of this fragment contains an open reading frame (ORF) which encodes a protein of 113 amino acids (aa). This aa sequence resembles that of other bacteriophage repressors and suggests that the N-terminal region forms a helix-turn-helix motif, typical of the DNA-binding domain of many bacterial regulatory proteins. The ORF is preceded by four 15-bp direct repeats, each of which contains an internal palindromic sequence, and by sequences resembling a SigA-dependent promoter. The nt sequence of an equivalent fragment from the PBSX thermoinducible strain has also been determined. There are three aa differences within the ORF compared to the wild type, one of which lies within the helix-turn-helix segment. This ORF encodes a repressor protein of PBSX.

TUF factor binds to the upstream region of the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae.

The upstream activation site of the pyruvate decarboxylase gene, PDC1, of Saccharomyces cerevisiae contains an RPG box, and mediates the increase in expression of a PDC1-lacZ fusion gene during growth on glucose. Oligonucleotide replacement experiments indicate that the RPG box functions as an absolute activator of expression, but other elements (possibly CTTCC repeats) are required for carbon source regulation, and maximal expression. Gel retardation and oligonucleotide competition experiments suggest that the DNA binding factor TUF interacts with the RPG box in the upstream region of PDC1. Binding of TUF factor is not carbon source dependent in in vitro experiments, and is probably not responsible for glucose induction of PDC1 expression.

The effect of phage T7 lysozyme on the production of biologically active porcine somatotropin in Escherichia coli from a gene transcribed by T7 RNA polymerase.

We have analyzed the inducible synthesis of recombinant porcine somatotropin (rPST) from the phage T7 gene 10 promotor on the vector pET3a [Rosenberg et al., Gene 56 (1987) 125-135]. Low-level synthesis of phage T7 lysozyme is crucial for high-level synthesis (40%) of rPST, which is greatly reduced if T7 lysozyme synthesis is absent or too high. The synthesis of rPST mRNA is optimized in those constructs coding for low levels of T7 lysozyme, with a reduction in mRNA levels in constructs coding for higher levels of T7 lysozyme or no lysozyme. The rPST can be readily purified following a single chromatographic step and is biologically active as determined by the tibia test following administration to hypophysectomized rats.

Characterization of PBSX, a defective prophage of Bacillus subtilis.

PBSX, a defective Bacillus subtilis prophage, maps to the metA-metC region of the chromosome. DNA (33 kilobases) from this region of the chromosome was cloned and analyzed by insertional mutagenesis with the integrating plasmid pWD3. This plasmid had a promoterless alpha-amylase gene (amyL) that provided information on the direction and level of transcription at the site of integration. Transcription under the control of the PBSX repressor proceeded in the direction metA to metC over a distance of at least 18 kilobases. Electrophoretic analysis of proteins produced by different integrant strains upon PBSX induction and by fragments subcloned in Escherichia coli allowed the identification of early and late regions of the prophage. A set of contiguous fragments directing mutagenic integration suggested that the minimum size of an operon that encodes phage structural proteins is 19 kilobases. The adaptation of PBSX transcriptional and replicational functions to a chromosomally based, thermoinducible expression system is discussed.

Autosomal dominant retinitis pigmentosa: exclusion of a gene from extensive regions of chromosomes 6, 13, 20, and 21.

Members of a large pedigree of Irish origin presenting with early onset Type I autosomal dominant retinitis pigmentosa (ADRP) have been typed for polymorphic DNA markers from chromosomes 6, 13, 20, and 21. For each marker close linkage to ADRP has been excluded by pairwise analyses. Using distances fixed from well-established genetic maps of these chromosomes and multipoint analyses with two or three contiguous markers, exclusion of ADRP was extended to the areas between markers, resulting in the exclusion of ADRP from extensive regions of each chromosome, totaling approximately 500 cM or 15% of the genome. The study indicates the large quantity of linkage/exclusion data obtainable using well-spaced highly polymorphic markers.