Inhibitors of kallikrein in human plasma.

Human plasma was fractionated by ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration to determine which method would give the greatest number of clearly separable kallikrein inhibitory peaks. With G-200 gel filtration three peaks could be separated which were demonstrated to contain alpha(2)-macroglobulin, C1 inactivator, and alpha(1)-antitrypsin. No other kallikrein inhibitors could be identified. The fractions containing C1 inactivator and alpha(2)-macroglobulin appeared to be more effective against kallikrein than that containing alpha(1)-antitrypsin. A patient with hereditary angioneurotic edema was shown to have an abnormal C1 inactivator protein capable of interfering with kallikrein's biologic, but not its esterolytic activity. Heat-treated human plasma, a commonly used source of kininogen for experiments with kallikrein, was shown to have kallikrein inhibitory activity.

Verification of a new model of the time course of RNA synthesis. Measurement of the rates of initiation and elongation.

The time course of RNA synthesis in vitro commonly starts with a lag followed by a linear phase. Differing from the earlier interpretation we have previously proposed that, under conditions where the initiation rate is low, the lag represents the time taken for the first RNA polymerase molecule to reach a termination site. During the linear phase, initiation is balanced by termination (Mahon, G.A.T., McWilliam, P., Gordon, R.L. and McConnell, D.J. (1980) J. Theor. Biol. 87, 483-515). We report the use of rifampicin as a further test of this new model. We show that it does apply under conditions of high ionic strength (0.3 M KCl), and under these conditions time courses may be analyzed to yield unbiased estimates of the initiation (Vi) and chain elongation (Vp) rates. We illustrate the application of the method of time course analysis and confirm some of its features by examining the effect of variation in the concentrations of RNA polymerase and nucleoside triphosphate on the estimates of Vi and Vp. The alternative interpretation of the time course applies under conditions of low ionic strength, where the initiation rate is high. (Chamberlin, M.J., Nierman, W.C., Wiggs, J. and Neff, N. (1979) J. Biol. Chem. 254, 10061-10069.) The advantages of each model in measuring Vi and Vp (the major parameters of the transcription reaction) are discussed.

Molecular cloning and expression of a Bacillus subtilis beta-glucanase gene in Escherichia coli.

A Bacillus subtilis gene coding for an endo-beta-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage lambda and plasmid vectors. The gene is contained within a 1.6-kb EcoRI-PvuI DNA fragment and directs the synthesis in E. coli of a beta-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect beta-glucanase activity in colonies on agar plates.

Molecular cloning of multiple bovine aspartyl protease genes.

Eight bovine genomic clones have been identified as members of the aspartyl protease gene family. The clones were prepared in phage vector lambda EMBL3 from Sau3AI partial digests of DNA from a single animal. Restriction maps show that seven of these clones are related and comprise at least five non-overlapping sequences. Allowing for allelic variation these probably represent three or more different genes. The nucleotide sequences show open reading frames (ORFs) corresponding closely to exons 6, 7 and 8 of human and porcine pepsin A. Comparison with other aspartyl proteases shows that these are multiple bovine pepsin A genes. The seven clones would encode at least two different but closely related forms of pepsin A. The 5' splice site at the end of exon 7 in all seven clones is the unusual sequence GC. The eighth clone contains an ORF homologous to exon 2 of the mammalian aspartyl proteases. The corresponding amino acid sequence is more closely related to bovine chymosin than to any of the other known sequences; it may be functionally homologous to chymosin but could be a novel mammalian aspartyl protease. The intron/exon boundaries seen in both this clone and in the bovine pepsin A clones are at the same positions as found in human pepsin A, bovine chymosin and human and mouse renins, further evidence that the general structure of mammalian aspartyl protease genes has been strongly conserved.

Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase.

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.

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.

Multigene families of Cellulomonas flavigena encoding endo-beta-1,4-glucanases (CM-cellulases).

Multiple genes coding for endo-beta-1,4-glucanases (CM-cellulases) have been isolated from a newly discovered highly cellulolytic strain of Cellulomonas flavigena. Clones of C. flavigena DNA were isolated in Escherichia coli and screened for gene expression on CM-cellulose plates staining with congo red. Six clones produced CM-cellulase activity as detected in liquid assays, and on activity gels. They fell into three groups within which the sequences cross-hybridised. There were small differences in the pH and temperature optima of the enzymes encoded by representatives of the three groups of clones.

Cloning and characterization of the gene for a methanol-utilising alcohol dehydrogenase from Bacillus stearothermophilus.

The cloning and characterization of the alcohol dehydrogenase (ADH) gene (adh) from Bacillus stearothermophilus strain DSM2334, an obligate aerobe, are described. The clone directed the synthesis of ADH as judged on Western blots, activity gels and tetrazolium plates. It specified an enzyme that oxidised methanol as well as ethanol. The enzyme was found to be encoded by a single gene in B. stearothermophilus which did not cross-hybridize to adh clones from Escherichia coli, yeast or maize. The cloned gene was expressed in E. coli but activity was not detected in Bacillus subtilis, despite stable maintenance of the recombinant plasmid in this host. The gene is catabolite-repressed in DSM2334.

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.

The isolation of human plasma prekallikrein.

1. The isolation of human plasma prekallikrein was achieved by fractionating human plasma on diethylaminoethyl cellulose (DEAE) in the presence of heparin.2. Heparin was shown to inhibit the activation of prekallikrein during the isolation procedure.3. The isolated prekallikrein fraction had some kallikrein activity which could be inhibited by diisopropylfluorophosphate (DFP) without affecting the ability of prekallikrein to be activated.4. The prekallikrein obtained was functionally pure in that it had no kallikrein inhibiting or activating activity. It was not physico-chemically pure, the major contaminant being the immunoglobulin IgG.

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.

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.

The DNA sequence at the T7 C promoter.

Restriction fragments of T7 DNA which selectively bind E. coli RNA polymerase have been identified. These include fragments located close to the beginning of gene 1 where according to Minkley and Pribnow (1973) there is a promoter called C. The smallest fragment from this region which binds RNA polymerase has been sequenced. It contains a promoter-like sequence, at an appropriate distance from the sequence TACA which Minkley and Pribnow suggested should lie at the initiation site of C. RNA synthesised in vitro from these fragments has been sequenced. The RNA sequence corresponds to the sequence to the right of the C promoter. The C promoter differs significantly from the A1 A2 and A3 promoters in sequence. Its structure and position suggest it plays a role in T7 infection.

Control sites in the sequence at the beginning of T7 gene 1.

The DNA sequence of the fragment Hind.30, 378 bases long, from the beginning of gene 1 of T7 is presented. It contains the C promoter, two in vitro transcriptional terminator sites and a sequence of 171 bases which probably codes for the N terminus of the T7 RNA polymerase. The sequence also codes for the RNase III cleavage site before gene 1. The overlaps with the transcriptional terminators, The RNA transcript of the sequence about the terminators can be arranged in a set of alternative double-stranded hairpin structures. It is suggested that conversion between these structures may have a role in termination; this may be influenced by interactions with ribosomes and RNase III. The region of the C promoter between genes 0.7 and 1 thus contains several sites which may be involved in the control of transcription and translation.

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.