Molecular characterization of a Galactomyces geotrichum lipase, another member of the cholinesterase/lipase family.

Geotrichum candidum secretes several lipase isoenzymes, differing in their selectively towards esters of long chain fatty acids with a cis-9 double bond. One group shows an absolute selectively towards these fatty acid esters, the other group has a more relaxed specificity and will also hydrolyze other long chain fatty acid esters. Galactomyces geotrichum secrets a lipase that has the same specificity as the latter group. The corresponding lipase gene was cloned from Galactomyces geotrichum. From an alignment of our enzymes' primary structure with those of different strains of Geotrichum candidum, remarkable conservation is evident and it is not yet possible to identify residues/structures responsible for differences in fatty acid specificity. Comparison of the GCL/GGL family with a variety of lipases from other sources, indicated that they are more related to mammalian than microbial lipases.

Conservation of binding site specificity of three yeast DNA binding proteins.

Sequence specific binding of protein extracts from 13 different yeast species to three oligonucleotide probes and two points mutants derived from Saccharomyces cerevisiae DNA binding proteins were tested using mobility shift assays. The probes were high affinity binding sites for GRF1/RAP1/ABF1 and CP1/CPF1. Most yeasts in the genus Saccharomyces showed specific binding to all three probes and also displayed similar sequence requirements when challenged by molar excesses of mutant probes. The affinities for the probes varied amongst the other yeasts tested, but in general, CPF1 binding activity was the most widespread, while the other two were more limited.

Thiamine repression and pyruvate decarboxylase autoregulation independently control the expression of the Saccharomyces cerevisiae PDC5 gene.

The Saccharomyces cerevisiae gene PDC5 encodes the minor isoform of pyruvate decarboxylase (Pdc). In this work we show that expression of PDC5 but not that of PDC1, which encodes the major isoform, is repressed by thiamine. Hence, under thiamine limitation both PDC1 and PDC5 are expressed. PDC5 also becomes strongly expressed in a pdc1delta mutant. Two-dimensional gel electrophoresis of whole protein extracts shows that thiamine limitation stimulates the production of THI gene products and of Pdc5p. Deletion of PDC1 only stimulates production of Pdc5p. We conclude that the stimulation of PDC5 expression in a pdc1delta mutant is not due to a response to thiamine limitation.

A survey of yeast ureases and characterisation of partially purified Rhodosporidium paludigenum urease.

Cell-free extracts of a selection of yeasts were analysed for urease activity. Species in the genera Filobasidiella, Rhodotorula and Rhodosporidium had the highest specific activities. Immune inactivation experiments showed widely different degrees of cross-reactivity between antiserum to jack bean urease and yeast ureases, with Rhodosporidium paludigenum (71%) the most and Schizosaccharomyces pombe (3%) the least affected. Only R. paludigenum urease was detected with anti-jack bean urease antiserum on Western blots. The urease of Rhodosporidium paludigenum was partially purified by column chromatography. The native enzyme was found to have a subunit size of 72 +/- 7 kDa probably in an octamer arrangement of 560 +/- 8 kDa, having a specific activity of 62.5 mumol urea hydrolysed min-1 (mg protein)-1. The enzyme was stable in the pH range 5-11 with optimum activity at pH 7.8. Vmax and Km values were determined as 65.2 +/- 3.8 mumol min-1 (mg protein)-1 and 3.81 +/- 0.47 mM, respectively.

PLATSAK, a potent antithrombotic and fibrinolytic protein, inhibits arterial and venous thrombosis in a baboon model.

Antiplatelet-antithrombin-staphylokinase (PLATSAK) is a chimeric protein that was recombinantly produced in Escherichia coli cells. The protein was designed to target haemostasis at three different levels. It consists of staphylokinase for activation of fibrinolyis, the Arg-Gly-Asp sequence for the prevention of platelet aggregation, and an antithrombotic peptide for the inhibition of thrombin. The in vivo activity of PLATSAK was evaluated by assessing its effect on platelet deposition in a baboon model of arterial and venous thrombosis. Dacron vascular graft segments and expansion chambers, inserted as extensions into permanent femoral arteriovenous shunts, were used to simulate arterial and venous thrombosis, respectively. PLATSAK (3.68 mg/kg) was administered as a bolus 10 minutes before placement of the thrombogenic devices. Platelet deposition onto the graft surface and in the expansion chamber was imaged in real time with a scintillation camera as the deposition of 111In-labeled platelets. After 2 hours, platelet deposition in the graft segments and expansion chambers was inhibited by 50% and 85%, respectively, when compared to control studies. The activated partial thromboplastin time was lengthened to greater than 120 seconds. Interestingly, the level of fibrinogen degradation products in plasma did not increase after administration of PLATSAK. These results demonstrate that PLATSAK effectively inhibited platelet deposition in both arterial- and venous-type thrombosis in an animal model.

Production of a recombinant antithrombotic and fibrinolytic protein, PLATSAK, in Escherichia coli.

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.

A physical analysis of the prophage of Proteus mirabilis PM5006.

Proteus mirabilis phage 5006M was investigated as a prophage by DNA hybridization. A physical map of the prophage was established and the site of integration was localized on the phage genome. Older reports that the phage is cryptic in P. mirabilis could not be verified.

Proteus mirabilis phages 5006M, 5006M HFT k and 5006M HFT ak: physical comparison of genome characteristics.

The genomes of proteus mirabilis phages 5006M, kanamycin resistance transducing variant 5006M HFT k and kanamycin-ampicillin resistance transducing variant 5006M HFT ak have been compared. Homo- and heteroduplex and partial denaturation mapping analyses were performed. The results confirm a sequential headful packaging mechanism, facilitate mapping of the ampicillin resistance marker, demonstrate a hairpin loop structure in both variants, reveal a common insertion site for 8 X 10(6) mol. wt. non-phage DNA in both variants and implicate a role for the non-inducible cryptic host strain prophage 5006M in the generatin cycle of the variant phages.

Proteus mirabilis phage 5006M: a physical characterization.

This report deals with physical characterization of the generalized transducing Proteus mirabilis phage 5006M. The morphology of the phage is presented, the buoyant density was determined (1.491 g/ml) and the G + C content of the phage DNA was found to be 44%. The phage genome has a length of 14.8 micrometers and mol. wt. of 30.7 x 10(6). Denaturation mapping revealed non-random circular permutation of the phage DNA. The genome exhibits 3.6% terminal redundancy as shown by homoduplex analysis. The existence of concatemeric precursors of phage 5006M DNA is inferred and the results are interpreted in terms of a sequential headful packaging mechanism.

Proteus mirabilis converting phage 5006Mpa has an oversized genome.

Proteus mirabilis phage 5006Mpa is a converting variant for ampicillin resistance of phage 5006M. We show here that the ampicillin resistance marker of transposon Tn1 is located on a 9.8% insertion with respect to the wild-type phage genome. This renders the 5006Mpa genome 5% oversized, albeit without loss of wild-type genetic material from the phage population.

Microdissection and cloning of the white locus and the 3B1-3C2 region of the Drosophila X chromosome.

Fragments from the 3B-3C region of the Drosophila X chromosomes were microdissected from salivary gland squashes and their DNA was cloned by the method developed by Scalenghe et al. (1981). These clones were used as starting points for a chromosome walk which covers 200 kb including bands 3B2 to 3C2. A number of deletion breakpoints were mapped on the cloned DNA allowing the localisation of several genes in the 3B region. The white locus in particular was isolated by microcloning the w insertion site. Two transcripts, of 2 and 2.4 kb, respectively, arise from the white region and its vicinity.

Telomerase activity and survival of late-stage South African esophageal carcinoma patients.

Squamous cell carcinoma of the esophagus is a cancer with a high incidence in South Africa. We have investigated the prognostic value of telomerase activity in tumors as well as nearby normal tissue. Biopsies from 98 patients (71 men and 27 women) were analyzed using an adaptation of the TRAP assay. We found all tumor biopsies to have moderate to high telomerase activity, while one third of biopsies from normal mucosa were negative. The telomerase activity level of the tumors had no prognostic value (P = 0.95) as determined by the log rank test. A P-value of 0.02 was found when the telomerase-negative and moderately positive normal biopsies were grouped together and compared to those with high activity. Our results show that telomerase activity of normal mucosa in the vicinity of the tumor can identify a population of patients with significantly worse prognosis, even in late stage patients.

Expression of a Bacillus alpha-amylase gene in yeast.

A recombinant plasmid, pSR11.3, containing the alpha-amylase gene (AMY) of Bacillus amyloliquefaciens was characterized and expressed in Bacillus subtilis. A 2.3 kilobase BamHI-BglII fragment carrying AMY was cloned into pBR322 (pEL322) and in both orientations into a multi-copy Escherichia coli-yeast shuttle vector YEp13 (pAM13) and expressed in E. coli HB101 and various Saccharomyces stains. We report on the successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals. Enzyme production in B. subtilis 1A297(pSR11.3), E. coli HB101(pEL322) and Saccharomyces JM2773-15B(pAM13) transformants was measured as 125, 22 and 123 U/ml, respectively. The molecular weight of the purified alpha-amylase secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B-(pAM13) was estimated to be 55 kDa. The pH and temperature optima for the alpha-amylase activities of the transformants were 6.5 to 8.0 and 50 to 65 degrees C, respectively. Amylose hydrolysis profiles of the alpha-amylases secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) indicate effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products.

G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae.

Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36-48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.

A Saccharomyces cerevisiae mutant defective in the kinesin-like protein Kar3 is sensitive to NaCl-stress.

Several mutants of Saccharomyces cerevisiae showing poor growth in the presence of elevated concentrations of NaCl were isolated to identify genes involved in the osmo-stress response. One of these mutants (WAY.5-4A-11; osr11) which showed a clear 2:2 segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain has been characterised. The mutation responsible for poor growth under salt-stress was recessive. The corresponding gene was cloned by complementation of the mutant phenotype and a 3.5-kb fragment was isolated. The sequence of this fragment matched that of KAR3, a gene previously identified to be involved in karyogamy and mitosis. Allelism of OSR11 to KAR3 was confirmed by tetrad analysis, and disruption mutants showed the same NaCl-phenotype as the original osr11 mutation. The disruption mutant was more sensitive to high sucrose concentrations than the original mutant was to high glucose concentrations. In a different genetic background (W303-1A), the kar3 disruptants were less sensitive to osmo-stress than the WAY.5-4A strain. Heat-stress, nitrogen-starvation and cultivation on ethanol failed to affect the growth of osr11 and kar3 mutants, pointing to a possible specific involvement of KAR3 in the osmotic-stress response. Microscopic studies showed that cell division of the kar3 mutants was impaired and NaCl-stress conditions aggravated the phenotype.