Degradation of 2-chloroethanol by wild type and mutants of Pseudomonas putida US2

A strain of Pseudomonas putida was isolated that was able to degrade 2-chloroethanol. The degradation proceeded via 2-chloroacetaldehyde and chloroacetate to glycolate. In crude extracts the enzymes for this degradation pathway could be detected. All enzymes proved to be inducible. The dehalogenase that catalyzed the dehalogenation of chloroacetate to glycolate was further characterized. It consisted of a single polypeptide chain with a molecular mass of 28 kDa. After induction the dehalogenase was expressed at a high level. In a mutant resistant to high concentrations of 2-chloroethanol the dehalogenase was no longer expressed. The mechanism of resistance seemed to be due to the inability to convert chloroacetate and export of this compound out of the cell.Non-standard abbreviations CEO 2-chloroethanol - DCPIP 2,6-dichlorophenolindophenol - FPLC fast protein liquid chromatography - PAGE polyacrylamide gelelectrophoresis - PES phenazine ethosulfate - PMS phenazine methosulfate - PQQ pyrroloquinoline quinone     

The cyanelle genome of Cyanophora paradoxa, unlike the chloroplast genome, codes for the ribosomal L3 protein.

We describe a 1132 bp sequence of the cyanelle genome of Cyanophora paradoxa containing the rpl3 gene. This gene, which is not chloroplast encoded in plants, is the first of a long cyanelle ribosomal operon whose organization resembles that of the S10 operon of E. coli. We have shown that the rpl3 gene is transcribed in cyanelles as a 7500 nucleotide precursor and that the 5'-end of the mRNA starts approximately 90 nucleotides upstream from the initiation codon. However, no typical procaryotic promoter could be found for this gene. We have detected, using anti E. coli L3 antibodies, the cyanelle L3 protein in cyanelle extracts and in E. coli cells transformed with the cyanelle rpl3 gene.     

Genetic heterogeneity in tuberous sclerosis

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by widespread hamartosis. Preliminary evidence of linkage between the TSC locus and markers on chromosome 9q34 was established, but subsequently disputed. More recently, a putative TSC locus on chromosome 11 has been suggested and genetic heterogeneity seems likely. Here we describe an approach combining multipoint linkage analysis and heterogeneity tests that has enabled us to obtain significant evidence for locus heterogeneity after studying a relatively small number of families. Our results support a model with two different loci independently causing the disease. One locus (TSC1) maps in the vicinity of the Abelson oncogene at 9q34 and a second locus (TSC2) maps in the region of the anonymous DNA marker Lam L7 and the dopamine D2 receptor gene at 11q23.     

Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1×10⁶ insect cells produced up to 3 g opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.Abbreviations dpi days post infection - pfu plaque forming units - AcNPV Autographa californica nuclear polyhedrosis virus     

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.     

A mechanical analysis of the closed Hancock heart valve prosthesis

In order to obtain mechanical specifications for the design of an artificial leaflet valve prosthesis, a geometrically non-linear numerical model is developed of a closed Hancock leaflet valve prosthesis. In this model, the fibre reinforcement of the leaflet and the viscoelastic properties of frame and leaflets are incorporated. The calculations are primarily restricted to 1/6 part of the valve and a time varying pressure load is applied. The calculations are verified experimentally by measuring the commissure displacements and leaflet centre displacement of a Hancock valve. The numerically obtained commissure displacements are found to be linearly dependent on the pressure load, and the slope of the curves is hardly dependent on loading type and loading velocity. Experimentally a difference is found between the three commissure displacements, which is also predicted numerically using a simplified asymmetric total valve model. Besides, experimentally a clear dependency of commissure displacements on frame size is found. For the leaflet centre displacement, a qualitative agreement exists between numerical prediction and experimental result, although the numerical predicted values are systematically higher. The numerically obtained stress distributions revealed that the maximum von Mises intensity in the membranes occurs in the vicinity of the commissure in the free leaflet area (0.2 N mm-2). Wrinkling of the membranes may occur in the coaptation area near the leaflet suspension. The maximum fibre stress is found near the aortic ring in the fibres which form the boundaries of the coaptation area (0.64 N mm-2). These locations seem to correlate with some common regions of tissue valve failure.     

The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Effects of medium composition on extracellular proteinase stability and yield in batch cultures of a Thermus sp.

Summary Thermus sp. Rt41A produces an extracellular proteinase that is produced concomitant with growth and l-glutamate catabolism. Calcium-chelating medium components were shown to decrease the half-life of the proteinase in growing cultures. Medium modifications avoiding these components resulted in an increase in the half-life and in the peak level of proteinase. By adding the inorganic phosphate requirement for growth in anabolic amounts to pH-controlled batch cultures, stability of the proteinase in the medium was greatly enhanced and there was consequent improvement in the total proteinase yield. This approach also allowed a balanced increase in substrate and phosphate concentrations to increase the cell and proteinase yield in batch culture in an almost stoichiometric manner.Offprint requests to: H. W. Morgan