Characterization of the activity of fatty-acid epoxide hydrolase in seeds of castor bean (Ricinus communis L.)

Epoxide hydrolase (EC 3.3.2.3) activity was measured with [1-¹⁴C]cis-9,10-epoxystearic acid as the substrate. Homogenates were prepared from the endosperm tissue of germinating seeds of castor bean (Ricinus communis L. zanzibariensis). The activity of fatty-acid epoxide hydrolase was characterized with respect to dependence on time, amount of protein, pH and temperature. Analyses of enzyme distribution in endosperm, cotyledons, root and hypocotyl showed the highest total activity in the endosperm, less in the cotyledons and low activity in the root and hypocotyl. The specific activity was similar for cotyledons and endosperm. Analysis of the temporal expression of the enzyme in the endosperm during germination revealed high activity already in the imbibed seed. Activity was maximal between days four to six and then decreased at the end of one week. Subcellular fractionation of endosperm revealed a dual distribution of activity between the glyoxysomal and the cytosolic fractions.     

Cloning and expression of the Vitreoscilla hemoglobin gene in pseudomonads: effects on cell growth

The gene (vgb) encoding the hemoglobin (VtHb) of Vitreoscilla sp. was cloned into a broad-host-range vector and stably transformed into Pseudomonas putida, Pseudomonas aeruginosa, and Xanthomonas maltophilia. vgb was stably maintained and expressed in functional form in all three species. When growth of the P. aeruginosa and X. maltophilia transformants in Luria-Bertani medium was compared with that of each corresponding untransformed strain, the VtHb-producing strains reached slightly higher maximum viable cell numbers, had significantly increased viability after extebded times in culture, and, like E. coli that produces VtHb, had significantly lower respiration rates. The VtHb-producing strain of P. putida also reached a slightly higher maximum viable cell number than its corresponding untransformed strain, but was significantly less viable after extended times in culture and, unlike the case in E. coli, had a generally higher respiration rate than the untransformed strain. When growth was monitored by absorbance, the results were similar to those obtained with viable cell counts.     

Tn552 transposase purification and in vitro activities.

The Staphylococcus aureus transposon Tn552 encodes a protein (p480) containing the 'D,D(35)E' motif common to retroviral integrases and the transposases of a number of bacterial elements, including phage Mu, the integron-containing element Tn5090, Tn7 and IS3. p480 and a histidine-tagged derivative were overexpressed in Escherichia coli and purified by methods involving denaturation and renaturation. DNase I footprinting and gel binding assays demonstrated that p480 binds to two adjacent, directly repeated 23 bp motifs at each end of Tn552. Although donor strand cleavage by p480 was not detected, in vitro conditions were defined for strand transfer activity with transposon end fragments having pre-cleaved 3' termini. Strand transfer was Mn(2+)-dependent and appeared to join a single left or right end fragment to target DNA. The importance of the terminal dinucleotide CA-3' was demonstrated by mutation. The in vitro activities of p480 are consistent with its proposed function as the Tn552 transposase.     

The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase.

In higher eukaryotes, the activity and specificity of the type 1 protein serine-threonine phosphatase (PP1) catalytic subunit is thought to be controlled by its association with a number of regulatory or targeting subunits. Here we describe the characterization of a gene encoding one such potential polypeptide in the yeast Saccharomyces cerevisiae. The gene which we have isolated (termed SDS22) encodes a product with a high degree of sequence identity to the fission yeast sds22 protein, a known regulator of the mitotic function of PP1 in Schizosaccharomyces pombe. Using two different criteria, we have demonstrated that Sds22p and the catalytic subunit of PP1 (Glc7p) interact in yeast cells. We have also generated a temperature-sensitive allele of GLC7 (glc7-12) which causes a block to the completion of mitosis at the restrictive temperature. Additional copies of SDS22 lead to allele-specific suppression of the glc7-12 mutant, strongly suggesting that the interaction between the two proteins is of functional significance. Sds22p is therefore likely to be the second example of a PP1 regulatory subunit identified in S. cerevisiae.     

Mutations in the Saccharomyces Cerevisiae Type 2a Protein Phosphatase Catalytic Subunit Reveal Roles in Cell Wall Integrity, Actin Cytoskeleton Organization and Mitosis

Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37°), cells dependent solely on pph22(ts) alleles for PP2A function displayed a rapid arrest of proliferation. Ts(-) pph22 mutant cells underwent lysis at 37°, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts(-) pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37°. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24° to 37°, Ts(-) pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37°, many Ts(-) pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts(-) pph22 mutant cells also accumulated aberrant microtubule structures at 37°, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts(-) pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.     

500 MHz H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

Sensitivity and adaptation in R7, an ultraviolet photoreceptor,in theDrosophila retina

Summary Receptor deficient mutants and chromatic adaptation were used to isolate the contribution of R7 to the electroretinogram (ERG) ofDrosophila. R7 was found to be a single-peaked ultraviolet (UV) receptor (Fig. 1). Photoconversion of the UV absorbing rhodopsin (R) to its stable 470–495 nm metarhodopsin (M) was shown to elicit a long-lived negative (depolarizing) afterpotential (Fig. 3) while inactivating R7. Photoreconversion ofM toR reactivates R7 (Fig. 2) and repolarizes the ERG (Fig. 3). The intensities of light needed to elicit afterpotentials by photointerconverting R7 photopigment were found to be about 2 log units greater than for R1-6 photopigment (Fig. 4). Vitamin A deprivation decreases R7 (as well as R8) sensitivity by about 2 log units (through decreased photopigment levels) without changing spectral sensitivity shape (Fig. 5). Vitamin A deprivation further eliminates the light-induced inactivation of R7 allowing experiments designed to characterize the in vivo spectral absorption of R7M. R7M was found to have UV and 495 nm maxima (Fig. 6). No polarization sensitivity was detected in the R7 ERG component. The adaptational properties of R7 are similar to the properties previously established for R1-6 but different from the properties of R8.Supported by NSF grants BMS-74-12817 and BNS 76-11921. I thank M. Chapin, R. Greenberg, K. Hu, A. Ivanyshyn, D. Lakin, G. Pransky, D. Sawyer, J. Walker and W. Zitzmann for technical assistance.