Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.     

Genetic structure of natural populations of the red-spotted newt,Notophthalmus viridescens

Genetic variation is described at 15 loci in 2 neotenic and 12 nonneotenic populations of red-spotted newts. Though high levels of genetic similarity (I=0.990) were found among all populations, allele frequencies at six of the eight most polymorphic loci show significant heterogeneity across populations. Change in allele frequencies at two of these loci (Pep-2 and Ldh-1) is significantly correlated with latitude. Interspecific homologies are established for newt peptidases based on substrate specificities and lactate dehydrogenases based on tissue distribution, thermal stability, and kinetic properties. Nonneotenic populations are highly variable (H=0.157) and neotenic populations are only slightly, but significantly, less variable (H=0.120). The high levels of heterozygosity detected in nonneotenic populations may result from large effective population size and/or environmental heterogeneity. The unexpectedly high heterozygosity values obtained for the neotenic populations may indicate adult dispersal or the presence of some previously undetected red efts at these localities. In any case, a major change in life history has apparently had little effect on the genetic structure of these populations.This research was supported by grants from the Blakeslee Fund of Smith College.     

Evidence for Plasmid Linkage of Raffinose Utilization and Associated alpha-Galactosidase and Sucrose Hydrolase Activity in Pediococcus pentosaceus

The ability to ferment the trisaccharide raffinose was linked with the presence of plasmid DNA in three strains of Pediococcus pentosaceus. Parental strains showed associated inducible alpha-galactosidase and sucrose hydrolase activities when grown in alpha-galactosides and sucrose, respectively. Derivative strains of PPE1.0, PPE2.0, and PPE5.0, which had lost 30-, 28-, and 23-megadalton plasmids, respectively, had no alpha-galactosidase or sucrose hydrolase activity.     

The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase

The kinetic mechanism of the phosphoribosyltransferase reaction is shown to be rapid equilibrium random bi bi with an enzyme-anthranilate-pyrophosphate abortive complex. We present a rate equation that not only predicts the observed kinetic patterns but also accommodates the fact that feedback inhibition is partial, even though tryptophan (Ki = 0.5 microM) and phosphoribosylpyrophosphate (Km = 50 microM) are competitive. Neither ligand completely abolishes the effect of the other. Instead, the binding of one ligand leads to a mutual elevation in the dissociation constant of the opposing ligand by a factor of two to three. Tryptophan inhibition is noncompetitive with respect to anthranilate (Km = 0.58 microM) and does not diminish the rate of interconversion of ternary complexes. Tryptophan cooperativity, with respect to the inhibition of phosphoribosyltransferase, conforms to the concerted Monod-Wyman-Changeux formulation (kinetic Hill coefficient = 2), whereas tryptophan as an inhibitor of anthranilate synthase more closely conforms to a Koshland model of sequential cooperativity with a kinetic Hill coefficient of 1.4. The aggregate contains only one class of tryptophan sites. Thus the first tryptophan molecule bound to the aggregate maximally inhibits both phosphoribosyltransferase active centers and one of the two anthranilate synthase catalytic sites. The remaining anthranilate synthase subunit thereupon is converted into a form with less (but not zero) affinity for chorismate and a greater affinity for a second molecule of tryptophan.     

Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins

Tubulin is a major substrate for endogenous Ca2+-calmodulin-dependent phosphorylation in synaptic cytoplasm. The present study details the purification to apparent homogeneity and characterization of a brain cytosolic Ca2+-calmodulin-dependent kinase which phosphorylates tubulin and microtubule-associated proteins as major substrates. The cytosolic kinase system, purified by sequential chromatography on phosphocellulose resin, calmodulin-affinity resin, and Fractogel TSK HW-55, chromatographs as a homogeneous complex of approximately 600,000 Da on Sephacryl S-300. This calmodulin-dependent kinase possesses a group of properties which specifically characterize this enzyme system: 1) the enzyme contains two calmodulin-binding doublets, rho and sigma, of approximately 52,000 and 63,000 Da, respectively; 2) both the rho and the sigma subunits demonstrate isoelectric points between 6.7 and 7.2; 3) both the rho and sigma subunits demonstrate autophosphorylation; 4) both the rho and sigma subunits show significant homologies as assessed by tryptic peptide fingerprints; 5) in the absence of substrate, both the rho and sigma subunits manifest lower mobility autophosphorylated species; 6) the kinase phosphorylates beta-tubulin equally on threonine and serine residues. Substrate specificity, kinetic parameters, calmodulin-binding properties, subunit composition, and subunit isoelectric points clearly differentiate this enzyme from other previously reported calmodulin-dependent kinases.     

Iron-Chelating Compounds Produced by Soil Pseudomonads: Correlation with Fungal Growth Inhibition

Strains of Pseudomonas putida, Pseudomonas sp., and Pseudomonas aeruginosa were examined for their ability to grow in the presence of the iron chelator, ethylenediamine-di-(o-hydroxyphenylacetic acid). In vitro fungal inhibition assays showed that the isolates varied in their ability to inhibit the growth of representative fungal plant pathogens. Fungal inhibition in vitro was superior to that of previously reported Pseudomonas sp. Studies with Fusarium oxysporum forma sp. lycopersici and a susceptible tomato cultivar demonstrated that Pseudomonas putida PPU3.1 was able to significantly reduce wilt disease.     

Cerebral Blood Flow and Oxidative Metabolism in Conscious Fischer-344 Rats of Different Ages

Abstract: The cerebral metabolic rates for O₂ and for glucose were measured in conscious, fasted male Fischer-344 rats at the ages of 3, 12, and 24 months, and cerebral blood flow was determined with ¹⁴C-iodoantipyrine. The metabolic rates for oxygen and glucose were obtained by multiplying blood flow by the O₂ and glucose concentration differences, respectively, between blood in the femoral artery and in the superior sagittal sinus. Mean cerebral blood flow and the metabolic rates for oxygen and glucose did not differ significantly (p > 0.05) between 3 and 12 or between 12 and 24 months. Nor did the arteriovenous differences for O₂ and for glucose change significantly with age. Because the superior sagittal sinus drains blood mainly from the cerebral cortex, the results indicate that average cerebral cortical oxidative metabolism, and the coupling ratios between the cerebral metabolic rate for oxygen and cerebral blood flow and between the cerebral metabolic rate for glucose and cerebral blood flow, do not change significantly with age in the Fischer-344 rat.