Identification and physical characterization of yeast glucoamylase structural genes

Summary Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta⁺ strains, each carrying a single genetically defined STA locus, were crossed with a Sta^– strain and the segregation behavior of the functional locus (i.e. Sta⁺) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5 and 3 homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta^–), but has retained extensive sequence homology to the 5 end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.     

Purification and properties of arylsulfatase B from high- and low-activity mouse strains

Arylsulfatase B (arylsulfate sulfohydrolase; EC 3.1.6.1) activities in C57BL/6J, SWR/J, and A/J mouse liver approximate a 5:3:1 ratio. Each enzyme was purified to apparent homogeneity, and the properties of the three purified enzymes were compared. The purified enzyme behaved as a monomer with an apparent molecular weight of 50,000. The purified enzyme catalyzed the hydrolysis of p-nitrocatechol sulfate (pNCS), 4-methylumbelliferyl sulfate (4MUS), and chondroitin-4-sulfate (C4S) heptasaccharide. Purified SWR/J arylsulfatase B possessed a higher relative electrophoretic mobility at pH 4.0 than the A/J and C57BL/6J isozymes, and the SWR/J enzyme was more thermostable than either the C57BL/6J or the A/J enzyme. No differences were observed among the three enzymes with respect to their Michaelis constants for 4MUS and pNCS, isoelectric points, responses to inhibitors, pH optima, or electrophoretic mobilities at pH 8.3. The relative in vivo rates of synthesis of C57BL/6J, A/J, and SWR/J arylsulfatase B were comparable.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Screening for Chlamydial Infections in Women Attending Family Planning Clinics—Evaluation of Presumptive Indicators for Therapy

Chlamydia trachomatis was recovered from the cervices of 9.8 percent (268/2,729) of women attending seven family planning clinics. The infection rate varied from 5.5 percent to 22.5 percent in different clinics. Chlamydial infection could be associated with younger age, nulliparity, being black and use of oral contraceptives. Most (70 percent) of the chlamydial infections were inapparent and presumptive indicators for antichlamydial therapy that are useful for symptomatic women will not make a major impact on this reservoir. It is concluded that chlamydial cultures are needed to deal with the high prevalence of these infections.     

Thymus-independent induction and antigen-dependent recovery of idiotype-specific suppression

BALB/c nude (nu/nu) mice and euthymic (nu/+) littermates were treated as neonates with anti-T15 antibody and challenged at various ages with either a thymus-independent, PC-Brucella abortus (PC-BA), or thymus-dependent, PC-keyhole limpet hemocyanin (PC-KLH), form of phosphorylcholine (PC). Nu/nu mice challenged with PC-KLH received KLH-primed splenic T cells prior to immunization. Neither neonatally anti-idiotype-treated nu/+ nor nu/nu mice responded with the production of T15-positive anti-PC antibodies after challenge with either form of PC antigen. It is concluded that neither induction nor maintenance of a state of T15-specific suppression requires thymus-matured T cells. Recovery of anti-PC responsiveness in suppressed nu/+ or nu/nu mice was similar and was found to be related to the form of antigen used to elicit the response. Immunization with PC-KLH revealed a long-lasting unresponsiveness (up to 16 weeks). In contrast, immunization with PC-BA elicited a full anti-PC response as early as at 6.5 weeks of age.     

Cells Persistently Infected with Newcastle Disease Virus III. Thermal Stability of Hemagglutinin and Neuraminidase of a Mutant Isolated from Persistently Infected L Cells

Data were obtained which indicated the possible cause of the defective elution from erythrocytes of the mutant virus (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus (NDV(o)). The chicken erythrocyte receptors for the mutant and wild-type viruses were equally sensitive to the action of Vibrio cholera filtrate neuraminidase; this suggests that the failure of NDV(pi) to elute from chicken erythrocytes is not due to a specific neuraminidase-resistant receptor for this virus on the erythrocyte membrane. There was no difference in the enzyme content of the intact virions of NDV(o) and NDV(pi) when tested with a soluble substrate, indicating that the inefficient elution of NDV(pi) was not due to a reduced enzyme content. The neuraminidase activity of intact NDV(pi) virions was significantly more stable at 55 C than the enzyme of NDV(o) virions, whereas the dissociated enzymes of the two viruses were inactivated at the same rate. On the basis of these findings, it seems likely there is a structural difference between the two viruses. The neuraminidase protein of the mutant NDV(pi) may be incorporated into the viral envelope in such a manner that it is prevented from reacting with the substrate in the erythrocyte membrane, although it can react with a soluble substrate. The hemagglutinin activity of both intact and disrupted NDV(pi) was significantly more resistant to thermal inactivation than that of the wild-type NDV(o). This finding suggests a genetic difference in the hemagglutinin protein of the two viruses.     

Properties of Mutants in Galactose Taxis and Transport

beta-Methylgalactoside (mgl) permease mutants of Escherichia coli, which are defective in three genes, mglA, mglB, and mglC, were assayed for galactose taxis and galactose transport. The mglB product is the galactose-binding protein. Previous evidence, supported by our new findings, shows that the galactose-binding protein is the recognition component for galactose taxis as well as for galactose transport. Most mutants defective in mglB showed strong effects on both chemotaxis and transport; however, a couple showed effects chiefly on one process or the other, thus allowing a separation of chemotaxis and transport. The mglA and mglC products have not yet been identified, but they must be components of the galactose transport machinery since mutants defective in mglA or mglC, or both, showed strongly reduced transport. Although some of these mutants showed little chemotaxis, most gave close to wild-type chemotactic responses. Thus, transport is not required for galactose taxis. The bacteria detect changes in the fraction of binding protein associated with galactose, not changes in the rate of transport.     

Antibacterial Action of Essential Oils of Artemisia as an Ecological Factor: II. Antibacterial Action of the Volatile Oils of Artemisia tridentata (Big Sagebrush) on Bacteria from the Rumen of Mule Deer

Rumen microorganisms of wild and captive deer were subjected to increasing amounts of volatile oils. The oils had a marked antibacterial effect on the rumen bacteria when the concentration reached approximately 16 muliters of oil per 10 ml of rumen fluid nutrient broth. The gross reactions of rumen bacteria obtained from wild, as well as captive, deer to the volatile oils seemed to be of the same magnitude; thus no adaptation by the bacteria to the oils was apparent.     

Interferon Production in Mice by Cell Wall Mutants of Salmonella typhimurium

The interferon response elicited by Salmonella typhimurium mutants in mice is not dependent on the presence of a complete cell wall lipopolysaccharide. In fact, a mutant (G30/C21) which has lost all the polysaccharide side chains and sugars of the O antigen and contains only 2-keto-3-deoxyoctonate and lipid is indistinguishable in its interferon-stimulating ability from the wild type which possesses a complete O antigen with polysaccharide side chains.