Lysosomal pool of free-amino acids

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20⁰ and 37⁰.     

Mutational Specificity of Ultraviolet Light in ESCHERICHIA COLI with and without the R Plasmid Pkm101

Plasmid pKM101 provides UV protection and increases the frequency of spontaneous and UV-induced mutations in Escherichia coli. By analyzing reversion patterns of defined trpA alleles, we showed that pKM101 altered the mutational specificity of UV-induced mutations. Certain UV-induced base-pair substitutions were strongly enhanced, while others were decreased in frequency in the presence of pKM101. This result suggests an interaction between cellular misrepair and an error-prone repair function(s) provided by pKM101. We have also examined UV mutational specificity in the absence of pKM101 and found the following: (1) UV preferentially enhances missense, as well as nonsense, intergenic suppressor mutations; (2) UV causes all possible base-pair substitutions as well as frameshift mutations; (3) G·C base pairs are more susceptible to UV mutagenesis than A·T base pairs at the same nucleotide positions; and (4) UV-induced mutations can occur at nucleotide positions that are not part of pyrimidine-pyrimidine sequences.     

Probe of beta-galactosidase structure with iodoacetate. Differential reactivity of thiol groups in wild-type and mutant forms of beta-galactosidase.

Carboxymethylation with 14 C-labeled iodoacetate of cysteine residues in wild-type beta-galactosidase from Escherichia coli and in a defective beta-galactosidase from deletion mutant strain M15 was investigated in order to determine accessible positions in the tetrameric wild-type form and the dimeric mutant M15 protein. The extent of carboxymethylation, the effects on biological activity, antibody activation, physical stability, and the labeling of particular residues were studied. The results distinguish three groups of spatial relationships for cysteine residues in the protein, define possible regions for subunit interactions, and confirm that no cysteine residue is specifically involved in catalysis. Residue 1019 and to a lesser extent 498 are accessible in the tetrameric protein and probably represent exposed areas. In the M15 protein, these two, and three additional residues, at 76,387 and 600, were found to react significantly with reagent. One or more of the latter are suggested to be in the dimer-dimer interface. Complementation and activation by antibody are inhibited by carboxymethylation of M15 protein.     

Pyruvate metabolism after in vivo exposure to oral arsenic

This study investigated altered pyruvate metabolism after prolonged oral arsenic exposure. Male rats were given access to deionized drinking water containing 0, 40 or 85 ppm sodium arsenate (As⁵⁺) for 3 weeks. Respiration studies with mitochondria isolated from treated animals indicated decreased state 3 respiration (with ADP) and decreased respiratory control ratios (RCR) for pyruvate/malate-mediated respiration, but not for succinate-mediated respiration, as compared to control respiration values. In addition, pyruvate dehydrogenase activity was measured, in both liver and intestine, before and after Mg-activation in vitro. After 3 weeks, the effects of arsenic at the highest dose level were pronounced on the basal pyruvate dehydrogenase activity (before activation) as well as the total pyruvate dehydrogenase (after activation). The inhibition of pyruvate dehydrogenase activity both before and after Mg-activation suggests an arsenic effect on mitochondrial pyruvate metabolism which, in part, involves inhibition of pyruvate decarboxylase. Evidence is also presented which may indicate an arsenic effect on the kinase and/or phosphatase which regulate pyruvate dehydrogenase activity.     

A new method of in situ hybridization

A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5–20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.Contribution number 5121 from the Department of Chemistry.     

Oncornaviral protein modulation in mouse uterine tissue by estrogen (38467).

Treatment of ovariectomized NIH Swiss mice with estrogens elevated the level of the murine leukemia virus group specific protein and the activity of an RNA-directed DNA polymerase in the uterus. The extent that these markers were raised was dependent on the relative biological potency of the estrogen and on the time interval following treatment. Increases in the levels of both viral marker proteins were evident within 24 hr of treatment and were highest at 48 hr. Subsequently, viral protein levels declined to pretreatment levels.     

Population studies in Artemisia tridentata ssp. vaseyana: Chromosome numbers and sesquiterpene lactone races

The sesquiterpene lactones and chromosome numbers for three chemical races of Artemisia tridentata ssp. vaseyana have been examined from four populations in western Montana. TLC analysis of the sesquiterpene lactones in the seeds and seed producing parents demonstrated that genetic exchange does occur between sympatric sesquiterpene lactone chemical races. However, other evidence suggests that introgression between these races is restricted to zones of sympatry. There appears to be no correlation between chromosome numbers and sesquiterpene lactone races.