α-Mannosidase-1 mutants of Dictyostelium dkoideum: Early aggregation-essential genes regulate enzyme precursor synthesis, modification, and processing

The lysosomal enzyme alpha-mannosidase-1 is one of the earliest developmentally controlled gene products in Dictyostelium discoideum. Although this enzyme is synthesized throughout the first 20 h of development, it is not required for complete morphogenesis, since structural gene (manA) mutants lacking activity develop normally. We isolated six strains deficient in alpha-mannosidase-1 activity which, unlike structural gene mutants, fail to aggregate. Fruiting revertants of these strains accumulate wild-type levels of alpha-mannosidase-1 activity, suggesting that both the enzymatic and morphological defects are caused by single mutations in nonstructural genes essential for early development. Direct genetic evidence for mutations outside of the structural gene was obtained by complementation analysis. We used alpha-mannosidase-1-specific monoclonal antibodies to analyze the biochemical defects in these mad (alpha-mannosidase-1-deficient) mutants. All mad mutants show a significantly reduced relative rate of enzyme precursor biosynthesis. The mad-404 mutation results in a complete lack of precursor biosynthesis, as well as a lack of functional alpha-mannosidase-1 mRNA. In some cases, however, the enzymatic defect results from improper post-translational modification which affects precursor processing. We conclude that a small number of aggregation-essential genes are involved in regulating the synthesis, modification, and processing of alpha-mannosidase-1 during development.     

Cellular location of heat-labile enterotoxin in Escherichia coli.

We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm. The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes. The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.     

Assay of glutamine phosphoribosylpyrophosphate amidotransferase using [1-C]phosphoribosylpyrophosphate

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-α- -ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction, 5-β-phosphoribosyl-1-amine, using [1-¹⁴C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent K_m values of the human lymphoblast enzyme are 0.46 m for glutamine and 0.71 m for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo- -norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity.     

Photosynthesis in Tall Fescue : IV. Carbon Assimilation Pattern in two Genotypes of Tall Fescue Differing in Net Photosynthesis Rates

We previously reported that the net photosynthetic rate of a decaploid genotype (I-16-2) of tall fescue (Festuca arundinacea Schreb.) was 32 to 41 versus 22 milligrams CO₂ per square decimeter per hour in a hexaploid genotype (V6-802) (Randall, Nelson, Asay Plant Physiol 59: 38-41). The high rate was later correlated with increases in total ribulose 1,5-bisphosphate carboxylase protein (17%) and activity (27%) (Joseph, Randall, Nelson Plant Physiol 68: 894-898). This report characterizes photosynthesis with respect to light saturation and early products of photosynthesis in an attempt to identify regulatory metabolic site(s) in these two genotypes. Analysis of the early products of photosynthesis indicated that both genotypes fixed CO₂ via the Calvin-Benson cycle with phosphoglyceric acid as the initial primary product. Both genotypes had similar ¹⁴C-labeled intermediates. Sucrose was the primary sink of ¹⁴CO₂ assimilation. After 10 min of ¹⁴CO₂ assimilation with attached leaves, sucrose accounted for 89% (decaploid) and 81% (hexaploid) of the total ¹⁴C incorporated. In 10 min, this amounted to 1.3 (decaploid) and 0.8 (hexaploid) μmol [¹⁴C]sucrose formed g fresh weight⁻¹ and reflected the observed differences in photosynthetic rates. There was limited labeling of starch (1%) and fructan (1%). Results of total nonstructural carbohydrates and P_i analysis also demonstrated sucrose was the predominant carbohydrate in fescue leaves. Quantitative differences in sucrose and P_i between the two genotypes may reflect changes in partitioning and this possibility is discussed.     

Soybean leaf urease: Comparison with seed urease

Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide-derived urea to NH₃ and CO₂. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 m M ). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent K_m (0.8 m M ), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross-reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.     

Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency: Rates of Legumin Synthesis and Levels of Legumin mRNA

It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in legumin breakdown.

Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S-deficient plants indicated a reduction of 60 to 70% in synthesis of legumin-related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyledons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants.

When restored to an adequate S supply, S-deficient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA.

     

Maternal effects and temperature-sensitive period of mutations affecting embryogenesis in Caenorhabditis elegans

We have used standard tests to investigate the nature of gene expression of a new set of temperature-sensitive mutants defining 30 emb genes (essential for embryogenesis) in the nematode Caenorhabditis elegans. The mode of gene expression as determined by progeny tests for parental effects divides the genes into four classes. For 18 genes maternal gene expression is necessary and sufficient for normal embryogenesis; for 2 genes zygotic expression is necessary and sufficient; for 7 genes either maternal or zygotic expression is sufficient; for 3 genes both maternal and zygotic expression are necessary. One mutant displayed partial paternal sufficiency. The results of temperature-shift experiments define two “execution stages,” corresponding to the limits of the temperature-sensitive period (TSP), and indicate the nature and the time of action or synthesis of the gene products. Most of the maternally expressed genes have very early execution stages indicating translation before fertilization, but some are temperature sensitive late in embryogenesis. Early execution stages for 2 zygotically necessary genes demonstrate that the zygotic genome can be active in the earliest stages of embryogenesis. All taken together, the mode of gene expression, TSP, and arrest stage (terminal phenotype) allow us to classify functionally and begin to order the genes essential for embryogenesis. The results indicate a preeminent role for maternal genes and gene products in embryogenesis, in agreement with the results of others.     

Relationship between foliar composition of red-pine and jack-pine seedlings and vulnerability to Lophodermium needle-cast disease

Summary The Mn and Al content of needles from two-year-old jack pine (Pinus banksiana) seedlings was found to be more than twice that of red pine (Pinus resinosa). It is speculated that the high Mn and Al content of jack pine seedlings may impart some degree of resistance to needle cast disease caused byLophodermium pinastri. Contribution from the Soils Dept., Univ. of Wis., Madison, in cooperation with and supported in part by the Wisconsin Dept. of Natural Resources. Publication approved by the Director of the Wisconsin Agricultural Experiment Station.     

Involvement of recA and exr Genes in the In Vivo Inhibition of the recBC Nuclease

When Escherichia coli cells are gamma irradiated they degrade their deoxyribonucleic acid (DNA). The DNA of previously gamma-irradiated T4 phage is also degraded in infected cells. The amount of degradation is not only dependent on the dose but also on the genotype of the cell. The amount of degradation is less in cells carrying a recB or a recC mutation, suggesting that most of the DNA degradation is due to the recB(+) and recC(+) gene product (exonuclease V). In some strains a previous dose of ultraviolet (UV) light followed by incubation renders the cells resistant to DNA degradation after gamma irradiation. We have shown this inhibition to take place for infecting T4 phage also. By using six strains of E. coli selected for mutations in the genes recA, exr (or lex), and uvrB, we have been able to show that the preliminary UV treatment produces no change in recA and exr cells for both endogenous DNA degradation and the degradation of infecting irradiated T4 phage DNA, i.e., inhibition was not detected in these strains. On the other hand, wild-type cells and strains carrying mutations of uvrB show inhibition in both types of experiments. Because the recA gene product and the exr(+) (lex(+)) gene product are necessary for the induction of prophage, it is possible that the phenomenon of inducible inhibition requires recA(+) and exr(+) presence. One interpretation of these results is that an inducible inhibitor may be controlled by the exr gene.