Sigeru Kuno

Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions.

The nematode Caenorhabditis elegans was cultured monoxenically with E. coli as a food source and the influence of the bacterial growth conditions on the life span was studied. When bacterial growth was restricted by reducing the concentration of bactopeptone, which was supplied as the energy source in nematode growth medium (NGM), the nematodes life span tended to be prolonged without a marked effect on postembryonic development. The effect of bactopeptone on the life span was clearly observed during the postreproductive period (that is, after the egg-laying stage of the wild-type C. elegans) rather than during the larval to young adult stage. Evidence is presented that this alteration of the life span was not brought about by any factor in the bactopeptone but by the concentration of bacteria.

Mutations Affecting Acetylcholine Levels in the Nematode Caenorhabditis elegans

Gene cha‐1 · unc‐17 of the nematode Caenorhabditis elegans is a complex gene, consisting of at least two complementation groups. One part (cha‐1 region) of the gene encodes the enzyme choline acetyltransferase (ChAT), but the function of the other part (unc‐17 region) is still unclear. We measured the ChAT activity and ACh levels of the cha‐1 and unc‐17 complex gene mutants. We show here that alterations in ACh levels, rather than the ChAT activity, reflect abnormal phenotypes accompanying cha‐1 · unc‐17 mutations, that is, the decreased ACh levels in cha‐1 mutations and abnormal accumulation in unc‐17 mutations. Our results suggest that the unc‐17 region may encode functions necessary for storage and/or release of ACh at the presynaptic level.

Single-strand-binding factor(s) which interact with ARS1 of Saccharomyces cerevisiae

Since plasmids containing autonomously replicating sequence(s) (ARS) can transform Saccharomyces cerevisiae cells at high frequency, ARS are considered to be the replication origins of chromosomes. To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors which interact with the ARS1 region located near the centromere of chromosome IV in S. cerevisiae. Using the gel-shift assay, we found protein factors which bound to a single-stranded, 97-bp fragment of the ARS1 region containing the core consensus. Competition experiments with various oligodeoxyribonucleotides (oligos) suggest that a site recognized by the factor(s) was within the element containing the core consensus and adjacent close matches to the core consensus of the minus strand. Indeed, when the oligo containing the minus strand of this element was used as a probe, two oligo-protein complexes were detected. Mutations in the core consensus reduced these binding activities. When the plus-strand oligo of the same region was used as a probe, a retarded band was also detected, but with less specific binding. Considering the fact that the core consensus and close matches to the core consensus are important for ARS function, these results imply that the protein factors detected in this experiment may participate in DNA replication.

Choline acetyltransferase from a temperature-sensitive mutant of caenorhabditis elegans.

A temperature dependent paralytic mutant of C. elegans was isolated and mapped to be an allele of the cha-1 gene that has been shown to be the structural gene for acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6; ChAT) (Hosono et al., J. Exp. Zool.235, 409-421, 1985; Rand and Russell, Genetics106, 227-248, 1984). In crude extracts from the mutant, ChAT activity was present when assayed at a permissive temperature but not detectable at a temperature that provoked abnormal phenotypes. The mutant ChAT was purified to a specific activity of 2.9 nmol of product min (-1) per mg of protein at 10 degrees C and its enzymatic properties were studied by comparison with the wild-type enzyme. The temperaturesensitivity of the mutant ChAT was so remarkable that no activity was detected over 20 degrees C. This inactivation at higher temperature appeared to be partly reversible. The Km values of the mutant enzyme for choline and acetyl-CoA were about twice of those in the wild-type enzyme, but increased 10- to 20-fold in the presence of high salt concentrations. The mutant enzyme was also more sensitive to sulfhydryl reagents. These findings indicate that depending upon changes in the physical environment, the mutant ChAT may lose the normal-conformation leading to inactivation.

Evidence for binding of at least two factors, including T-rich strand-binding factor(s) to the single-stranded ARS1 sequence in Saccharomyces cerevisme

SummaryTo study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors interacting with the ARS1 region located near the centromere of chromosome IV in Saccharomyces cerevisiae. Using the gel shift assay, we found protein factor(s) which specifically bound to the T-rich strand of the region containing the core consensus and its flanking sequences in ARS1, but not to the opposite strand. We designated this factor ATS (ARS1, T-rich strand-binding factor(s)). Similar specific complexes were also detected with oligonucleotide probes specific for the H4 or C2G1 ARS. As we have previously identified another binding factor, we conclude that at least two factors bind to the single-stranded ARS1 sequence.

Practical application of microorganism for pulp wastewater treatment

Abstract Microorganism, which can grow in pulp wastewater, was isolated by the enrichment culture method. The microorganism was able to utilize most organic compounds, except lignin derivatives, in the pulp waste. Effects of environmental conditions, such as temperature, pH, concentration of wastewater, and others on the treatment have been studied in a batch culture. Maximal values in growth rate and in degree of utilization of organic materials with 10 to 40 % pulp wastewater were obtained at 37° and pH of 7.0. The extent of decomposition of organic materials in 10 % wastewater reached 85 to 90 %, as measured by reducing sugar content. In order to obtain optimal design, operation, and control for practical application, an equational expression which was based on an estimation of macromolecule synthesis in individual cell with different cell age, was adopted for calculation of growth and removal of nutrient in balanced and unbalanced growth states. The calculated values according to this model with several parameters showed satisfactory agreement with experimental data, such as number of cells, concentration of cell components, and concentration of nutrient in the wastewater, during the course of treatment.

Selective isolation of a thymine-requiring mutant of phage T4

A simple method for the selective isolation of a thymine-requiring mutant of phage T4 (thy- T4) was devised. The method is based on the fact that thy− T4 can grow in a thymine-requiring mutant of Escherichia coli in the presence of thymine and aminopterin, but wildtype T4 cannot.