Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Refined Mapping of a Gene Responsible for Fukuyama-Type Congenital Muscular Dystrophy: Evidence for Strong Linkage Disequilibrium

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common form of childhood muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy associated with an anomaly of the brain. After our initial mapping of the FCMD locus to chromosome 9q31-33, we further defined the locus within a region of ~5 cM between loci D9S127 and CA246, by homozygosity mapping in patients born to consanguineous marriages and by recombination analyses in other families. We also found evidence for strong linkage disequilibrium between FCMD and a polymorphic microsatellite marker, mfd220, which showed no recombination and a lod score of (Z) 17.49. A “111-bp” allele for the mfd220 locus was observed in 22 (34%) of 64 FCMD chromosomes, but it was present in only 1 of 120 normal chromosomes. This allelic association with FCMD was highly significant (χ² =50.7; P<.0001). Hence, we suspect that the FCMD gene could lie within a few hundred kilobases of the mfd220 locus.     

Isolation and Classification of Temperature-Sensitive Mutants of Influenza B Virus

We isolated 25 temperature-sensitive mutants of B/Kanagawa/73 strain generated by mutagenesis with 5-fluorouracil and classified them into seven recombination groups by pair-wise crosses. All mutants showed a ratio of plaquing efficiency at the nonpermissive temperature (37.5 C) to the permissive temperature (32 C) of 10^–4 or less. At 37.5 C most of group I, II, and III mutants did not produce appreciable amounts of protein, but all other group mutants were protein synthesis-positive. A group VII mutant produced active hemagglutinin (HA) and neuraminidase (NA) at the nonpermissive temperature, but Group V mutants produced only active NA and were defective in the HA molecule. The other group mutants, including group IV mutants with mutation only in the NA gene (8, 10), lacked both activities at the nonpermissive temperature. One of nine influenza B virus isolates in 1989 had EOP 37.5/32 of 1/3 × 10^–2 and belonged to recombination group VII.     

Two novel protein kinase C-related genes of fission yeast are essential for cell viability and implicated in cell shape control.

Two novel protein kinase C (PKC)-like genes, pck1+ and pck2+ were isolated from fission yeast by PCR. Both contain common domains of PKC-related molecules, but lack a putative Ca(2+)-binding domain so that they may belong to the nPKC group. Gene disruption of pck1+ and pck2+ establishes that they share an overlapping essential function for cell viability. Cells of a single pck2 deletion display severe defects in cell shape; they are irregular and sometimes pear-like instead of cylindrical. In contrast, the induced overexpression of pck2+ is lethal, producing multiseptated and branched cells. These results suggest that fission yeast PKC-like genes are involved in the polarity of cell growth control. We show that pck2 is allelic to sts6, a locus we have previously identified by its supersensitivity to staurosporine, a potent protein kinase inhibitor [Toda et al. (1991) Genes Dev., 5, 60-73]. In addition, the lethal overexpression of pck2+ can be suppressed by staurosporine, indicating that fission yeast pck1 and pck2 are molecular targets of this inhibitor.     

Requirement for PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase is lessened by the dosage increase of a novel gene sds23+.

Ubiquitin-dependent proteolysis is required for the onset of anaphase. We show that protein dephosphorylation by protein phosphatase 1 (PP1) is also essential for initiating anaphase in fission yeast. PP1 may directly or indirectly regulate the 20S cyclosome/APC (anaphase-promoting complex) required for anaphase-promoting proteolysis. Using anti-phosphopeptide antibodies, PP1 is shown to be dephosphorylated at the C-terminus, upon the onset of anaphase, for reactivation. sds23+, a novel gene, is a multicopy suppressor for mutations in PP1 and the 20S cyclosome/APC, implying that the gene dosage increase can relieve the requirement for PP1 and the cyclosome/APC for the onset of anaphase. The sds23+ gene is not essential for cell viability, but a mutant with the gene deleted cannot form colonies at 22 and 36 degrees C. In the sds23 deletion mutant, the progression of anaphase and cytokinesis is retarded and cell shape is aberrant. These defects are overcome by plasmids carrying the genes encoding subunits of the 20S cyclosome/APC or PP1. These results demonstrate functions other than promoting anaphase for the components of the 20S cyclosome/APC and also a close functional relationship of Sds23 with PP1 and 20S cyclosome/APC.     

Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation.

The Schizosaccharomyces pombe sts1+ gene, identified by supersensitive mutations to a protein kinase inhibitor, staurosporine, was isolated by complementation by the use of a fission yeast genomic library. Nucleotide sequencing shows that the sts1+ gene encodes a 453 amino acid putative membrane-associated protein that is significantly similar (26% identity) to the chicken lamin B receptor. It is also highly related (53% identity) to a budding yeast ORF, YGL022. These three proteins contain a similar hydrophobicity pattern consisting of eight or nine putative transmembrane domains. By gene disruption we demonstrate that the sts1+ gene is not essential for viability. These disruptants exhibit pleiotropic defects, such as cold-sensitivity for growth and at the permissive temperature, a supersensitivity to divalent cations and several unrelated drugs including staurosporine, caffeine, chloramphenicol, sorbitol, and SDS. Disruption of the sts1+ gene does not lead to a sensitivity to thiabendazole or hydroxyurea.     

Lysosulfatide (Sulfogalactosylsphingosine) Accumulation in Tissues from Patients with Metachromatic Leukodystrophy

We describe here a sensitive assay method for lysosulfatide (sulfogalactosylsphingosine) in human tissues using HPLC. The method involves extraction of lipids, saponification, isolation using a C18 Sep-Pak column, derivatization with o-phthalaldehyde, and detection of the fluorescent lysosulfatide using HPLC. In control subjects, a small amount of lysosulfatide was detected in the cerebral white matter (9-35 pmol/mg of protein), spinal cord (35 pmol/mg of protein), sciatic nerve (14 pmol/mg of protein), and kidney (approximately 2 pmol/mg of protein) but not in the cerebral gray matter and liver. A marked accumulation of the lipid was noted in tissues from six patients with metachromatic leukodystrophy (MLD). The concentration of lysosulfatide was high in the cerebral white matter, spinal cord, and sciatic nerve (223-1,172 pmol/mg of protein). Even in the cerebral gray matter, kidney, and liver, where lysosulfatide was hardly detected in the control sample, a considerable amount (3-45 pmol/mg of protein) accumulated in MLD patients. The concentration and distribution pattern of lysosulfatide were similar to those of galactosylsphingosine (psychosine) accumulated in patients with Krabbe disease. Therefore, the accumulation of lysosulfatide may explain the demyelination in patients with MLD, as is the case with Krabbe disease.