From oocyte maturation to the in vitro cell cycle: the history of discoveries of Maturation-Promoting Factor (MPF) and Cytostatic Factor (CSF)

This article briefly reviews the classical cell cycle studies using oocytes and zygotes of mainly amphibians in the past century. The discussions are focused on the investigations into the cytoplasmic factors that regulate meiosis during oocyte maturation and the initiation of mitosis during fertilisation, which were carried out in the author's lab between 1967 and 1987. This chronicle traces the development of the problems and the direction in which their solutions were attempted in the course of these investigations. The author tries to answer the following questions: why he decided to study oocyte maturation, how he discovered progesterone as a maturation-inducing hormone, how he discovered and characterised the cytoplasmic regulators of the cell cycle, Maturation-Promoting Factor (MPF) and Cyto-Static Factor (CSF), and how he invented the method of observing cell cycle processes in a cytoplasmic extract in vitro.     

Effect of calcium and F-actin on the rate of SH2 modification in skeletal myosin

The rate constant of modification of a specific thiol group, SH₂, with N-ethylmaleimide (NEM) has been used to estimate the conformational change in the local area containing SH₂ (SH₂ region) of skeletal myosin as a structural probe. The rate of Mg²⁺-ATP-induced SH₂ modification of subfragment-1 (S-l) isozymes was regulated by Ca²⁺ in the pCa range below 6.4 and was not regulated in the pCa range above 6.4. No substantial difference between S-1 containing alkali light chain, A1, (S-1(A1)) and S-1 containing alkali light chain, A2, (S-1(A2)) was observed in the Ca²⁺-dependent rate of SH₂ modification. Due to the presence of this Ca²⁺ regulation in myosin (absence in S-1 isozymes) in the pCa range above 6.4, absence of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) light chain in S-1 isozymes, and high affinity of Ca²⁺ for DTNB light chain, this Ca²⁺ regulation in the pCa range above 6.4 is possibly related to the Ca²⁺ binding to DTNB light chain. F-Actin, which is entirely free from tropomyosin and troponin, enhanced the rate of Mg²⁺-ATP-induced SH₂ modification of S-1 isozymes equally and of myosin, and reduced the Ca²⁺ sensitivity with an increase in F-actin concentration.     

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells

Summary The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH₂-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH₂-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.     

Distribution of 59- and 55-kDa Catalase in Dark- and Light-grown Pumpkin and Various Other Plant Tissues

The catalase molecule in germinating pumpkin cotyledons is synthesizedas a precursor (59-kDa) form, whose relative molecular massis larger than the mature enzyme (55-kDa). Although both typesof molecules are localized in the microbodies, the 59-kDa specieshas been shown to be present predominantly in the leaf peroxisomesisolated from green cotyledons, while the 55-kDa species ispredominantly in the glyoxysomes from etiolated cotyledons [Yamaguchiet al. (1984) Proc. Natl. Acad. Sci. USA, 81: 4809]. We examinedthe distribution of the 59- and 55-kDa catalase molecules indark- and light-grown tissues of pumpkin seedlings as well asin other plant species, using the immunoblotting technique.The ratios of the 59- and 55-kDa catalase species differed inthe pumpkin tissues examined. Light interferes with the conversionof the 59-kDa precursor to the 55-kDa form, especially in thecotyledons. The effect of light was less pronounced in the rootsand hypocotyls, indicating that the light regulation of theconversion is tissue-specific. Dark- and light-grown cotyledonsfrom cucumber and watermelon seedlings showed a similar lightregulation, suggesting that cucurbitaceous plants possess similarlight-regulatory mechanism. From the analysis of catalase proteinfrom various plant tissues, a limited correlation between molecularforms of catalase and different microbody populations was observed. (Received September 6, 1986; Accepted December 4, 1986)     

Metabolic switching patterns provide definitive evidence of substrate-specificity and delineate subtle differences in positioning at the aromatase active site [Poster 06]

We found that extensive metabolic switching can be triggered in aromatization. Up to 20–35% of (19-³H₃)-labeled substrate is diverted to non-estrogenic product, namely 1β- and 2β-hydroxy androgens, negating usefulness for [³H]water assays. The strikingly substrate-specific metabolite patterns observed reflect positioning at the active site, and the large kinetic isotope effects involved. This switching is unusual in that it involves: 1) tritium labeling, 2) a multistep enzymic process, and 3) the potential unraveling of an enzymic mechanism.     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Taxonomic studies ofAsarum sensu lato

The floral vascular anatomy of 12 species representing each ofAsarum s. str.,Asiasarum, Geotaenium, Heterotropa andHexastylis are compared to clarify intergeneric relationships. The five genera have basically similar structures in floral morphology and vasculature, and consistently have a six-carpelled compound ovary and the associated similar placental vasculature. They show, however, a significant difference in the position and the constituent of the “ventral” carpellary bundles in the placental axis betweenAsiasarum-Heterotropa-Hexastylis andAsarum-Geotaenium. InAsiasarum, Heterotropa andHexastylis the ventral bundles of each carpel are basically free and antilocular as expected in the least specialized compound ovary of angiosperms; in contrast, inAsarum andGeotaenium the ventral carpellary bundles are antiseptal and heterogenous (i.e., formed by the lateral fusion of ventral bundles of adjacent carpels). Shared probable apomorphic floral vasculature, as well as shared single style-column, suggests the closest mutual relationships betweenAsarum andGeotaenium. In terms of floral morphology and anatomy,Asiasarum, Heterotropa andHexastylis retain plesiomorphies. Possible chromosomal evolution in the related genera is also discussed.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.