Search

Maize genomic fragments containing the regulatory and coding regions of a zein gene for a low size class 23‐kd protein have been inserted in an interspecific Escherichia coli‐Saccharomyces cerevisiaeexpression vector in different constructions. The presence of the inducible GAL1‐10upstream activation site (UAS) allows us to regulate differentially by carbon sources the transcription of the zein gene both under the plant promoter and under the yeast CYC‐1promoter. We found that the zein promoter region is properly recognized at the correct transcription start, while different termination points occur during transcription. The yeast UAS was also shown to function as a typical eukaryotic enhancer regardless of its distance or orientation with respect to the plant promoter. Yeast cells transformed by a plasmid containing a zein sequence fused to a short piece of the CYC‐1gene produced a fused polypeptide, of expected mol. wt, in variable amount from 0.2 to 5% depending on the growth phase conditions.

The localization of acrosin (EC 3.4.21.10) activity in mammalian spermatozoa was investigated by use of the fluorescent site-directed acrosin inhibitor, dansylalanyllysylchloromethyl ketone (DALCK). Fluorescence microscope preparations revealed, after the spermatozoa were subjected to a specific treatment, that acrosin activity is confined specifically to the inner acrosomal membrane (IAM). Spectrofluorometric and fluorescence polarization investigations verified that the fluorescent probe, once it is specifically bound to the treated spermatozoa, lies in a very hydrophobic environment and shows a remarkable reduction of rotational freedom. These results are compatible with the hypothesis that, under the experimental conditions used, active acrosin is tightly bound to the IAM and that the "specificity site" of the acrosin-active center is probably of a highly hydrophobic nature.

Recently the polyphosphoinositides (PI) turnover has been related to the control of growth and cell cycle also in Saccharomvces cerevisiae, and the RAS2and RAS1gene products have been shown to be involved in the stimulation of PI turnover in G0/G1 arrested yeast cells. Here we show that addition of glucose to previously glucose-starved cells, stimulates the PI turnover with fast kinetics also in yeast cells that were not arrested in the G0/G1 phase of the cell cycle. In addition PI turnover is equally stimulated in temperature sensitive cdc25-1and cdc25-5strains at restrictive temperature, as well as in ras1, ras2strain, suggesting that PI turnover stimulation is not dependent on the CDC25-RASmediated signal transduction pathway.

A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented. At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type. The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C. Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type. Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media. The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media. CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway. Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.

Models able to describe the events of cellular growth and division and the dynamics of cell populations are useful for the understanding of control mechanisms and for theoretical support for the automated analysis of flow cytometric data and of cell volume distributions. This paper reports on models that have been developed by the Authors with this aim, describing in a rather unitary frame the cell cycle of eukaryotic cells, like mammalian cells and yeast, and of prokaryotic cells.

Several biological parameters have been measured in populations of budding yeast growing in a glucose-limited chemostat, both in steady state and during perturbed growth conditions. The population structure has been determined with a segregated data analysis performed on protein distribution and on volume distributions. A new structured model for budding yeast population is presented, in which the entire system is described only in terms of critical cell mass parameters. This approach allows to integrate in a fairly simple way the population model with the bioreactor performance and to describe transient states of growth.

We used two different anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins in a germ cell, the boar spermatozoon. Ejaculated spermatozoa presented three major polypeptides, of Mr 43000, 40000 and 36000, respectively, that were immunorecognized on Western blots. These proteins were selectively enriched in the Triton X-100-soluble fraction and were released neither after an A23187-induced acrosome reaction nor after sperm homogenization. These findings suggest the presence of the three proteins in plasma membrane regions not involved in the acrosomal vésiculation. When epididymal boar spermatozoa were investigated, Western blot analysis of the detergent-soluble fractions from caput sperm did not reveal any detectable 43000, 40 000 and 36 000 Mr proteins cross-reactive with phosphotyrosine antibodies, whereas the detergent-soluble fractions from cauda sperm yielded very strong immunoreactive signals. Labelling of freshly ejaculated spermatozoa with [32P] orthophosphate yielded a wide range of labelled phosphoproteins, but we failed to identify specific tyrosine phosphorylation under the experimental conditions employed. Tyrosine phosphorylation occurred when specific synthetic polymers of tyrosine, commonly used for studying tyrosine protein kinases, were assayed as substrates against both the Triton-soluble and Triton-insoluble sperm fractions. This is the first immunological and biochemical report on the presence of phosphotyrosine-containing proteins and protein kinase activities that phosphorylate tyrosine residues in a mammalian mature spermatozoon.

Recent technological developments allow to measure segregated parameters of cell populations (cell size; protein, DNA, RNA content) on significant samples of the population (10 - 10 cells). To exploit these information and to develop control approaches based on these parameters, which are more directly related to the growth metabolism, it is necessary to use structured mathematical models of cell population. In the present paper a structured model able to describe cell growth and unequal division of budding yeast Saccharomyces cerevisiae is presented and from it age and size distributions for populations in exponential steady states of growth are derived. In several conditions the experimental protein distributions, obtained by flow cytometry, are shown to accurately fit those predicted by the model and to contain relevant information on the conditions of growth of the microbial biomass. These findings may have a predictive value on process dynamics being very sensitive to changes in synthesis and division rates, and may find a utilization in the development of new control approaches for microbial reactors.

Two C-terminal fragments (334 and 509 amino acid residues) of CDC25, a Saccharomyces cerevisiaeGDP/GTP exchange factor, and the RAS2 protein were purified from E. coli, using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length RAS2 (42kDa) into 37 and 30kDa forms. Of the two CDC25 fragments containing the conserved catalytic domain, only CDC25-509 could enhance the guanine nucleotide exchange on RAS2. Comparison of the activities of RAS2-42/37kDa and RAS2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by CDC25-509. RAS2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. CDC25-509 displayed a higher specific activity than the catalytic domains of the two CDC25-like proteins: S. cerevisiaeSDC25 and mouse CDC25Mm.

The timing of the nuclear division cycle in Neurospora crassa was studied by inhibiting DNA synthesis with hydroxyurea added to exponentially growing cultures at concentrations that do not inhibit cytoplasmic growth. After the addition of hydroxyurea only the nuclei that have passed the .S’-phase divide, and therefore by counting the increase in the average number of nuclei per hypha, the stage in the cycle in which completion of S phase occurred was determined in different nutritional conditions of exponential growth. The stage at which DNA replication terminates is different in different conditions of exponential growth; however, when the length of Gt+ M phases is calculated a constancy of these phases is shown. Our data indicate that in N. crassa the coordination between cytoplasmic growth and the nuclear division cycle is achieved mainly through a growth-rate-dependent expansion of the G, phase, according to a sizer-plus-timer model of control of the nuclear division cycle.

Treatment with picolinic acid blocked Neurospora crassa nuclei in G1, and recovery from the treatment allowed a synchronous wave of deoxyribonucleic acid synthesis to occur. Nuclei, which appeared as compact globular bodies during the period of blockage, assumed a ring shape during the following S phase, which was also maintained in the G2 phase. The proportion of compact globular nuclei was much higher in hyphae growing at lower rates, whereas that of ring nuclei increased when the hyphae were growing at higher rates. Horseshoe nuclei (probably mitotic nuclei) and double ring nuclei were also observed in growing hyphae, but their frequencies were low and fairly independent of the rate of growth. The length of the S phase of the Neurospora nuclear division cycle was determined to be about 30 min. From the frequencies of the phase-specific nuclear shapes, the durations of the G1 phase and the combined S plus G2 phases were calculated. The results showed that variations in the growth rates of the mycelia were mainly coupled with variations in the G1 phase of the nuclear division cycle. For mycelia growing in minimal sucrose, the lengths of all of the phases of the nuclear division cycle were estimated.

In exponentially growing cultures of Neurospora crassa, the basal rate of protein degradation increases as the constant of the rate of growth decreases, so that in slow growing cells (mu = 0.13) the rate of protein degradation is about 25% of the rate of protein accumulation. During glucose starvation and shift-down transition of growth, the rate of protein degradation is greatly enhanced, and a moderate reduction (about 30%) of the ATP level is observed. Treatment of glucose-starved cells with 2-deoxyglucose reduces the ATP content by 70% and blocks protein degradation. The addition of cycloheximide, given at the onset of glucose starvation, prevents the enhancement of protein degradation; instead cycloheximide is without effect if added when proteolysis has already started. At a supraoptimal temperature (42 degrees C) the basal rate of protein degradation is not stimulated, contrary to the behavior observed in bacteria. Guanosine nucleotides, which appear to have a regulatory role for protein degradation in bacteria, are not found in N. crassa.