Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The activity of a model heterologous protein in pep4-3 mutants of Saccharomyces cerevisiae

Summary The bacterial lacZ gene was introduced into two sibling strains of Saccharomyces cerevisiae, one a wild-type strain with normal proteinase activity and the other a pep4-3 mutant strain. The pep4-3 mutation resulted in 90% reduced activity of the four major vacuolar proteinases. By comparing the activity of the lacZ gene product (-galactosidase) in both strains the degradative effect of the major vacuolar proteinases on a heterologous protein was estimated. The mutant strain with reduced proteinase activity had higher -galactosidase activity under all the test conditions. In the most productive case the pep4-3 mutant had 55% higher -galactosidase activity than the wild-type. Batch cultures of the two strains were evaluated for growth characteristics. The strain with reduced proteinase activity grew to higher optical densities than the wild-type. Upon further examination it was found that not only were the optical densities of pep4-3 cultures greater but the cell numbers were much greater than expected due to the smaller size of pep4-3 cells. It is concluded that the strain lacking vacuolar proteinases maintained increased levels of -galactosidase and is physiologically as healthy as the wild-type.Offprint requests to: J. M. Wingfield     

A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons.

Cerebellar long-term depression (LTD) is a model of synaptic plasticity in which conjunctive stimulation of parallel fiber and climbing fiber inputs to a Purkinje neuron induces a persistent depression of the parallel fiber-Purkinje neuron synapse. We report that an analogous phenomenon may be elicited in the cultured mouse Purkinje neuron when iontophoretic glutamate application and depolarization of the Purkinje neurons are substituted for parallel fiber and climbing fiber stimulation, respectively. The induction of LTD in these cerebellar cultures requires activation of both ionotropic (AMPA) and metabotropic quisqualate receptors, together with depolarization in the presence of external Ca2+. This postsynaptic alteration is manifest as a depression of glutamate or AMPA currents, but not aspartate or NMDA currents. These results strengthen the contention that the expression of cerebellar LTD is at least in part postsynaptic and provide evidence that activation of both ionotropic and metabotropic quisqualate receptors are necessary for LTD induction.     

Foliar flavonoid distribution during Spinacia chloroplast isolation

The flavonoid glycoside accumulation level was compared in class A and in class C spinach chloroplast suspensions. Class A chloroplasts (up to 97% intact) contained about 0.4% of the total flavonoid glycosides present in leaves. Further purification of 97%-intact chloroplast suspensions, through a Percoll gradient, reduced the glycoside level to less than 0.15%. On the other hand, class C chloroplasts (100% broken plastids) contained between 10 to 30 times more flavonoids than intact Percoll purified chloroplasts. These results indicate that chloroplasts could bind vacuolar glycosides during their period of isolation. This hypothesis was confirmed by controlled contamination experiments using a cell-free supernatant as a source of vacuolar glycosides. Furthermore, the level of flavonoids in chloroplasts could be decreased to a level close to that obtained in intact Percoll purified chloroplasts by washing with soluble polyvinylpyrolidone. The results presented in this paper demonstrate the importance of maintaining the physiological integrity of plastids during the course of organelle isolation when investigating flavonic compartmentation in leaves.     

Regulation of thymidine kinase synthesis during the cell cycle of Physarum polycephalum by the heat-sensitive system which triggers mitosis and S phase

Temperature shifts from 22 to 32 °C perturb one of the systems responsible for mitosis triggering in the plasmodia of Physarum (Myxomycetes). In order to determine if the same regulatory mechanism could also be involved in some other cell cycle events, the effects of temperature shifts on the peak of thymidine kinase (EC 2.7.1.21, ATP : thymidine 5′-phosphotransferase) synthesis have been studied. At 22 °C, the increase in thymidine kinase (tdk) activity begins shortly before mitosis and is thus always associated with the end of the G2 phase, the mitosis and the beginning of the S phase. The consequences of temperature shifts depend upon their position in the cell cycle. In all cases, a peak of tdk occurs concomitantly with the 32 °C mitosis. But, when the temperature shift is applied 90-15 min before the control metaphase at 22 °C, another peak of tdk is observed at 32 °C in absence of mitosis, but at the same time as the control mitosis at 22 °C. These results indicate that the increase in the synthesis of tdk is controlled by the heat-sensitive regulatory system which plays a role in the onset of mitosis and S phase. We further suggest that the increase in the synthesis of tdk and the triggering of mitosis are both controlled by the amount of a heat-sensitive effector. But the former takes place when the amount of the effector reaches a critical value lower than the value necessary to trigger mitosis.     

Decouverte de Septorella brachycera grambast et de Septorella ultima Grambast (Charophytes,Clavatoracees) dans le Maestrichtien superieur des Petites Pyrenees. Consequences stratigraphiques

The discovery of an assemblage of Clavatoraceae including Septorella brachycera and S. ultima in the “Marnes d'Auzas” (upper Maestrichtian) of the Petites Pyrénées constitutes the first element of comparison between the upper Cretaceous of the Petites Pyrénées and Provence, but creates a problem with regard to the subdivision of the provencial Rognacian, based, in part, on the superposition of these two species.     

A study of the oxidation of butan-1-ol and propan-2-ol by nicotinamide-adenine dinucleotide catalysed by yeast alcohol dehydrogenase.

1. The kinetics of oxidation of butan-1-ol and propan-2-ol by NAD+, catalysed by yeast alcohol dehydrogenase, were studied at 25 degrees C from pH 5.5 to 10, and at pH 7.05 from 14 degrees to 44 degrees C, 2. Under all conditions studied the results are consistent with a mechanism whereby some dissociation of coenzyme from the active enzyme-NAD+-alcohol ternary complexes occurs, and the mechanism is therefore not strictly compulsory order. 3. A primary 2H isotopic effect on the maximum rates of oxidation of [1-2H2]butan-1-ol and [2H7]propan-2-ol was found at 25 degrees C over the pH range 5.5-10. Further, in stopped-flow experiments at pH 7.05 and 25 degrees C, there was no transient formation of NADH in the oxidation of butan-1-ol and propan-2-ol. The principal rate-limiting step in the oxidation of dependence on pH of the maximum rates of oxidation of butan-1-ol and propan-2-ol is consisten with the possibility that histidine and cysteine residues may affect or control catalysis.     

DEVELOPMENT OF THE EPIPHYLLOUS INFLORESCENCE OF HELWINGIA JAPONICA (HELWINGIACEAE)

The inflorescence of Helwingia japonica (Thunb.) Dietr. is initiated adjacent to the leaf axil on the adaxial side of the base of a leaf primordium during its second plastochron. The inflorescence which develops from the resulting primordium comes to be situated on the midrib of the mature fertile leaf, through the action of a basal, intercalary meristem. In fertile leaves this meristem develops beneath, as well as above, the insertion of the inflorescence primordium on the leaf primordium. The same meristem is present in sterile leaves as well. A separate, adaxial vascular bundle departs from the leaf trace in the base of the petiole and leads to the inflorescence, in the mature fertile leaf. This adaxial vascular bundle is absent in sterile leaves. It is argued that the vascular anatomy does not conclusively confirm the hypothesis that the epiphyllous inflorescence is the congenital fusion product of a leaf and an axillary inflorescence. Instead, it is suggested that the interplay of changes in the position of primordium initiation, and intercalary growth, offers an ontogenetic explanation of the situation, which in turn may be related to the phylogeny of the species in question. It appears to be misguided and futile to look for homologies (i.e., 1:1 correspondences) between fertile and sterile leaves, since 1:1 correspondences do not exist in this case.