Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin

The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.     

Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme

We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.     

Coordinate induction of alcohol dehydrogenase 1, aldolase, and other anaerobic RNAs in maize

Anaerobiosis results in the selective synthesis of a particular set of polypeptides in the maize root including the two alcohol dehydrogenases (Sachs, M. M., Freeling, M., and Okimoto, R. (1980) Cell 20, 761-768), pyruvate decarboxylase (Wignarajah, K., and Greenway, H. (1976) New Phytol. 77, 575-584; Laszlo, A., and St. Lawrence, P. (1983) Mol. Gen. Genet. 192, 110-117), glucose phosphate isomerase (Kelley, P. M., and Freeling, M. (1984) J. Biol. Chem. 259, 673-677) and aldolase (Kelley, P. M., and Freeling, M. (1984) J. Biol. Chem. 259, 14180-14183). This report describes the identification and characterization of cDNA clones to five different mRNA species induced upon anaerobic shock. Immunoprecipitation of hybrid-selected translation polypeptides has determined the identity of the cDNA clone for fructose-1,6-diphosphate aldolase mRNA. Quantitative hybridization analysis of anaerobic mRNAs using the cDNA clones has shown that there is not a simultaneous accumulation of anaerobic mRNAs. Upon reintroduction of air, the anaerobic mRNAs disappear rapidly and at approximately the same rate. A translocation line that generates progeny that contain 1, 2, and 3 doses of the long arm of chromosome one (1L) allowed us to test for clustering of the anaerobic genes; two of the anaerobic genes tested do not reside with Adh 1 and Phi 1 on the long arm of chromosome 1.     

Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph

A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment.     

Conformational transitions of thioredoxin in guanidine hydrochloride

Spectral and hydrodynamic measurements of thioredoxin from Escherichia coli indicate that the compact globular structure of the native protein is significantly unfolded in the presence of guanidine hydrochloride concentrations in excess of 3.3 M at neutral pH and 25 degrees C. This conformational transition having a midpoint at 2.5 M denaturant is quantitatively reversible and highly cooperative. Stopped-flow measurements of unfolding in 4 M denaturant, observed with tryptophan fluorescence as the spectral probe, reveal a single kinetic phase having a relaxation time of 7.1 +/- 0.2 s. Refolding measurements in 2 M denaturant reveal three kinetic phases having relaxation times of 0.54 +/- 0.23, 14 +/- 6, and 500 +/- 130 s, accounting for 12 +/- 2%, 10 +/- 1%, and 78 +/- 3% of the observed change in tryptophan fluorescence. The dominant slowest phase is generated in the denatured state with a relaxation time of 42 s observed in 4 M denaturant. Both the slowest phase observed in refolding and the generation of the slowest phase in the denatured state have an activation enthalpy of 22 +/- 1 kcal/mol. These features of the slowest phase are compatible with an obligatory peptide isomerization of proline-76 to its cis isomer prior to refolding.     

Androgen-induced alterations in vocalizations of femaleXenopus laevis: modifiability and constraints

Summary We examined effects of exogenous androgen (testosterone and dihydrotestosterone) on vocalizations of ovariectomized, adult female South African clawed frogs,Xenopus laevis. When paired with sexually active males, all ovariectomized females exhibited ticking, the unreceptive or release call. Ticking consists of low amplitude, regularly spaced clicks with a mean interclick interval of 154 ms. When androgen-treated and paired with sexually active males, these ovariectomized females also exhibited an aberrant call (atypical ticking) in which click multiples replaced the single clicks of ticking. Mean ICI's for atypical ticking were 37 ms for click doublets and 22 ms for click quadruplets. Androgen treatment decreased the total time spent vocalizing (typical and atypical ticking) by ovariectomized females.All androgen-treated females were then tested repeatedly with sexually receptive females in an attempt to elicit the male-typical vocalization, mate calling. Six of 17 females did not vocalize at all, even when gonadotropin injected. Eight females gave rapid (mean ICI, 36 ms) trains of clicks in an irregular temporal pattern (tick-like calls). Three females gave brief trills with alternating fast and slow components. Comparison of mate calllike vocalizations of androgen-treated females to mate calling of males reveals that calls in females are considerably shorter in duration (female: 0.32 min versus male: 45 min) and slower in tempo (ICI's; fast trill, female: 21 ms, male: 14 ms; slow trill, female: 36 ms, male: 28 ms). Incomplete masculinization of the vocal pattern of females by androgen treatment in adulthood may be due to developmental constraints on the modifiability of the neurons and muscles responsible for calling.Abbreviations C cholesterol - DHT dihydrotestosterone - HCG human chorionic gonadotropin - IBI interburst interval - ICI interclick interval - ovx ovariectomized - T testosterone     

Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli

Escherichia coli K-12 mutants constitutive for the synthesis of the enzymes of fatty acid degradation (fad) synthesize significantly less unsaturated fatty acid (UFA) than do wild-type (fadR+) strains. The constitutive fadR mutants synthesize less UFA than do fadR+) strains both in vivo and in vitro. The inability of fadR strains to synthesize UFAs at rates comparable to those of fadR+ strains is phenotypically asymptomatic unless the fadR strain also carries a lesion in fabA, the structural gene for beta-hydroxydecanoyl-thioester dehydrase. Unlike fadR+ fabA(Ts) mutants, fadR fabA(Ts) strains synthesize insufficient UFA to support their growth even at low temperatures and, therefore, must be supplemented with UFA at both low and high temperatures. The low levels of UFA in fadR strains are not due to the constitutive level of fatty acid-degrading enzymes in these strains. These results suggest that a functional fadR gene is required for the maximal expression of UFA biosynthesis in E. coli.     

Mutagenesis in Oocytes of DROSOPHILA MELANOGASTER. I. Scheduled Synthesis of Nuclear and Mitochondrial DNA and Unscheduled DNA Synthesis

As a model system for studying mutagenesis, the oocyte of Drosophila melanogaster has exhibited considerable complexity. Very few experiments have been conducted on the effect of exposing oocytes to chemical mutagens, presumably due to their lower mutational response relative to sperm and spermatids. This lower response may be due either to a change in probability of mutation induction per adduct due to a change in the type of DNA repair or to a lower dose of the mutagen to the female germ line. To study molecular dosimetry and DNA repair in the oocyte, the large number of intracellular constituents (mtDNA, RNA, nucleic acid precursors and large quantities of proteins and lipids) must be separated from nuclear DNA. In this paper we present results showing reliable separation of such molecules enabling us to detect scheduled nuclear and mitochondrial DNA synthesis. We also, by understanding the precise timing of such events, can detect unscheduled DNA synthesis (UDS) as a measure of DNA repair. Furthermore, by comparing the UDS results in a repair competent (Ore-R) vs. a repair deficient (mei-9^L1 ) strain, we have shown the oocyte capable of DNA repair after treatment with ethyl methanesulfonate (EMS). We conclude that the important determinant of mutation induction in oocytes after treatment with EMS is the time interval between DNA alkylation and DNA synthesis after fertilization, i.e., the interruption of continuous DNA repair.