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H Heslot

Publications and source records attributed to H Heslot.

At least 19 recordsLinked to original sources
PubMed1998

Artificial fibrous proteins: a review.

Several kinds of natural fibrous proteins have been chosen as models: silk fibroin from Bombyx mori, silks from various species of spiders and collagens. The dragline silk of the spider Nephila clavipes is able to stretch by 30% before breaking and has a high tensile strength. It is stronger per unit weight than high tensile steel. Although the partial sequence of the two components of dragline silk is known, its molecular structure is still far from being clearly established. It is however demonstrated that it contains beta-sheet crystals composed of polyalanine residues. Artificial fibrous proteins have been prepared in vivo using either Escherichia coli or the yeast Pichia pastoris. As these proteins contain repetitive sequences, there is a risk of deletion at the DNA level. This difficulty has been solved by making use of the genetic code degeneracy. One group has successfully synthesized silk-like polymers; prolastin polymers containing both silk-like and elastin-like blocks; proNectin polymers containing the RGD triplet coming from fibronectin and able to fix numerous mammalian cell types; and synthetic collagen analogs. Some of these polymers have been spun into fibers that, up-to-now, do not display any measurable molecular orientation. Another group has studied artificial fibrous proteins able to form beta-sheet crystals of defined thickness and bearing functional groups at their surface, for instance Glu residues, selenomethionine or p-fluorophenylalanine. Apart from university laboratories, a venture capital society, an industrial research center and a US army research center are quite active in this field. A number of patents has been deposited.

Amino Acid Sequence↗
PubMed1989

Product of the Lactococcus lactis gene required for malolactic fermentation is homologous to a family of positive regulators.

Malolactic fermentation is a secondary fermentation that many lactic acid bacteria can carry out when L-malate is present in the medium. The activation of the malolactic system in Lactococcus lactis is mediated by a locus we call mleR. Induction of the genes necessary to perform malolactic fermentation occurs only in bacteria with a functional copy of mleR. The mleR gene consists of one open reading frame capable of coding for a protein with a calculated molecular mass of 33,813 daltons. The amino acid sequence of the predicted MleR gene product is homologous to that of positive activators in gram-negative bacteria: LysR, IlvY gene products of Escherichia coli, MetR, CysB of Salmonella typhimurium, AmpR of Enterobacter cloacae, NodD of Rhizobium sp., and TrpI of Pseudomonas aeruginosa.

Amino Acid Sequence↗
PubMed1988

Construction of an expression vector for the fission yeast Schizosaccharomyces pombe.

We have isolated and characterized a S. pombe promoter using a functional heterologous gene product assay. Random S. pombe genomic fragments were cloned upstream from the promoterless 'lacZ gene and tested in vivo for their efficiency to promote expression of the beta-galactosidase protein in the fission yeast. An efficient S. pombe promoter called 54/1 was isolated and shown to drive up to 5% of total protein synthesis as beta-galactosidase. The structure and nucleotide sequence of this promoter were determined, precise localization of its mRNA transcriptional start points established. Translational fusion of the Pseudomonas putida XylE gene with the 54/1 gene was shown to allow expression of catechol oxidase activity in S. pombe. An expression vector suitable for transcriptional fusions was then constructed from engineered 54/1 promoter sequences and used to drive expression of the E. coli Tn5 ble gene, thus confering resistance to the fission yeast against bleomycin and phleomycin antibiotics.

Amino Acid Sequence↗
PubMed1988

Role of malolactic fermentation in lactic acid bacteria.

Although decarboxylation of malate to lactate by malolactic enzyme does not liberate biologically available energy (e.g., ATP, NADH), the growth rate of many malolactic bacteria is greatly enhanced by malolactic fermentation. The deacidification of the medium due to malate dissipation cannot fully account for this situation. The chemiosmotic theory postulates that another form of energy could generated by translocation of protons through the membrane coupled to end-product efflux. Konings et al. showed that this theory is indeed applicable to lactate efflux in Streptococcus cremoris at pH 7.0. A similar mechanism could account for the observed increased activity in malolactic bacteria. The study in wild type and mutant strains of Streptococcus lactis unable to carry out malolactic fermentation led us to the following conclusions: (1) under glucose non-limiting conditions, malolactic fermentation helps to maintain pH of the medium at a certain level; (2) during glucose limited growth, malolactic fermentation could be coupled with an energetic process independent from that mentioned above.

Energy Transfer↗
PubMed1987

Selection of Streptococcus lactis Mutants Defective in Malolactic Fermentation.

An enrichment medium and a new sensitive medium were developed to detect malolactic variants in different strains of lactic bacteria. Factors such as the concentration of glucose and l-malate, pH level, and the type of indicator dye used are discussed with regard to the kinetics of malic acid conversion to lactic acid. Use of these media allowed a rapid and easier screening of mutagenized streptococcal cells unable to ferment l-malate. A collection of malolactic-negative mutants of Streptococcus lactis induced by UV, nitrosoguanidine, or transposonal mutagenesis were characterized. The results showed that several mutants were apparently defective in the structural gene of malolactic enzyme, whereas others contained mutations which may either inactivate a putative permease or affect a regulatory sequence.

Journal Article↗
PubMed1986

Estimation of compartmentation of lysine inside the cells of Yarrowia lipolytica.

We studied the compartmentation of lysine in vivo by the tracer method of Subramanian et al. (J. Bacteriol. 115 (1973) 284-290). To calculate the size of the different lysine pools and lysine fluxes inside the cell, a mathematical model was designed enabling quantitative estimation of these data from experimental measurements. This technique was applied to a lysine-accumulating strain, 15901.7, and to its low lysine pool derivatives, ply-1, ply-4 and ply-9, which were selected for their low polyphosphate pools. In the mother strain, 15901.7, it appeared that lysine was stored mainly in a non-cytoplasmic compartment. In the mutants, the efflux from this compartment was higher and accounted for the higher percentage of lysine in the cytoplasm of these strains. Simulation with calculated parameters fitted the experimental curves very well. This was good evidence for the pertinence of the model. The biological significance of these results are discussed.

Cytoplasm↗
PubMed1983

Transformation of Kluyveromyces lactis by killer plasmid DNA.

Some strains of Kluyveromyces lactis contain two linear double-stranded DNA plasmids, k1 and k2. The presence of the two plasmids confer on the cell a "killer" character, due to the production of a toxin that kills the sensitive cells. We have used one of these linear DNA molecules as a gene vector to transform K. lactis cells. Hybrid plasmids containing parts of the k1 plasmid and the URA3 gene of Saccharomyces cerevisiae have been constructed. We have found that the hybrid plasmids were able to transform a uracil-requiring strain of K. lactis (uraA mutant) to a prototrophic form. The transformed phenotype cosegregated with the hybrid plasmids. The transforming plasmids contained the sequence of one or both ends of the linear k1 DNA, but they were integrated into a circular molecule.

Ascomycota↗
PubMed1982

Wild-type and mutant forms of homoisocitric dehydrogenase in the yeast Saccharomycopsis lipolytica.

Homoisocitric dehydrogenase (EC 1.1.1.155) has been purified 525-fold from the yeast Saccharomycopsis lipolytica with a yield of 25%. The preparation was judged to be homogeneous by electrophoresis under denaturing and non-denaturing conditions and by isoelectric focusing; it consisted of a single protein with molecular weight of 48000. In the presence of homoisocitric acid, a higher molecular weight was observed, suggesting a dimeric structure for the native enzyme. Complementing mutants devoid of homoisocitric dehydrogenase activity mapped at two closely linked loci (lys9 and lys10). Lys10 mutants displayed NAD-reducing activity, whereas lys9 mutants retained some carboxylating activity. Our results are best explained by the assumption that the active enzyme is a dimer of identical subunits involved in successive dehydrogenation and decarboxylation steps.

Alcohol Oxidoreductases↗
PubMed1979

[Expression of a bacterial gene, cloned in the yeast, Saccharomyces cerevisiae].

Vectors allowing cloning of foreign D.N.A. in the yeast Saccharomyces cerevisiae have been recently described. We have introduced in this yeast the lac Z gene of the bacteria Escherichia coli. An active beta-galactosidase, which is absent in the recipient strain, has been detected in transformed yeast. We thus conclude that the bacterial lac Z gene is expressed in yeast. We further showed that the enzyme found in the transformed yeast is identical to the bacterial enzyme with respect to size and immunological criteria.

Cloning, Molecular↗
PubMed1979

Genetic control of lysine permeases in Saccharomycopsis lipolytica.

In order to obtain strains of Saccharomycopsis lipolytica impaired in the active transport of L-lysine, mutants resistant to a mixture of L-canavanine, L-4-5-transdehydrolysine and L-S-amino ethylcysteine, taken either all three or two by two, were isolated. These compounds were shown previously to be competitive inhibitors of L-lysine uptake. The resistance patterns and excretion capacity of the mutants were established. All mutants behaved as monogenic. Recombination tests indicated that four genes at least were involved. All mutants were impaired in both high and low affinity L-lysine transport systems. Several hypotheses on the functions of these genes are put forward and discussed.

Ascomycota↗
PubMed1979

Evidence for mutations in the structural gene for homocitrate synthase in Saccharomycopsis lipolytica.

Eight strains devoid of homocitrate synthase activity were found among lysine requiring mutants of the yeast Saccharomycopsis lipolytica. Genetic analysis of these strains showed that they were all affected at the same locus LYS 1. Three lines of evidence suggest that this locus defines a structural gene for homocitrate synthase. First, the mutations show various degrees of intragenic complementation; it could be shown in some cases that the hybrid enzyme formed in vivo displayed modified properties in vitro. Second, reversion of some of these mutations can result in a modified enzyme (desensitized). Third, a feedback mutant of homocitrate synthase was directly isolated from the wild type strain, and shown to carry a single mutation at of near LYS 1. We also present here the first attempts at genetic fine mapping in Saccharomycopsis lipolytica.

Genes↗
PubMed1979

High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid.

By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block. In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain. The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.

Chimera↗
PubMed1979

General and lysin specific control of saccharopine dehydrogenase levels in the yeast Saccharomycopsis lipolytica.

Lysine supplementation of the growth medium of a wild type strain of the yeast Saccharomycopsis lipolytica specifically results in saccharopine dehydrogenase repression. Starvation of the strain for histidine triggers a general depression of various histidine, leucine, arginine and lysine biosynthetic enzymes, including saccharopine dehydrogenase. These two types of control, specific and general, act independently on saccharopine dehydrogenase expression, since mutants which fail to respond to the specific control still are sensitive to the general one. These mutants were first selected as unable to catabolize lysine, suggesting that a link may exist between saccharopine dehydrogenase specific regulation and activity of the catabolic pathway.

Ascomycota↗
PubMed1978

Physiology of lysine permeases in Saccharomycopsis lipolytica.

Two active lysine transport systems were detected in Saccharomycopsis lipolytica. No excretion of lysine out of the cells could be obtained, even by chasing with L-lysine or by poisoning with sodium azide. The kinetic properties of one of the permeases, the high-affinity lysine permease, were studied in detail. Its Km was 1.91 +/- 0.23 X 10(-5) M. It proved highly specific, the only potent competitive inhibitors being (i) arginine and its analogs L-canavanine and L-ornithine, and (ii) the lysine analogs L-5 aminoethylcysteine and L-4,5-transdehydrolysine. It is suggested that the high-affinity lysine permease is common to L-lysine, L-ornithine, and L-arginine. The other amino acids tested behaved as noncompetitive inhibitors. The variation of uptake during a growth cycle was studied on ammonia-rich, ammonia-poor, and ammonia-free media. In each case, the uptake exhibited a peak in the early exponential growth phase. No new permease activity was detected during the lag phase or the stationary phase. Ammonia ions competitively inhibited the uptake and also decreased the Vmax value.

Amino Acids↗
PubMed1977

Recombination after protoplast fusion in the yeast Candida tropicalis.

Candida tropicalis protoplasts obtained by snail enzyme treatment were induced to fuse by the use of polyethylene-glycol. Heterokaryons formed by two auxotrophic strains were selected by complementation on minimal medium. These heterokaryons were unstable and readily dissociated into their nuclear components. Under appropriate conditions, the parental nuclei of an heterokaryon fused. The homokaryon so obtained was unstable and segregated into various types of auxotrophic and prototrophic recombinants.

Candida↗
PubMed1976

A kinetic study of homocitrate synthetase activity in the yeast Saccharomycopsis lipolytica.

1. A rapid method for estimating the activity of the first enzyme of lysine biosynthesis in yeasts (acetyl-coenzyme A: 2-ketoglutarate C-acetyl transferase, EC 4.1.3.21) is described. 2. In the wild type strain, the fixation of one substrate, S-acetyl coenzyme A, shows sigmoidal saturation kinetics. The initial rate experiments indicate that the reaction obeys an ordered mechanism, 2-ketoglutaric acid binding before S-acetyl coenzyme A. 3. The activity is completely inhibited in vitro by lysine and by some lysine analogs, which all show cooperative binding and have an heterotropic effect on 2-ketoglutaric binding sites. A second class of affectors is found, including 2-aminoadipic acid, pipecolic acid and dipicolinic acid, which all affect the cooperativity of S-acetyl coenzyme A binding sites. 4. Two types of mutations which modify these inhibition patterns without affecting the catalytic activity are described. One results in a desensitization towards lysine and lysine analogs only. The other entirely abolishes the susceptibility towards the second type of inhibitors, without affecting the susceptibility to lysine. 5. No variations of the specific activity could be detected in the wild type strain at all; mutants showing an increased or a reduced activity were isolated. 6. Our results do not support the existence of isoenzymes at the level of homocitrate synthetase in this yeast.

Acetyl Coenzyme A↗