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.

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.

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.

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.

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.

In vitro RNA synthesis by viruses isolated from the yeast Yarrowia lipolytica.

Viruses isolated from the yeast Yarrowia lipolytica possess a DNA-independent RNA polymerase activity which is inhibited by ethidium bromide and by sodium pyrophosphate but not by actinomycin D. RNA synthesis is maximum at pH 8.0 and at 30 degrees C. Newly synthesized RNA molecules are largely released from the particles, are single-stranded and are able to hybridize with denatured viral genomic RNA.

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.

Evidence for the control of a mutation in lysine catabolism by the mating type in Yarrowia lipolytica.

Reversions of a mutation lye 1.5 blocking the first step of the lysine catabolic pathway were isolated. These reversions mapped inside the LYC1 locus. They exhibited an alteration of the regulation of the lysine pool after nitrogen and phosphorus starvation. This phenomenon did not appear in sexual diploids homozygous for the reversion. Diploids homozygous for the mating type were constructed by protoplast fusion. They displayed a pattern similar to that of the haploids. We conclude that the expression of this mutation is under the control of the physiological state of the mating type.

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.

Replication and recombination of 2-µm DNA in Schizosaccharomyces pombe.

Any one of the inverted sequences present on the 2-µm DNA from Saccharomyces cerevisiae can promote replication of chimeric plasmids in Schizosaccharomyces pombe. When however a complete 2-µm molecule is present on the transforming plasmids, these are very unstable and systematically rearranged in S. pombe. Two types of transformation are observed in this case. One results from chromosomal integration of the incoming DNA. The second is dependent on a site specific recombination event between two molecules of the incoming DNA and results in a stably replicating dimeric structure. The choice between both pathways seems to depend on the expression of 2-µm function(s) in S. pombe.

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.

Genetic and functional analysis of the complex locus ade10 in Schizosaccharomyces pombe.

The complex locus ade10 of Schizosaccharomyces pombe was subjected to genetical fine structure analysis and characterized for enzymatic activities. Out of twenty alleles isolated, fifteen were found to complement and to be localized in two adjacent regions. Complementing alleles mapping in region I lack AICAR-formyltransferase activity while complementing alleles mapping in region II lack IMP-cyclohydrolase activity. In both cases, the remaining activity was generally normal. Three of these alleles were identified as missense and one as nonsense. Interallelic complementation was found to occur inside each region. The five other alleles are completely pleiotropic, suggesting that the two regions belong to a single transcription unit. These alleles are distributed over both regions. Two of them appear to be frameshift. The genetic map shows the occurrence of map expansion.

r-DNA plasmid from Schizosaccharomyces pombe: Cloning and use in yeast transformation.

1. an extrachromosomal, extramitochondrial DNA circle of about 3 µm length, occurring in S. pombe, was cloned in pBR322 and a detailed restriction map of the cloned sequence constructed. 2. Hybridization of this DNA with the ribosomal DNA from S. cerevisiae revealed regions of homology which were mapped in the cloned S. pombe DNA. 3. A transformation system was developed using the URA3 gene of S. cerevisiae and the 3 µm molecule. It was shown that the r-DNA part of the hybrid plasmid promotes replication in S. pombe.

Cloned ß-galactosidase gene of Escherichia coli is expressed in the yeast Saccharomyces cerevisiae.

The expression of the lacZ gene of E. coli in S. cerevisiae has been studied. The enzymatic activity coded by the lacZ gene in E. coli, ß-galactosidase, is detectable in yeast cells harboring a chimeric plasmid carrying the gene. On the basis of size and immunological criteria, no difference was detected between the Coli-in-yeast ß-galactosidase and the E. coli enzyme.

[Expression of a bacterial gene, cloned in the yeast, Saccharomyces cerevisiae]. / Sur l'expression d'un gène bactérien cloné dans la levure 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.

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.

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.

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.