Geotrichum candidum gene expression and metabolite accumulation inside the cells reflect the strain oxidative stress sensitivity and ability to produce flavour compounds.

Geotrichum candidum is a fungus-like yeast widely used as a starter culture for cheese ripening for its proteolytic and lipolytic activities and its contribution to the cheese flavours. The sequenced strain G. candidum CLIB 918 was isolated from cheese Pont-L'Evêque. This strain's ability to produce volatile compounds was compared to the ability of a known strong sulphur compound producer G. candidum strain (Gc203). The aminotransferase-coding genes BAT2 and ARO8 were identified to be involved in methionine catabolism. The production of volatile compounds indicated that the sequenced strain was a moderate producer compared to the strong producer strain. The major volatile compounds were produced from sulphur amino acid, branched-chain amino acid and fatty acid metabolisms. Metabolite content of the cells showed that the ability of the strain to produce volatile compounds was inversely proportional to its ability to store amino acids inside the cells. Reduced glutathione, hypotaurine and taurine intracellular concentrations and volatile fatty aldehyde production indicated the role of oxidative stress sensitivity in flavour production. The increase in expression of several genes in a Reblochon-type cheese at the end of ripening confirmed that oxygen and iron were key factors regulating cheese flavour production.

Cloning the Yarrowia lipolytica homologue of the Saccharomyces cerevisiae SEC62 gene.

Yarrowia lipolytica SEC62 cDNA was cloned by functional complementation of a thermo-sensitive sec62 Saccharomyces cerevisiae mutant strain. The Y. lipolytica SEC62 promoter region was amplified by the inverse polymerase chain reaction (PCR). The cDNA codes for a 396 amino-acid protein with two potential trans-membrane domains. Y. lipolytica Sec62p behaves as an integral membrane protein as shown by Western blotting. Y. lipolytica SEC62 cDNA is able to complement a S. cerevisiae sec62 null mutant strain confirming functional conservation, although only 53.6% amino-acid similarity is observed between Y. lipolytica and S. cerevisiae Sec62.

Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery.

The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p. Gene disruption studies have shown that the SEC61 is required for viability in both S. pombe and Y. lipolytica demonstrating that the essential nature of this protein is not unique to S. cerevisiae. Moreover, heterologous complementation studies indicate that the Y. lipolytica SEC61 gene can complement a null mutation in S. cerevisiae. Sequence comparisons between the various eukaryotic Sec61p homologues reveal a number of highly conserved domains, including several transmembrane sequences and the majority of cytosolic loops. These comparisons will provide an important framework for the detailed analysis of interactions between Sec61p and other components of the translocation machinery and between Sec61p and translocating polypeptide chains.

Structure of the tilapia (Oreochromis mossambicus) prolactin I gene.

The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its transcript (3,677 bases long) begins with a guanine and is organized in five exons and four introns like the other known prolactin genes. Analysis of the 1,555-bp 5'-flanking region suggests that pituitary-specific expression of the gene could be regulated through a trans-factor related to the mammalian pituitary-specific factor Pit-1. Two potential binding sites for such a factor were found in the first intron, suggesting a possible regulatory role for this region. Moreover, two potential Z-DNA regions are located at positions -837 to -812 and -246 to -179 from the transcription start site. These two regions could play an important role in the regulation of PRL gene expression.

Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins.

Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/l. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.

Tilapia prolactin: molecular cloning of two cDNAs and expression in Escherichia coli.

We have isolated cDNA clones encoding tilapia prolactin (tiPRL) from a cDNA library prepared from tilapia (Oreochromis niloticus) anterior pituitary glands. A trout PRL cDNA fragment was used as hybridization probe to select the recombinant plasmids carrying the tiPRL coding sequence. Two types of PRL cDNA were isolated and their complete nucleotide sequence determined. The larger cDNA (tiPRL-I) codes for a polypeptide of 212 amino acids, including a putative signal sequence of 24 amino acids, and contains a 3' untranslated region of 787 bp. The second prolactin cDNA (tiPRL-II) encodes a polypeptide of 200 amino acids, including a presumptive signal peptide of 23 amino acids, and contains a noncoding region of 512 bp. tiPRL-I and tiPRL-II cDNA sequences are 81% similar, whereas the encoded proteins share 69% amino acid identity at optimal alignment. Mature tiPRL-I was efficiently expressed in Escherichia coli carrying a plasmid in which the tiPRL-I cDNA was under the control of the phi 10 promoter of T7 bacteriophage. The new recombinant protein representing about 45% of the total cellular proteins was found in inclusion bodies and cross-reacted with salmon PRL antiserum.

Tilapia growth hormone: molecular cloning of cDNA and expression in Escherichia coli.

A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterior pituitaries. The recombinant clones carrying the cDNA sequence of tilapia growth hormone (tiGH) were selected using a fragment of the trout growth hormone (tGH) cDNA as hybridization probe. The nucleotide sequence of the full-length tiGH cDNA was determined. This cDNA encodes a protein of 204 amino acids, including the putative signal peptide of 17 amino acids. Mature tiGH cDNA was inserted in an Escherichia coli expression vector which led to the production of tiGH protein with a yield estimated to be 20% of the total bacterial proteins.

Molecular cloning and characterization of two forms of trout growth hormone cDNA: expression and secretion of tGH-II by Escherichia coli.

We constructed a cDNA library using mRNA isolated from rainbow trout pituitaries. Two types of cDNA clones encoding growth hormone (GH) were isolated and their complete nucleotide sequences determined. Twenty seven nucleotide substitutions in the coding region and 108 in the noncoding region distinguish the cDNAs of trout GH-I and II. Both cDNAs encode polypeptides of 210 amino acids, including a putative signal peptide of 22 amino acids, which differ by 12 residues. In both trout and salmon, GH-I mRNA is predominant, which suggests that the variation in the amount of secreted GH originates from a transcriptional event. Moreover, comparison of rainbow trout and chum salmon GH reveals that, in both cases, the predominant GH-I has mutated less than its GH-II counterpart. Mature tGH-II was expressed in Escherichia coli using the pIN-III-ompA-Hind secretion vector.

Rainbow trout prolactin cDNA cloning in Escherichia coli.

We describe the isolation and characterization of a cDNA for trout prolactin (tPrl). An extensive analysis of tPrl recombinant clones by restriction analysis and sequencing revealed the presence of only one form of tPrl mRNA. The deduced protein sequence consists of 210 amino acids, including a signal peptide of 23 amino acids. The amino acid sequence of the mature protein is compared among teleosts and mammals, showing two domains of strong similarity that may be involved in biological activity.