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Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

A C-terminally truncated form of the hepatitis C virus (HCV) putative envelope glycoprotein E2 was expressed in two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, using a yeast signal peptide sequence to direct the viral glycoprotein to the endoplasmic reticulum (ER) pathway of secretion. Characterization of secreted E2 showed that the protein is endoglycosidase-H-sensitive in both yeasts. Moreover, in vivo inhibition of glycosylation with tunicamycin prevented secretion of E2 and showed that, of its 11 putative N-linked glycosylation sites, at least eight were core-glycosylated. Analysis of the heterologous glycoprotein by SDS-PAGE under nonreducing conditions and by gel filtration demonstrated the formation of multiple disulphides, which resulted in secretion of heterogeneous aggregates with an average molecular mass of 770-1000 kDa in both yeasts. However, variations were observed in the binding of the glycoprotein secreted by the two yeasts to a mannose-specific lectin, and also in its reactivity with anti-E2-specific antibodies. This denotes differences between the two yeasts in folding and/or modification of the E2 glycoprotein.

A mutation (op1) in the Saccharomyces cerevisiae AAC2 gene, which codes for the most abundant ADP/ATP carrier isoform, results in lack of mitochondrial-dependent growth and in an as yet unexplained petite-negative phenotype. A gene from the petite-negative yeast Kluyveromyces lactis has been isolated by complementing in multicopy the op1 mutation of S. cerevisiae. This gene, designated KIAAC, can complement the petite-negative phenotype of op1 as well as its inability to grow on nonfermentable carbon sources. KIAAC contains a 915-base pair open reading frame coding for a protein of 305 amino acids which shows a high degree of identity to AAC2. The K. lactis ADP/ATP carrier also shares identity with other known ADP/ATP carrier sequences. In particular, the degree of identity of KIAAC is higher with the Neurospora crassa carrier (80.1%) than with AAC1 (76.6%). The nucleotide sequence upstream of the KIAAC coding region was found to contain a long DNA segment with no coding potential, but presenting features of highly regulated promoter sequences.

In Saccharomyces cerevisiae a number of chemical agents induce synthesis of cytochrome P450. A cytochrome P450 gene has been well characterized in this yeast: CYP51, which codes for a constitutive enzyme involved in the 14 alpha-demethylation of lanosterol, a key step in the biosynthesis of ergosterol. In this work, we have analysed the level of transcription of the CYP51 gene in correlation with cytochrome P450 enzymatic activity after treatment with several chemical agents known to interact with cytochrome P450. Using as a probe a DNA fragment whose identity to the CYP51 gene was established by sequence analysis and mapping on chromosome VIII, a unique RNA species was observed in all treatment samples. The increased level found for this transcript in cells treated with ethanol, 20% glucose, phenobarbital or 5-methoxypsoralen correlates with the levels of induction in cytochrome P450 enzymatic activity measured in cells grown under the same conditions, indicating that induction of cytochrome P450 by these treatments is regulated at the transcriptional level.

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.

We have determined the entire sequence of the plasmid K2 from Kluyveromyces lactis which is involved in the maintenance of both killer plasmids in the cell. K2 shares many of the characteristics of the smaller killer plasmid K1: high A+T content (74.7%) and very compact genomic organization. K2 contains ten open reading frames. Some of them overlap on different strands and some on the same strand. Northern blotting of K2 transcripts shows that at least eight ORFs are transcribed. Analysis of the predicted aminoacid sequence of ORF2 from K2 reveals homology with the aminoacid sequence of ORF 1 from K1 and with several viral DNA polymerases. The sequence of K2 from Saccaromyces cerevisiae F102-2 was also determined. Only one nucleotide difference was found between the K2 sequence from the two yeasts. This mutation does not change the genome organization of the plasmid and has only minimal effect on the structure of the encoded proteins.

Killer strains of Kluyveromyces lactis secrete a toxin which presumably is processed during secretion from a larger precursor. Analysis of the sequence of the K. lactis killer toxin gene predicts that the first 16 amino acids at the amino terminus of the protein should represent its leader peptide. We have tested the capability of this leader peptide to direct secretion of a protein fused to it by inserting a synthetic oligonucleotide identical to the sequence of the putative leader peptide into a yeast expression vector. Subsequently, the cDNA coding for the secreted active portion of the human interleukin 1 beta (IL-1 beta) was fused to the leader peptide sequence of the killer toxin. This construction in Saccharomyces cerevisiae is capable of directing synthesis and secretion of correctly processed IL-1 beta into the culture medium.

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60,204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.

Intra-species fusion products of Saccharomyces cerevisiae, Saccharomyces unisporus and Torulopsis glabrata have been isolated following polyethylene glycol-induced fusion of protoplasts and selection for prototrophic colonies. Staining with lomofungin showed that all fusion products were uninucleate. Measurement of DNA content mostly gave values between haploid and diploid levels indicating that the majority of fusion products were aneuploid, Nevertheless fusion products of S. cerevisiae and S. unisporus were, as expected, more resistant to X-irradiation than their haploid parents. By contrast, the X-ray dose-response curve of all T. glabrata fusion products was indistinguishable from their progenitors despite the fact that mitotic segregants could be recovered amongst the survivors to X-rays. A possible explanation for the behaviour towards X-rays of T. glabrata fusion products is that this species lacks a DNA repair pathway involving recombination between homologous chromosomes. We conclude from this study that the shape of the X-ray dose-response curve should not be taken to indicate the ploidy of new yeast isolates without supporting data.