No abstract

We have previously reported the isolation of the gene coding for a 25‐kDa polypeptide present in a purified yeast QH2:cytochrome c oxidoreductase preparation, which was thus identified as the gene for the Rieske ironsulphur protein [Van Loon et al. (1983) Gene 26, 261–272]. Subsequent DNA sequence analysis reported here reveals, however, that the encoded protein is in fact manganese superoxide dismutase, a mitochondrial matrix protein. Comparison with the known amino acid sequence of the mature protein indicates that it is synthesized with an N‐terminal extension of 27 amino acids. In common with the N‐terminal extensions of other imported mitochondrial proteins, the presequence has several basic residues but lacks negatively charged residues. The function of these positive charges and other possible topogenic sequences are discussed. Sequences 5′ of the gene contain two elements that may be homologous to the suggested regulatory sites, UAS 1 and UAS 2 in the yeast CYC1‐gene [Guarente et al. (1984) Cell 36, 503–511]. The predicted secondary structures in manganese superoxide dismutase appear to be very similar to those reported for iron superoxide dismutase, suggesting similar three‐dimensional structures. Making use of the known three‐dimensional structure of the Fe enzyme, the Mn ligands are predicted.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (

Stability of HIV-1 RNA in blodd during specimen handling and storage prior to amplification by NASBA-QT Bruisten, S.M.; Oudshoorn, P.; van Swieten, P.; Boeser-Nunnink, B.; van Aarle, P.; Tondreau, S.P.; Cuypers, H.T.M.

The nucleotide sequence of a 2.5 x 103-base segment of yeast nuclear DNA, containing the structural gene for the 40-kDa subunit I1 of the ubiquinol: cytochrome-c oxidoreductase, has been determined. The region contains only one single reading frame of length sufficient to encode a protein of the size of subunit 11. The mature protein is predicted to have a length of 352 amino acids, with a molecular mass of 38714 Da. It is predominantly hydrophilic, with an overall polarity of 45%. Comparison of the sequence of the reading frame with that derived from direct sequence analysis of the N terminus of the mature 40-kDa protein shows that subunit I1 is synthesized as a longer precursor and shows that the extension is N-terminal. The presequence is 16 amino acids long and it contains a number of positively charged residues and lacks acidic ones. It is also rich in neutral, polar amino acids. S1 nuclease protection analysis of DNA . RNA hybrids identifies two major and one minor transcript of the gene, whose 5' termini map approximately 55, 65 and 75 nucleotides upstream of the initiation codon. Sequences 5' of these termini lack obvious homology to the regulatory sequences of other imported mitochondrial proteins, whose synthesis is controlled by oxygen and by catabolite repression. A mutant lacking a functional subunit I1 gene has been constructed by a one-step gene-disruption procedure. This mutant grows only slowly on glycerol and still displays a low level of QH,: cytochrome-c oxidoreductase activity (approx. 5% of that of wild type). The implications of this finding for the possible role of subunit I1 in the complex are discussed.QH, : cytochrome-c oxidoreductase is a proton-translocating enzyme of the respiratory chain in mitochondria. In yeast it consists of eight to ten subunits [l -31, of which only one, apocytochrome b, is mitochondrially encoded [4, 51. The remaining subunits are encoded in the nucleus. The dual genetic origin of the enzyme poses questions concering the coordinate synthesis, import and assembly of individual subunits. To address these, molecular information about the genes and the subunits is required. This is so far available for subunits I11 (cytochrome cl), IV (cytochrome b) and VI-VIII [6-91 (Maarse, unpublished results). We describe here an analysis of the nuclear gene for subunit 11. Comparison of the nucleotide sequence with a partial amino acid sequence of the N terminus of the mature protein reveals an amino-terminal extension which displays several features common to the presequences of other imported mitochondrial proteins. The remaining protein sequence is predominantly hydrophilic, consistent with previous suggestions that the bulk of the subunit protrudes from the mitochondrial inner membrane into the matrix space MATERIALS AND METHODS Strains and mediaSaccharomyces cerevisiae strains DL1 (a, his3, leu2, ura3) and HR2 (a, his3,leu2, trpl) were used for most experiments. Transformation was carried out as described by Klebe et al. [Ill and transformants were selected on minima...