The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron.

The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message. Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon. A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection. Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides. The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns. One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron. The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.     

ATP4, the structural gene for yeast F0F1 ATPase subunit 4

A plasmid containing the gene coding for the Saccharomyces cerevisiae F0F1 ATPase subunit 4 was isolated from a yeast genomic DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13 tg 130 and M13 tg 131 phage vectors. A 732-base-pair open reading frame encoding a 244-amino-acid polypeptide is described. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein with a hydrophilic and basic 35-amino-acid leader sequence. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 Da. This subunit presents amphiphilic behaviour with two distinct domains. A high alpha-helix content of 77% was predicted from the sequence. Subunit 4 shows homology with the b subunit of Escherichia coli ATP synthase.     

Differential regulation of the two genes encoding Saccharomyces cerevisiae cytochrome c oxidase subunit V by heme and the HAP2 and REO1 genes.

In Saccharomyces cerevisiae, the COX5a and COX5b genes encode two forms of cytochrome c oxidase subunit V, Va and Vb. We report here that heme increases COX5a expression and decreases COX5b expression and that the HAP2 and REO1 genes are involved in positive regulation of COX5a and negative regulation of COX5b, respectively. Heme regulation of COX5a and COX5b may dictate which subunit V isoform is available for assembly into cytochrome c oxidase under conditions of high- and low-oxygen tension.     

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.     

Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III.     

Isolation and structure of a rat cytochrome c gene

We screened a Charon 4A-rat genomic library using the cloned iso-1 cytochrome c gene from Saccharomyces cerevisiae as a specific hybridization probe. Eight different recombinant phages homologous to a coding region subfragment of the yeast gene were isolated. Nucleotide sequence analysis of a 0.96-kilobase portion of one of these established the existence of a gene coding for a cytochrome c identical in amino acid sequence with that of mouse. The rat polypeptide chain sequence had not previously been determined. In contrast to the yeast iso-1 and iso-2 cytochrome c genes, neither of which have introns, the rat gene contains a single 105-base pair intervening sequence interrupting glycine codon 56. The overall nucleotide sequence homology between cytochrome c genes of yeast and rat is about 62%, with areas of greater homology coinciding with four regions of functionally constrained amino acid sequences. Two of these regions displayed 85-90% DNA sequence homology, including the longest consecutive homologous stretch of 14 nucleotides, corresponding to amino acids 47-52 of the rat protein. Somewhat less homology was observed in the DNA-specifying amino acids 70-80, which are invariant residues in most known cytochrome c molecules. Thermal dissociation of the yeast probe from the homologous rat DNA was at about 58 degrees C in 0.39 M Na+. These results establish that cytochrome c genes may be isolated by interspecies hybridization between widely divergent organisms.     

Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component

Complementary and genomic DNA clones corresponding to the human C-reactive protein (CRP) mRNA and structural gene have been analyzed and compared. Nucleotide sequencing of the coding regions of both cDNA and genomic DNA revealed an additional 19 amino acid peptide not described in the published CRP amino acid sequence. The CRP gene contains a single 278 base pair intron within the codon specifying the third residue of mature CRP. The intron contains a repetitive sequence (GT)15G(GT)3 which is similar to structures capable of adopting the Z-DNA form. A comparison of CRP coding and amino acid sequences with those of serum amyloid P component revealed striking overall homology which was not uniform: a region of limited conservation is bounded by two highly conserved regions.     

Structure of the human cytochrome c oxidase subunit Vb gene and chromosomal mapping of the coding gene and of seven pseudogenes.

Subunit Vb of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) is encoded by a nuclear gene and assembled with the other 12 COX subunits encoded in both mitochondrial and nuclear DNA. We have cloned the gene for human COX subunit Vb (COX5B) and determined the exon-intron structure by both hybridization analysis and DNA sequencing. The gene contains five exons and four introns; the four coding exons span a region of approximately 2.4 kb. The 5' end of the COX5B gene is GC-rich and contains many HpaII sites. Genomic Southern blot analysis of human DNA probed with the human COX Vb cDNA identified eight restriction fragments containing COX Vb-related sequences that were mapped to different chromosomes with panels of human x Chinese hamster somatic cell hybrids. Because only one of these fragments hybridized with a 210-bp probe from intron 4, we conclude that there is a single expressed gene for COX subunit Vb in the human genome. We have mapped this gene to chromosome 2, region cen-q13.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

Cloning and analysis of the nuclear genes for two mitochondrial ribosomal proteins in yeast

Summary Two mitochondrial ribosomal proteins of yeast (Saccharomyces cerevisiae) were purified and their N-terminal amino acid sequences determined. The sequence data were used for the synthesis of oligonucleotide probes to clone the corresponding genes. Thus, the genes for two proteins, termed YMR-31 and YMR-44, were cloned and their nucleotide sequences determined. From the nucleotide sequence data, the coding region of the gene for protein YMR-31 was found to be composed of 369 nucleotide pairs. Comparison of the amino acid sequence of protein YMR-31 and the one deduced from the nucleotide sequence of its gene suggests that it contains an octapeptide leader sequence. The calculated molecular weight of protein YMR-31 without the leader sequence is 12792 dalton. The gene for protein YMR-44 was found to contain a 147 bp intron which contains two sequences conserved among yeast introns. The length of the two exons flanking the intron totals 294 nucleotide pairs which can encode a protein with a calculated molecular weight of 11476 dalton. The gene for protein YMR-31 is located on chromosome VI, while the gene for protein YMR-44 is located on either chromosome XIII or XVI.     

Deletion of the Leader Peptide of the Mitochondrially Encoded Precursor of Saccharomyces Cerevisiae Cytochrome C Oxidase Subunit II

Cytochrome c oxidase subunit II (Cox2p) of Saccharomyces cerevisiae is synthesized within mitochondria as a precursor, pre-Cox2p. The 15-amino acid leader peptide is processed after export to the intermembrane space. Leader peptides are relatively unusual in mitochondrially coded proteins: indeed mammalian Cox2p lacks a leader peptide. We generated two deletions in the S. cerevisiae COX2 gene, removing either the leader peptide (cox2-20) or the leader peptide and processing site (cox2-21) without altering either the promoter or the mRNA-specific translational activation site. When inserted into mtDNA, both deletions substantially reduced the steady-state levels of Cox2p and caused a tight nonrespiratory phenotype. A respiring pseudorevertant of the cox2-20 mutant was heteroplasmic for the original mutant mtDNA and a ρ(-) mtDNA whose deletion fused the first 251 codons of the mitochondrial gene encoding cytochrome b to the cox2-20 sequence. The resulting fusion protein was processed to yield functional Cox2p. Thus, the presence of amino-terminal cytochrome b sequence bypassed the need for the pre-Cox2p leader peptide. We propose that the pre-Cox2p leader peptide contains a targeting signal necessary for membrane insertion, without which it remains in the matrix and is rapidly degraded.     

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

Identification of Reo1, a Gene Involved in Negative Regulation of Cox5b and Anb1 in Aerobically Grown Saccharomyces Cerevisiae

In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration. In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V. The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources. By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions. Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression. The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase. reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated. These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen. The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions. Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.