Study of a region on yeast chromosome XIII that complements pet G 199 mutants ( COX 7 ) and carries a new non-essential gene

The mutants of Saccharomyces cerevisiae assigned to complementation group G199 are deficient in mitochondrial respiration and lack a functional cytochrome oxidase complex. Recombinant plasmids capable of restoring respiration were cloned by transformation of mutants of this group with a yeast genomic library. Sequencing indicated that a 2.1-kb subclone encompasses the very end (last 11 amino acids) of the PET111 gene, the COX7 gene and a new gene (YMR255W) of unknown function that potentially codes for a polypeptide of 188 amino acids (about 21.5 kDa) without significant homology to any known protein. We have shown that the respiratory defect corresponding to group G199 is complemented by plasmids carrying only the COX7 gene. The gene YMR255W was inactivated by one-step gene replacement and the disrupted strain was viable and unaffected in its ability to grow in a variety of different test media such as minimal or complete media using eight distinct carbon sources at three pH values and temperatures. Inactivation of this gene also did not affect mating or sporulation.


Introduction
In the yeast Saccharomyces cerevisiae, cytochrome oxidase is composed of eleven different polypeptides (1) and the enzyme contains two spectrally distinct heme a groups and two copper atoms.The three large subunits (COX1, COX2, and COX3) which are generally believed to represent the major catalytic centers of cytochrome oxidase are encoded by mitochondrial DNA and possess significant similarity in their primary sequence to the three subunits of prokaryotic cytochrome oxidases (2).All additional subunits are encoded by the nuclear genome.These subunits are thought to function in the regulation of catalysis or in the assembly of the holoenzyme (3).The main function of cytochrome oxidase, the terminal enzyme in the respiratory chain, is energy conservation.This hetero-oligomeric lipoprotein complex of the mitochondrial inner membrane is a molecular pump that uses redox chemistry to drive protons from the mitochondrial matrix across the membrane (4).Besides the eleven enzyme subunits, about 60 more nuclear genes are known to be necessary for the activity of cytochrome oxidase (5,6), an unexpected and exciting problem for study.
In order to define the role of one more nuclear-encoded subunit, we have characterized a pet mutant assigned previously to complementation group G199.Pet mutants are yeast strains which, due to a recessive mutation in a nuclear gene affecting mitochondrial function, have lost the ability to grow on non-fermentable carbon sources, but grow normally on fermentable substrates (5).
The G199 gene was cloned by complementation of the inability to grow on glycerol/ethanol in mutant E880/U1, after transformation with a yeast genomic plasmid library.After subcloning, a 2.1-kb HindIII fragment still had the ability to restore respiration in the mutants.The sequence of this DNA segment contained the end of the PET111 gene (7), the COX7 gene (8,9) and a new ORF.A search at the Saccharomyces Genome Database (SGD), Stanford, CA, confirmed our results and showed that this region is located between coordinates 777570 and 779664 on yeast chromosome XIII.The function of this new ORF (YMR255W) was explored by phenotypic analysis after gene disruption.

Yeast strains and media
The genotypes and sources of yeast strains used in this study are described in Table 1.The nuclear pet mutants were isolated by mutagenesis of the respiratory competent haploid S. cerevisiae strain D273-10B/A1 with ethyl methanesulfonate as described previously (5).

Transformation of S. cerevisiae
Mutant E880/U1 originated from a haploid spore derived from the sporulation of a diploid resulting from the mating of E880 and W303-1A.It was grown in 100 ml YPGal to an approximate density of 10 6 cells/ ml and the entire culture was transformed by the method of Schiestl and Gietz (10) with a yeast genomic library.The library (provided by Dr. Marion Carlson, Department of Human Genetics, Columbia University) con-  (11).Uracil-independent clones were selected on minimal glucose medium and were subsequently checked for respiratory competence by the ability to grow on glycerolrich medium (YEPG).Plasmid DNA was isolated from independent uracil + and glycerol + transformants and amplified in Escherichia coli RR1.One plasmid (pG199/T1) that was checked as positive after transformation of the original recipient was used to subclone and characterize the nuclear DNA insert.

Subcloning and DNA sequencing
Restriction fragments from the plasmid pG199/T1 were subcloned into YEp352/351 (12) and pKS+ (Bluescript ® II KS (+) phagemid, Stratagene) and the DNA was sequenced by the method of Maxam and Gilbert (13).All restriction sites used for 5'-end labeling were crossed from neighboring sites and the sequence was confirmed from the complementary strands.The ends of the insert were sequenced by the method of Sanger et al. (14) using primers (New England Biolabs) adjacent to the BamHI cloning site in the YEp24 vector.

Construction of a deletion strain at ORF YMR255W
A 1.1-kb TaqI fragment (from pG199/ T5) containing the entire ORF was treated with Pfu DNA polymerase (Stratagene) to generate blunt ends beginning 336 bp before the ORF's ATG and extending 211 bp after the termination codon.The fragment was ligated to the pKS+ vector previously digested with ApaI, BstXI and the Pfu DNA polymerase (for blunting), generating plasmid pKS/T8.To construct a disrupted allele of the YMR255W gene, plasmid pKS/T8 was digested with BstEII and EcoRV, a procedure that removes 137 bp internal to the ORF.The deleted plasmid was ligated to a 1.1-kb HindIII fragment containing the yeast URA3 gene.The disrupted orf::URA3 allele was recovered from this construction as a linear 2.0-kb PvuII fragment and was used to transform both the haploid W303-1A strain and the isogenic diploid strain W303 by the procedure of Rothstein (15).

DNA manipulation procedures and miscellaneous methods
Standard methods were used for the transformation of E. coli, the preparation of recombinant plasmid DNA, digestion of DNA with restriction endonucleases, isolation and ligation of DNA fragments, and nick translation (16).The isolation of yeast nuclear DNA and the conditions for Southern analysis have been described elsewhere (17).

Functional analysis
Media used to test the growth of gene disruptants included complete glucose medium (YPD) or synthetic medium with glucose (WO) and also both media supplemented with the following carbon sources replacing glucose: 2% fructose, 2% galactose, 2% maltose, 2% raffinose, 2% potassium acetate, 2% lactate, and 2% glycerol at three different pH values (pH 3.5, pH 6.6 and pH 8.5).Plates with the test media were incubated at three different temperatures (18, 30 and 37 o C).The proper haploid derivatives were mated to generate diploids homozygous for the disruption of YMR255W.The sporulation of these cells was induced and examined as described by Rockmill et al. (18).

Phenotype of COX7 mutants
E880 is the mutant from the pet mutant collection of Tzagoloff and Dieckmann (5) carrying the mutation allelic to the comple-mentation group G199.The mutant is unable to grow on non-fermentable carbon sources and is complemented by mating to a rho o tester strain, indicating that the respiratory deficiency is caused by a recessive mutation in a nuclear gene.The phenotype of this mutant suggests that the respiratory deficiency stems from a specific defect in cytochrome oxidase.Mitochondria of the mutants lack cytochromes a and a 3 but have the absorption bands corresponding to cytochromes b, c and c 1 (data not shown).

Cloning and sequencing of COX7 and ORF YMR255W
To clone the gene carrying the mutation in E880, strain E880/U1 was transformed with a yeast genomic library.Five of the uracil-independent and respiratory-competent clones obtained from the transformation were found to have plasmids with related inserts of yeast nuclear DNA.The nuclear DNA insert of those plasmids was ascertained to be approximately 9 kb long.An XbaI fragment of 6.9 kb was obtained from the original recombinant plasmid YEp24 and ligated to the XbaI site in the polylinker of plasmid YEp352 originating recombinant plasmid pG199/T2 (Figure 1).Initially, a total of three subclones were generated from this XbaI fragment (T3, T4 and T5; Figure 1).T3 is a 2.9-kb EcoRV fragment, T4 is a 3.2-kb XbaI-HindIII fragment and T5 is a 2.1-kb HindIII fragment.The DNA fragments were cloned in YEp352 and their ability to complement the respiratory-deficient phenotype of G199 mutants was investigated.The 2.1-kb recombinant plasmid pG199/T5 was the smallest complementing clone and its sequence was determined.It was found that this region contained the Cterminal end of the PET111 gene, the COX7 gene and another open reading frame of sufficient length to qualify as a protein-coding gene.Three new subclones were generated from pG199/T5.Subclone pG199/T6 (EcoRV-HindIII, 1.3 kb; Figure 1), that contained the entire COX7 gene and part of gene YMR255W, complemented the respiratory defect in the mutants.Subclone pG199/T7 (HindIII-EcoRV, 0.7 kb; Figure 1) contained part of gene YMR255W and did not complement mutants.Subclone pG199/T8 was constructed (1.1 kb, TaqI fragment from pG199/ T5; Figure 1) to contain the entire YMR255W gene including its upstream (promoter) region.The sequence of this region is shown in Figure 2 with the centromere proximal region located at the beginning and the telomere proximal region at the end of the sequence.The ORF of YMR255W encodes a polypeptide of unknown function with 188 amino acids and a calculated molecular mass of 21,584 Da (Figure 2) without significant homology to any known protein.

In situ disruption of the YMR255W gene
Plasmid pKS/T8 was constructed to explore the function of this new gene by onestep gene disruption.A null allele was created by insertion of the yeast URA3 gene replacing the BstEII-EcoRV fragment (137 bp) internal to the coding sequence (Figure 3).The disrupted allele was isolated on a linear (PvuII) fragment and used to transform the respiratory-competent haploid strain W303-1A and the isogenic diploid strain W303.Southern analysis showed that the uracil-independent clones obtained from the transformation had acquired the disrupted ymr255w::URA3 allele.Nuclear DNA from the parental strains, as well as from each transformant, was digested with HindIII and probed with a 833-bp KpnI-SacI fragment of pKS/T8.The probe recognizes a 2.1-kb fragment in the genomic DNA of the parental strain.The genomic DNA of the knockout mutant strain has a new species cross-hybridizing at about 3.1 kb, the size expected after the manipulations used in the construction and after the insertion of the 1166-bp fragment bearing the URA3 gene (Figure 3).

Phenotypic analysis of ORF YMR255W disruptants
The YMR255W gene was successfully disrupted in a haploid strain.Consequently the gene knockout is viable.Growth behavior of the disruptant in the different media and under the conditions listed in the Material and Methods section failed to disclose a behavior distinct from the wild-type strain.The disruptants also presented a normal frequency in the generation of spontaneous cytoplasmic petite mutants.

Discussion
The HindIII fragment characterized by sequencing is located between coordinates 777570 and 779664 on the right arm of chromosome XIII.The gene order (Figure 1) is centromere proximal, YMR255W, YMR 256C (COX7), and YMR257C (PET111).Our sequence analysis placed these genes together for the first time.This has been fully confirmed by the results of the systematic yeast genome sequencing project.The phenotype of the mutant E880 that originated     this study was respiratory incompetence.The yeast strains belonging to this group were unable to grow on non-fermentable substrates and upon spectral and enzymatic examination revealed the absence of cytochrome c oxidase activity and a lack of the spectral bands assigned to this respiratory complex (heme a-a 3 ).The activities of the other membrane enzymes were present (data not shown).
Here we demonstrate that the mutant phenotype could be completely rescued by transformation of the mutant with plasmid pG199/ T6 carrying a 1.4-kb fragment that contained only the COX7 gene and its promoter region.This gene has been sequenced previously (8,9) and the sequence of the fragments amplified by PCR using degenerate primers corresponded to the amino-and carboxylterminal regions of the sequenced subunit VII polypeptide.We now used a genetic method that independently led to the cloning of the same gene.Although cytochrome oxidase in yeast or mammals is a complex enzyme with 11 to 13 subunits, studies with the corresponding Paracoccus denitrificans enzyme showed that the mininal unit required for electron transfer-linked proton pumping consists of subunits I and II (4).Current research aims to understand the function of the other subunits.The recent elucidation of the three-dimensional structure of the mammalian enzyme (19) will certainly direct new attempts to define the role of the accessory polypeptides such as subunit VII (COX7).Another interesting problem is the detection and study of the many nuclear genes that affect cytochrome oxidase activity without being part of the purified active enzyme (6).
One of the genes adjacent to COX7 is PET111 (7).This is also a respiratory gene that functions as a translational factor for the mitochondrially encoded COX2 mRNA re-quiring the 5'-untranslated portion of the messenger (20).The presence of two neighboring respiratory genes made us wonder if the next gene YMR255W, of unknown function, had a mitochondrial role.To that effect a specific null mutant was generated.The disruption of this gene behaved as many other inactivated yeast genes (21), i.e., its knockout does not result in an observable phenotype as examined by growth in rich and minimal media with different carbon sources and at different temperatures.The stability of the mitochondrial genome was found to be normal and was estimated by measuring the spontaneous generation of petite mutants.Also, the null mutant did not affect mating or sporulation in tests carried out with the appropriate haploids or the homozygous diploid yeast.The usual assumption in those cases is that the genome harbors at least one functional equivalent of the disrupted gene or that the experimental conditions tested in the laboratory do not include the physiological demanding conditions that would require this gene product.
Examination of the protein sequence encoded by YMR255W as analyzed by the Yeast Proteome Database shows that the 188-amino acid basic polypeptide (calculated pI of 9.95) is probably a gene with low abundance mRNA as suggested by the codon adaptation index of 0.138 (22) and low codon bias (0.039) (23).The sequence does not show any transmembrane domains but there are regions with the potential to generate coiledcoil domains (24) and potential phosphory-lating sites for Cdc28 kinase and cAMPdependent protein kinase (Yeast Proteome Database) as well as putative N-glycosylation sites (Figure 4).There is no extensive homology to any known protein.A similarity search within the yeast genome using the FASTA algorithm (25) through the SGD revealed 13 ORFs, 8 belonging to genes of unknown function and 5 to genes already named, all with 20 to 29% identity in overlaps that range from 120 to 188 amino acids.Another search using the National Center for Biotechnology Information (NCBI) BLAST program (26) against all non-redundant sequences deposited showed two or three regions (10 to 38 amino acids) of similarity in three additional yeast genes: TAF61, a subunit of transcription factor TFIID (27), ROD1, resistance to O-dinitrobenzene and ions such as zinc and calcium (28), and MLP1, a probable coiled-coil protein with myosin-like motifs and possible involvement in DNA repair (29).These proteins have similarity to YMR255W in its central region (residues 66 to 104 approximately) and also in the Cterminal region.Such similarities could eventually resolve the problem of finding a function for this gene.Further phenotypic testing (30), over-expression of the gene and a search for a synthetic lethal mutant (31) could provide important new clues.

Figure 1 -
Figure 1 -Partial restriction maps of pG199/T2 and derivative plasmids.The larger insert (T2) is a 6.9-kb fragment of nuclear DNA ligated at the Xba I site of vector YEp352.The locations in YEp352 of the URA3 and the ßlactamase genes are shown.The bars in the upper part of the figure represent the size and position of the indicated subclones relative to the larger (T2) insert.The arrows below the restriction map represent the main ORFs present in this segment of chromosome XIII.The two Taq I sites that define subclone T8 are indicated.The plus signs denote the recombinant plasmids that complement the respiratory deficiency of group G199 mutants.

Figure 2 -
Figure 2 -Nucleotide sequence of YMR255W and flanking regions.The sense DNA strand from the Taq I fragment (pG199/ T8) and the translated reading frame corresponding to YMR 255W are shown (GenBank accession number AF007064).Restriction enzyme sites used for cloning, disruption and probe preparation are identified.
865 CTT ATC CTT CAA CGT TTG AAG ACC TCT TTG AAA ATA TGA TCC CTC CCA 912 913 TCA CAT GGA CAC TTT TAA GCA CGT TTT GCT TGA AGA GAC ATT AGT TTA 960 961 CAC CAT TAT AAT TGT TTA ATA AAT ACT AGA CAT ACT TCA AGT GAG AAA 1008 1009 ATC AAT CAC ACC TTT TTC AAT GAT TAG CAA ATC TTT ATT GCC AGG CCT 1056 1057 AAC TGA AAC GAT TTA TTA CTG CGC AAG AAA ACA AAG ATG GAA AAG

Figure 3 -
Figure 3 -Southern hybridization analysis of yeast genomic DNA from representative isolates of strain W303 after disruption of YMR255W.The location of the restriction sites is marked along the line that represents the DNA.The ORFs and respective orientations are shown below.A, Map of the YMR255W region indicating the BstEII and EcoRV sites used to delete a 137-bp internal fragment of the gene before the insertion of a 1166-bp DNA fragment carrying the URA3 gene.B, Map of the same chromosomal region after gene replacement with the construction explained above.C, Electrophoretogram of total yeast DNA prepared from the control strain W303-1A (lanes 1 and 5) or from individual transformants from experiments done with the W303-1A haploid strain (lanes 2, 3 and 4) or with the diploid W303 strain (lanes 6, 7 and 8).About 0.2 µg of DNA was digested with HindIII, fractionated by electrophoresis on 1% agarose gel and blotted onto Hybond N. The Southern blot was hybridized to the labeled TaqI-SacI fragment (probe) and exposed to X-ray film.The size of the fragments detected is indicated on the left-hand margin.

Figure 4 -
Figure 4 -Derived amino acid sequence corresponding to the coding region of gene YMR 255W.Sequence motifs identi- fied by the Yeast Proteome Database as putative targets for Nglycosylation (A), protein kinase A (B) or Cdc28 kinase (C) are underlined.Heptad sequences that represent potential coiledcoil structures have been identified (24) and are displayed in bold characters.