YMC1, a yeast gene encoding a new putative mitochondrial carrier protein

We have cloned and sequenced a Saccharomyces cerevisiae gene coding for a protein with significant similarities to the mitochondrial carrier family. The gene we termed YMC1 (yeast mitochondrial carrier) is located on chromosome XVI, closely downstream of ARO7 encoding chorismate mutase.     

The complete sequence of the 8.2 kb segment left of MAT on chromosome III reveals five ORFs, including a gene for a yeast ribokinase

We report here the DNA sequence of a segment of chromosome III extending over 8.2 kb. The sequence was determined using the random clone strategy followed by oligonucleotide-directed sequencing. The segment contains five long open reading frames, YCR521, 522, 523, 524 and 526, with only short distances between them. YCR523 (333 codons) encodes a ribokinase, a new function for yeast. YCR526 originates inside the MAT cassette, which is in continuity with the present segment, and extends over 358 codons outside of MAT. YCR524 (923 codons) codes for a putative membrane protein. YCR521, 522 and 524, have each been disrupted by insertion of a URA3 cassette and are non-essential genes. An active ARS element is located within YCR523 or its vicinity.     

QCR9, the nuclear gene encoding a small subunit of the mitochondrial cytochrome bc1 complex,maps to the right arm of chromosome VII in Saccharomyces cerevisiae

We present here mapping data for QCR9, a nuclear gene encoding a subunit of the ubiquinol-cytochrome c oxidoreductase complex. Deletion of QCR9 results in the inability of cells to grow on non-fermentable carbon sources at 37°C. Thus, qcr9 mutants can be scored by growing cells on YPE/G at 37°C, or followed by the URA3 marker, which was inserted when making the qcr9 deletion strain, JDP1. The location of QCR9 on the right arm of chromosome VII with respect to the previously mapped genes ADE3, SER2 and PET54 is given.     

The carboxyl-terminal extension on fungal mitochondrial DNA polymerases: identification of a critical region of the enzyme from Saccharomyces cerevisiae

Fungal mitochondrial DNA (mtDNA) polymerases, in comparison to their metazoan counterparts, harbour unique carboxyl-terminal extensions (CTEs) of varying lengths and unknown function. To determine the essential regions of the 279 residue CTE of the yeast enzyme (Mip1p), several CTE-truncation variants were expressed in Saccharomyces cerevisiae. The respiratory competence of mip1delta175 cells, in which Mip1p lacks the C-terminal 175 residues, is indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351 and Mip1pdelta279 rapidly lose mtDNA. Approximately one in six mip1delta216 transformants grew on glycerol, albeit poorly. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and the rapid loss of mtDNA during fermentative, but not respiratory, growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. Comparison of this essential segment with the sequences of other fungal mtDNA polymerases revealed novel features shared among the mtDNA polymerases of the Saccharomycetales.     

Development of a rapid mitochondrial DNA extraction method for species identification in milk and milk products

Isolation of mitochondrial DNA (mtDNA) from milk offers an effective way to monitor aspects of quality control and traceability to ensure food safety. A few methods of DNA isolation from milk have been reported, but many of them are time consuming and expensive. Here, we report a rapid, simple, and efficient method of mtDNA extraction from raw and processed milk (pasteurized, retorted, and UHT milk) to generate substrate for analysis using any PCR analysis platform. Various techniques used for the separation of mitochondria were explored and combined with a sodium dodecyl sulfate method for mtDNA extraction from raw and processed milk. The optimized protocol supports the efficient amplification of mtDNA independent of sample origin and is sufficiently straightforward to allow its widespread adoption by industry.     

The complete sequence of K3B, a 7.9 kb fragment between PGK1 and CRY1 on chromosome III, reveals the presence of seven open reading frames.

We have determined the nucleotide sequence of a segment from chromosome III of Saccharomyces cerevisiae extending over 7.9 kb between the PGK1 and CRY1 loci. The fragment contains seven open reading frames, YCR241, YCR242, YCR243, YCR244, YCR245, YCR246 and YCR247, of more than 70 codons. The study of the effects of a global disruption of YCR242, YCR243, YCR244, YCR245 and YCR247 shows that they are not essential for growth and division.     

The complete sequence of a 7.5 kb region of chromosome III from Saccharomyces cerevisiae that lies between CRY1 and MAT.

We report the sequence of a 7.5 kb region lying between the CRY1 and MAT loci of chromosome III from Saccharomyces cerevisiae. This region lies in the overlap between two major contigs used for the generation of the complete nucleotide sequence of this chromosome. Comparison of this sequence with those reported previously for this overlap [Thierry et al. (1990) Yeast 6, 521; Jia et al. (1991) Yeast 7, 413] reveals 38 nucleotide differences, 45% of which generate changes in the amino acid sequences of the four genes in this region (YCR591, YCR592, YCR521 and YCR522). These differences appear to reflect true sequence polymorphisms between the two yeast strains used to generate the clones used in the sequencing project. Three of the four genes in this region display weak homologies to proteins in the PIR database. Some properties of YCR521 are analogous to those of ribosomal protein genes. However, the functions of all four genes remain obscure.     

Structure and expression of yeast DPR1, a gene essential for the processing and intracellular localization of ras proteins

The ras proteins represent a unique example of membrane proteins which apparently do not utilize the secretory pathway for their membrane localization. Instead, it is believed that palmitic acid, covalently attached to the protein, acts as an anchor to the membranes. Recent identification of yeast mutants defective in the processing of the ras proteins has provided a novel approach for defining these biosynthetic processes. We report here the characterization of yeast DPR1, a gene essential for the processing of the ras proteins. The sequence of the gene indicates that it encodes a protein of 431 amino acids which contains no significant homology with any known proteins. It is a relatively hydrophilic protein with no apparent hydrophobic stretches. The C-terminal half of the encoded protein has an unusually high content of cysteine. The DPR1 gene product has been identified in a cell-free translation system as a protein having an apparent molecular weight of 43 kd. This represents the first step in the investigation of a novel protein-processing pathway, one that is distinct from the secretory pathway.     

Nucleotide sequence of the cytochrome oxidase subunit 2 and val-tRNA genes and surrounding sequences from Kluyveromyces lactis K8 mitochondrial DNA

The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial DNA is reported. Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene. A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of val-tRNA. This sequence is absent from regions between val-tRNA-cox2 and cox2-cox1. In addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene. Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K. lactis, but is not present upstream of other genes. Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K. lactis is closely related to that of Saccharomyces cerevisiae.     

The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing,but cytosolic,protein that is essential for growth on methanol

Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65·3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dakΔ mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dakΔ mutant and the import of peroxisomal proteins was not disturbed. The dakΔ mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dakΔ mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dakΔ mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198. © 1998 John Wiley & Sons, Ltd.     

A mutant for the yeast scERV1 gene displays a new defect in mitochondrial morphology and distribution

The yeast scERV1 gene is the best characterized representative of a new gene family found in different lower and higher eukaryotes. The gene product is essential for the yeast cell and has a complex influence on different aspects of mitochondrial biogenesis. The homologous mammalian ALR(A ugmenter of L iver R egeneration) genes from man, mouse and rat are important at different developmental stages of the organism as, for example, in spermatogenesis and liver regeneration. In this study the influence of scERV1 on the morphology of mitochondria and its submitochondrial localization are investigated. A temperature‐sensitive mutant of the gene was stained with a mitochondria‐specific dye and fluorescence was inspected at the permissive and restrictive temperature. A new phenotype for morphological defects of mitochondria was identified. Already at the permissive temperature mitochondrial vesicles accumulate at defined positions in the cell. After shift to the restrictive temperature, morphological changes, and finally complete loss of mitochondrial structures, are observed. Ultrastructural studies confirm these findings and demonstrate the loss of the mitochondrial inner membrane and at the final stage a drastic reduction or complete absence of mitochondria from the cell. GFP fusion experiments with the scERV1 gene and subcellular localization by fractionation experiments identify the gene product inside mitoplasts and the cytosol. Re‐investigation of the mutant phenotype demonstrates that after longer incubation of the mutant at the restrictive temperature an irreversible defect of the cells, even on glucose complete medium, is found that is in accordance with a complete loss or irreversible damage of mitochondria. Copyright © 1999 John Wiley & Sons, Ltd.     

X. Yeast sequencing reports. The sequence of a 36 kb segment on the left arm of yeast chromosome X identifies 24 open reading frames including NUC1, PRP21 (SPP91), CDC6, CRY2, the gene for S24, a homologue to the aconitase gene ACO1 and two homologues to chromosome III genes

A 36 kb fragment from the left arm of chromosome X, located at about 50 kb from the telomere, was sequenced and analysed. The segment contains a new putative ARS, a new tRNA for threonine, remnants of a solo delta and 24 open reading frames (ORFs) numbered from J0310 to J0355. Six of them, NUC1, PRP21 (also called SPP91), CDC6, CRY2, the gene encoding the ribosomal protein S24 and the gene coding for a hypothetical protein of 599 amino acids, have been sequenced previously. Three ORFs show high homology to the yeast gene ACO1 encoding mitochondrial aconitase and to the chromosome III genes YCR34W and YCR37C of unknown function. Three other ORFs show lower but significant homology: a first one to UNP, a gene related to the tre-2 oncogene from mouse and to the gene coding for the yeast deubiquitinating enzyme DOA2; a second one to SLY41, a suppressor of the functional loss of YPT1 and a third one to the gene encoding the proline utilization activator PUT3. The complete nucleotide sequence of 36 016 bp was submitted to the EMBL database (accession number X77688).     

Nucleotide sequence of 9.2 kb left of CRY1 on yeast chromosome III from strain AB972: evidence for a Ty insertion and functional analysis of open reading frame YCR28.

We report the 9210 bp sequence from a segment of yeast chromosome III cloned from strain AB972 in lambda PM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24-24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24-24A and cause a quite significant rearrangement of the whole region. We performed functional analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24-24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain-dependent effect on sensitivity to amino acid balance and to 3-aminotriazole, when cells were grown in synthetic medium.     

Effects of different feeding levels during a 14-week preweaning phase in dairy heifer calves on telomere length and mitochondrial DNA copy number in blood

Telomeres cap the ends of eukaryotic chromosomes, and the telomere length (TL) is related to cellular age. The mitochondrial DNA copy number (mtDNAcn) reflects the abundance of mitochondria in a cell. In addition to generating energy, mitochondria are also the main producers of reactive oxygen species, which in turn can accelerate TL attrition and impair mitochondrial function. Nutrition in early life could influence mtDNAcn and TL in later life. In the present study, we investigated the effects of feeding different levels of milk replacer (MR) on TL shortening and energetic status by examining mtDNAcn of heifers during their first year of life. In this study, whole blood samples were obtained from German Holstein heifer calves 36 to 48 h after birth (wk 1) and at wk 12 and wk 16 of life (n = 37), as well as from 31 calves when reaching 1 yr of age. Calves were fed either a high level of MR (14% solids) at 10 L/d (1.4 kg of MR/d; n = 18) or a restrictive low level at 5.7 L/d (0.8 kg of MR/d; n = 19) until linear weaning in wk 13 to 14 of life. Additional whole blood samples were taken from their respective dams 36 to 48 h after calving. Relative TL (qT) and mtDNAcn in cells from whole blood were measured by multiplex quantitative PCR. The greatest qT values were observed in neonates (36–48 h after birth), with decreasing qT values thereafter. Delta qT values were calculated as ΔqT = qT (first year of life) ? initial qT (36–48 h after birth). We found no effect of the feeding regimen on qT values, but qT decreased with age. The mtDNAcn was lowest in neonates, increased until wk 12 of life, and then remained at a constant level until after weaning (wk 16). After the first year of life, mtDNAcn was decreased and returned to levels comparable to those of the neonatal stage. No differences in mtDNAcn were detectable between feeding groups within each time point. When comparing the values of qT and mtDNAcn between the calves and their dams after calving (36–48 h after birth and after calving), greater values were observed in calves than in dams. Delta qT values were negative in all but 2 calves (on the restricted diet), indicating that the change in TL with age was not uniform among individual animals, whereas no difference in mean ΔqT values occurred between the feeding groups. Additional analyses of the correlation between qT, mtDNAcn, and various indicators of oxidative status from birth until wk 16 of life did not indicate major interactions between oxidative status, qT and mtDNAcn. The results of this study support an age-dependent decrease of TL in calves independent of the MR feeding level and show the dynamic changes of mtDNAcn in early life.     

基于线粒体编码基因差异对食品中牛肉与猪肉成分的鉴别

市场上有不法厂商使用深加工后的猪肉冒充或添加于牛肉进行销售,但针对熟制食品中猪肉和牛肉成分的辨别方法仍不够成熟。为建立灵敏、可靠的食品中猪肉和牛肉成分的鉴定方法,分别根据线粒体基因组和细胞基因组编码基因的差异设计多组物种特异性引物,应用PCR技术对加工食品中的猪肉和牛肉成分进行鉴别。结果表明,两组基于线粒体编码基因差异所设计的引物各具优势,均可有效地进行食品中猪肉和牛肉成分的鉴定,检测灵敏度达纳克级DNA,且鉴定效果显著优于使用基于细胞基因组编码基因差异的引物。对市售食品样本的鉴别验证了方法的实用价值。总之,建立并优化了可用于深加工熟制食品中猪肉和牛肉成分鉴别的PCR方法,为肉制品质量控制提供了新的途径。