Characterization of DNA-Binding Proteins Involved in the Assembly of Mitochondrial Nucleoids in the Yeast Saccharomyces cerevisiae

Mitochondrial nucleoids (mt-nucleoids) isolated from the yeastSaccharomyces cerevisiae were analyzed to identify the proteincomponents that are involved in the compact packaging of mtDNA.The isolated mt-nucleoids were disassembled by the additionof 2 M NaCl and the disassembled mt-nucleoids were reassembledonce again into compact structures by dialysis against a bufferthat contained NaCl at concentrations below 0.1 M, as monitoredby staining of the DNA with 4',6-diamidino-2-phenylindole. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa,50 kDa, 38 kDa, 30 kDa and 20 kDa were separated from isolatedmt-nucleoids by column chromatography on DNA cellulose afterdigestion of mt-nucleoids by DNase I in the presence or absenceof 2 M NaCl. Purified mtDNA was compactly packaged into nucleoid-likestructures upon the addition of fractions that contained DNA-bindingproteins and subsequent dialysis to reduce the concentrationof NaCl. Five proteins, with molecular masses of 67 kDa, 52kDa, 50 kDa, 38 kDa and 30 kDa, respectively, had lower affinityfor double-stranded DNA than that of the 20-kDa protein. Thefraction that contained the five DNA-binding proteins otherthan the 20-kDa protein was also able to fold mtDNA compactlyinto nucleoid-like structures. By contrast, the combinationof the 20-kDa protein and mtDNA resulted in formation of lesstightly packed, string-of-bead structures. These results suggestthat at least six different DNA-binding proteins are involvedin the organization of the mt-nucleoids. (Received April 7, 1995; Accepted July 10, 1995)     

Isolation, Purification, and Characterization of Catalase from the Methylotrophic Yeast Pichia pastoris

Catalase (CAT_pp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CAT_pp solutions and of its membrane-concentrated form (CAT_pp-conc) were studied. Rates of H₂O₂ decomposition and kinetic characteristics K _m and k _cat of CAT_pp and CAT_pp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30°C, as well as the effective constant k _in of the enzyme inactivation rate during the catalysis and the constant k ₂ of the interaction rate of the Complex I catalases with H₂O₂. Thermal inactivation of CAT_pp in solutions at 45°C was characterized by the effective rate constant k _in ^*, and the low-frequency (27 kHz) ultrasonic inactivation of CAT_pp at 20°C was characterized by the firstorder rate constant k _in (US). All spectral and kinetic characteristics of CAT_pp and CAT_pp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CAT_cb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CAT_pp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of k _cat/K _m, thermal stability comparable with the thermal stability of CAT in terms of k _in ^*, the minimal k _in, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm².     

Isolation and Partial Characterization of DNA Topoisomerase I from the Nucleoids of White Mustard Chloroplasts

DNA topoisomerase was isolated for the first time from nucleoids of white mustard (Sinapis alba L.) chloroplasts. The enzyme had a molecular weight of 70 kDa; was ATP-independent, required the presence of mono- (K⁺) and bivalent (Mg²⁺) cations, and was capable of relaxing both negatively and positively supercoiled DNA. These results suggest that the enzyme isolated belongs to the type IB DNA topoisomerases.     

Preparation of a Monoclonal Antibody Specific for a 48-kDa Protein from Mitochondrial Nucleoids of the Yeast, Saccharomyces cerevisiae

Monoclonal antibodies (mAbs) were raised against yeast mitochondrialnucleoids (mtnucleoids). In an analysis by a combination ofimmunofluorescence microscopy and staining with 4',6-diamidino-2-phenylindole(DAPI), one of them, designated YMN-1, distinctly stained mtnucleoids,which were visible as dots, in spheroplasts and in isolatedmitochondria. However, staining of isolated mt-nucleoids wasrather weak. YMN-1 mAb recognized a 48-kDa protein in immunoblotsof both mitochondrial and mt-nucleoid proteins. The 48-kDa proteinwas a minor component of mt-nucleoid proteins and was separatedfrom extract of both mitochondria and mt-nucleoids by immunoamnitychromatography. The affinity-purified 48-kDa protein reassociatedwith mt-nucleoids when mixed with isolated mt-nucleoids, asmonitored by immunofluorescence microscopy. The results suggestthat a large amount of 48-kDa protein is associated with mt-nucleoidsin vivo, and that lysis of mitochondria by the treatment withdetergent releases a considerable amount of this protein frommt-nucleoids during the isolation of mt-nucleoids. (Received June 25, 1992; Accepted November 16, 1992)     

Identification of the genes GPD1 and GPD2 of Pichia jadinii.

Two genes coding for proteins with a high degree of sequence similarity to glycerol-3-phosphate dehydrogenases have been isolated from the yeast Pichiajadinii. Fragments of the genes were PCR-amplified with degenerated primers from genomic DNA of P. jadinii. Clones containing the full-length genes PjGPDI and PjGPD2 were isolated by screening genomic libraries. DNA sequencing revealed open reading frames (ORFs) of 1182 bp and 1185 bp for PjGpdlp and PjGpd2p, respectively. In a complementation study PjGPD1 rescued the growth defect of a Saccharomyces cerevisiae Agpdl mutant strain under osmotic stress, while complementation by PjGPD2 is temperature sensitive. The sequences of the PjGPD1 and PjGPD2 ORFs have been submitted to the EMBL Nucleotide Sequence Database under Accession No. AJ632339 and AJ632340, the sequences of the corresponding genomic DNA fragments under Accession No. AJ632341 and AJ635370, respectively.     

毕赤酵母发酵液的脱色和重组水蛭素的分离

用毕赤酵母表达重组水蛭素的发酵液中含有大量色素和一些杂蛋白。本文对发酵上清的脱色和重组水蛭素的分离进行了探讨。鉴于水蛭素的稳定性 ,用加热法预处理发酵上清取得了满意的效果。对离子交换、疏水层析、凝胶过滤、羟基磷灰石吸附层析以及它们的优化组合进行了研究 ,结果表明 ,阳离子交换层析、凝胶过滤和阴离子交换层析的组合对发酵上清的脱色和重组水蛭素的分离是有效的。     

Isolation and Characterization of Amber Suppressors in Yeast

Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome c in cyc1-179 strains suppressed with these efficient amber suppressors.     

Isolation and Characterization of a Thermotolerant Methanol-Utilizing Yeast

A yeast capable of growth on methanol as its sole carbon-energy source was isoalted from soil samples and identified as a strain of Hansenula polymorpha. A continuous enrichment culture at 37 C with a simple mineral salts medium was used to select this organism. The isolate, designated DL-1, has a maximal specific growth rate of 0.22 per h, at pH 4.5 to 5.5 and temperatures of 37 to 42 C, in simple mineral salts medium with methanol (0.5%), biotin, and thiamine. Growth occurred in a chemostat at temperatures up to 50 C, with strong growth at 45 C. The maximal growth yield of the yeast on methanol was 0.36 g of dry cell weight per g of methanol, and the yield on oxygen was 0.37 g of dry cell weight per g of O(2). Protein content of the isolate is 46%, and total nucleic acid content varies from 5.0 to 7.0% with increasing growth rate from 0.08 to 0.20 per h. The amino acid profile of this yeast protein indicates that it could serve as a good source of food protein. Feeding studies with rats show the yeast to have no toxic effects.     

前列腺特异膜抗原在Pichia pastoris酵母中的表达及鉴定

前列腺特异膜抗原是一种具有高度前列腺特异性的糖蛋白 ,其表达的增高与肿瘤的术后复发、激素抵抗及较差的预后正相关 ,在前列腺癌的诊断和治疗中有广泛的应用前景 .从前列腺癌组织提取总RNA ,利用RT PCR技术获得PSMA基因的全长序列 ,构建了重组酵母表达载体pPIC3.5K PSMA .电击法转化Pichiapastoris酵母 ,通过表型筛选和PCR鉴定证实该基因已稳定整合入Pichiapastoris酵母基因组中 .SDS PAGE显示 ,获得的PSMA蛋白分子量约 10 0kD ,表达产物经West ern印迹证实可特异地与PSMA单克隆抗体 4G5结合 .结果表明 ,成功地获得PSMA编码的cDNA并在酵母细胞中获得表达 .     

毕赤氏酵母醇氧化酶-2基因启动子突变体的分离和鉴定

巴斯毕赤我苯酵母表达系统已被广泛用于生产外源蛋白的寄主菌。利用该系统将外源基因整合交换到染色体上时,ＡＯＸ１基因被破力的甲醇利用缓慢,给发本报生产千古 定影响。在不改变现有表达系统前提下,从ＡＯＸＩ功能缺陷 株分离出Ｍｕｔ＾＋自发突变化突变体,通过突变体在甲醇培养基中生长曲线的测定,ＨＳＡ表达产物的聚丙烯酰胺凝胶电泳检测,证明突变体的甲醇利用能力和蛋白表达比原始菌株大大提高,突变体ＡＯＸ２基因上游     

Isolation and Characterization of Omnipotent Suppressors in the Yeast Saccharomyces Cerevisiae

Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the psi+ extrachromosomal determinant. The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers. The suppressors were further distinguished by differences in growth on paromomycin medium, hypertonic medium, low temperatures (10 degrees), nonfermentable carbon sources, alpha-aminoadipic acid medium, and by their dominance and recessiveness. Genetic analysis of 12 representative suppressors resulted in the assignment of these suppressors to 6 different loci, including the three previously described loci SUP35 (chromosome IV), SUP45 (chromosome II) and SUP46 (chromosome II), as well as three new loci SUP42 (chromosome IV), SUP43 (chromosome XV) and SUP44 (chromosome VII). Suppressors belonging to the same locus had a wide range of different phenotypes. Differences between alleles of the same locus and similarities between alleles of different loci suggest that the omnipotent suppressors encode proteins that effect different functions and that altered forms of each of the proteins can effect the same function.     

Isolation and Characterization of Chromosome-Gain and Increase-in-Ploidy Mutants in Yeast

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37°. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37°. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth.     

Whole Cell Formaldehyde Cross-Linking Simplifies Purification of Mitochondrial Nucleoids and Associated Proteins Involved in Mitochondrial Gene Expression

Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data.     

Production and Characterization of Recombinant Phanerochaete chrysosporium Cellobiose Dehydrogenase in the Methylotrophic Yeast Pichia pastoris

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.     

Isolation and Characterization of Pichia heedii Mutants Defective in Xylose Uptake

To investigate the role of xylose uptake in xylose metabolism in yeasts, we isolated a series of mutated strains of the yeast Pichia heedii which are defective in xylose utilization. Four of these demonstrated defects in xylose uptake. Overlaps between the functional or regulatory mechanisms for glucose and xylose uptake may exist in this yeast since some of the mutants defective in xylose uptake were also defective in glucose transport. None of the mutants were defective in xylose reductase or xylitol dehydrogenase activities.     

Isolation and Partial Characterization of a Cytochrome b Complex and Cytochrome Oxidase from Yeast Mitochondria

A cytochrome b complex and cytochrome oxidase have been purified 14- and 20-fold respectively from yeast submitochondrial particles by a simple procedure involving their spontaneous precipitation from a deoxycholate extract. The recovery of both proteins was almost quantitative. The specific heme contents were 11 and 8 nmoles/mg protein for the cytochrome b complex and cytochrome oxidase respectively and both were spectrally pure. Sodium dodecyl sulfate gel electrophoresis resolved the cytochrome b complex into seven distinct subunits with molecular weights 42, 000, 33, 000, 27, 500, 23, 000, 15, 500, 13, 000 and 10, 500. Cytochrome oxidase contained five bands with molecular weights 42, 000, 26, 500, 21, 000, 14, 000 and 10, 500. Much of the cytochrome b complex (and all of the cytochrome oxidase) could be resolubilized in aqueous buffer following precipitation from the deoxycholate extract. The fraction of the cytochrome b preparation which remained insoluble appeared identical to the soluble protein in terms of polypeptide composition but contained less phospholipid and bound detergent, suggesting that insolubility may result from interaction between hydrophobic regions otherwise occupied by amphiphiles. The soluble cytochrome b complex migrated as a single species upon analytical ultracentrifugation and column chromatography, and during electrophoresis on polyacrylamide gels. Triton X-100, urea, or bile salts, failed to dissociate the complex. These findings suggest that the subunits are tightly associated in situ.