Characterization of the SAC3 gene of Saccharomyces cerevisiae

A temperature-sensitive mutation (act1-1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC3 gene. A DNA fragment containing the SAC3 gene was sequenced. SAC3 codes for a 150 kDa hydrophillic protein which does not show any significant similarities with other proteins in the databases. Sac3 therefore is a novel yeast protein. A nuclear localization of Sac3 is suggested by the presence of a putative nuclear localization signal in the Sac3 sequence. A SAC3 disruption mutation was constructed. SAC3 disruption mutants were viable but grew more slowly and were larger than wild-type cells. In contrast to the sac3-1 mutation, the SAC3 disruption was not able to suppress the temperature sensitivity and the osmosensitivity of the act1-1 mutant. This demonstrates that act1-1 suppression by sac3-1 is not the result of a simple loss of SAC3 function. Furthermore, we examined the act1-1 and the sac3 mutants for defects in polarized cell growth by FITC-Concanavalin A (Con A)-labelling. The sac3 mutants showed a normal ConA-labelling pattern. In the act1-1 mutant, however, upon shift to non-permissive temperature, newly synthesized cell wall material, instead of being directed towards the bud, was deposited at discrete spots in the mother cell.     

Lunasin induces apoptosis and modifies the expression of genes associated with extracellular matrix and cell adhesion in human metastatic colon cancer cells

Scope: Lunasin is an arginine‐glycine‐aspartic acid (RGD) cancer preventive peptide. The objective was to evaluate the potential of lunasin to induce apoptosis in human colon cancer cells and their oxaliplatin‐resistant (OxR) variants, and its effect on the expression of human extracellular matrix and adhesion genes. Methods and results: Various human colon cancer cell lines which underwent metastasis were evaluated in vitro using cell flow cytometry and fluorescence microscopy. Lunasin cytotoxicity to different colon cancer cells correlated with the expression of α₅b₁ integrin, being most potent to KM12L4 cells (IC₅₀ = 13 μM). Lunasin arrested cell cycle at G2/M phase with concomitant increase in the expression of cyclin‐dependent kinase inhibitors p21 and p27. Lunasin (5–25 μM) activated the apoptotic mitochondrial pathway as evidenced by changes in the expressions of Bcl‐2, Bax, nuclear clusterin, cytochrome c and caspase‐3 in KM12L4 and KM12L4‐OxR. Lunasin increased the activity of initiator caspase‐9 leading to the activation of caspase‐3 and also modified the expression of human extracellular matrix and adhesion genes, downregulating integrin α₅, SELE, MMP10, integrin β₂ and COL6A1 by 5.01‐, 6.53‐, 7.71‐, 8.19‐ and 10.10‐fold, respectively, while upregulating COL12A1 by 11.61‐fold. Conclusion: Lunasin can be used in cases where resistance to chemotherapy developed.     

Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y

We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH₂-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene. Using the S. cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P. pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned. The open reading frame of the P. pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids. The molecular mass of the protein is calculated to be 59·44 kDa without sugar chains. The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites. The NH₂-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant. There is 61% identity between the amino acid sequences of P. pastoris Prc1p and S. cerevisiae Prc1p. Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity. Over-expression of the PRC1 gene under regulation of the P. pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY. The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987.     

Development of the methylotrophic yeast Pichia methanolica for the expression of the 65 kilodalton isoform of human glutamate decarboxylase

We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD₆₅) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD₆₅ at levels approaching 0·5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD₆₅ was purified to greater than 90% purity. © 1998 John Wiley & Sons, Ltd.     

ISOLATION AND CHARACTERIZATION OF THE 11S GLOBULIN FROM AMARANTH SEEDS

Globulins of Amaranthus hypochondriacus were extracted with two different buffer systems which varied in ionic strength. SDS-PAGE analysis demonstrated that subunit patterns were different between the two extracting systems. Amarantin, the native 11S globulin of amaranth, and its subunits were purified by gel filtration chromatography and preparative electrophoresis. The native amarantin exhibited two heterogeneous forms of MW 330 and 400 kDa, which yielded the same subunit composition after reduction. SDS-PAGE analysis showed that prior to reduction, the 50–52 kDa subunit was the major band, and that after reduction, two new bands of 32–34 kDa and 22–24 kDa appeared. This is a typical characteristic of 11S proteins whose subunits consist of an acidic polypeptide (27–37 kDa) and a basic polypeptide (20–24 kDa) linked by a disulfide bond. Ultracentrification analysis showed that amarantin has a 11.9S sedimentation coefficient whereas DSC demonstrated that the denaturation temperatures in the presence of H₂O, Tris-HCl and K₂HPO₄-KH₂PO₄ are 99C, 99.8C and 103C, respectively.     

Sequence and functional analysis of a 7·2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II

In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79·2 kDa (corresponding gene YBR156c), 12·1 kDa (YBR157c), 62·7 kDa (YBR158w) and 38·7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grr1 (Cat80) protein with respect to the leucine-rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains. The DNA sequence was deposited at the EMBL data base with accession numbers Z36025 (YBR156c), Z36026 (YBR157c), Z36027 (YBR158w) and Z36028 (YBR159w).     

XV. Yeast sequencing reports. Nucleotide sequence of the GDS1 gene of Saccharomyces cerevisiae

We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae. The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database. GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms. Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product. The sequence has been deposited in the GenBank data library under Accession Number U18262.     

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein is similar to models proposed for other integral mambrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1·5 kb. The mae1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5′ to the MFm1 gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid. The sequence of the S. pombe mae1 gene is available in GenBank under Accession Number U21002.     

RIPENING‐ASSOCIATED PROTEOLYSIS OF A 27‐kDa MAJOR INTRINSIC PROTEIN (MBP27) IN TOMATO FRUIT1

Major intrinsic proteins (MIPs) are believed to contribute towards the maintenance of structural integrity, osmoregulation, and responses to water and salt stresses in higher plants. In this work we identified a 27‐kDa MIP (MIP27) in the microsomal membranes from tomato fruit using affinity purified antibodies to MIP27 from Beta vulgaris L. Sucrose gradient centrifugation analysis of microsomal membranes showed that MIP27 was associated with the plasma membrane and tonoplast fractions of tomato fruit. MIP27 aggregated to a 45‐kDa protein upon SDS‐polyacrylamide gel electrophoresis in the absence of dithiothreitol, a property characteristic of MIP proteins. MIP27 was degraded to a 25‐kDa protein as tomato fruit progressed through the various stages of ripeness. The proteolytic degradation of MIP27 to the 25‐kDa protein was also observed when microsomal membranes were treated with Pronase E. Treatment of microsomal membranes with thermolysin plus digitonin resulted in the complete degradation of MIP27. MIP27 was insensitive to treatments with trypsin and carboxypeptidase Y. The proteolytic degradation of MIP27 may play a role in the structural integrity and textural properties of tomato fruit during ripening.     

Honey major protein characterization and its application to adulteration detection

The major proteins in honey have different molecular weights depending upon the honeybee species. To confirm the origin of major honey proteins, honey protein produced by Apis cerana or Apis mellifera were purified and analyzed by MALDI-TOF. Two major proteins were identified as a major royal jelly protein 1. Although two major proteins shared primary structure, they showed different molecular weights of 56 and 59 kDa, respectively. To discriminate the honeybee species producing honey using SDS–PAGE, artificial marker proteins, 56 and 59 kDa, were produced from Escherichia coli. Two artificial marker proteins were co-electrophoresed with honey samples and the difference in molecular weight was readily distinguished by SDS–PAGE. Therefore, the measurement of major proteins in honey is a useful method to discriminate the honey that produced from different honeybee species.     

Sequence analysis of the CEN12 region of Saccharomyces cerevisiae on a 43·7 kb fragment of chromosome XII including an open reading frame homologous to the human cystic fibrosis transmembrane conductance regulator protein CFTR

In the framework of the European Union BIOTECH project for systematically sequencing the Saccharomyces cerevisiae genome, we determined the nucleotide sequence of a 43·7 kb DNA fragment spanning the centromeric region of chromosome XII. A novel approach was the distribution of sublibraries prepared by the DNA coordinator (J. Hoheisel, Heidelberg, FRG), using a new hybridization-based DNA mapping method, in order to facilitate ordered sequencing. The sequence contains 22 open reading frames (ORFs) longer than 299 bp, including the published sequences for ATS/DPS1, SCD25, SOF1, DRS1, MMM1, DNM1 and the centromeric region CEN12. Five putative ORF products show similarity to known proteins: the leucine zipper-containing ABC transporter L1313p to the yeast Ycf1p metal resistance protein, to the yeast putative ATP-dependent permease Yhd5p, to the yeast putative proteins Yk83p and Yk84p, to the human cystic fibrosis transmembrane conductance regulator protein (hCFTR) and to the human multidrug resistance-associated protein hMRP1; L1325p to the Drosophila melanogaster Pumilio protein, to the putative yeast regulatory protein Ygl3p and to the yeast protein Mpt5p/Htr1p; L1329p to human lipase A and gastric lipase, to rat lingual lipase and to the putative yeast triglyceride lipase Tgl1p; L1341p to the putative yeast protein Yhg4p; and the leucine zipper-containing L1361p to the two yeast proteins 00953p and Ym8156.08p and to the Arabidopsis thaliana protein HYP1. Eight ORFs show no homology to known sequences in the database, three small ORFs are internal and complementary to larger ones and L1301 is complementary overlapping the ATS/DPS1 gene. Additionally three equally spaced ARS consensus sequences were found. The nucleotide sequence reported here has been submitted to the EMBL data library under the accession number X91488.     

Analysis of the peroxisomal acyl‐CoA oxidase gene product from Pichia pastoris and determination of its targeting signal

Acyl‐CoA oxidase (Pox1p) is involved in the β‐oxidation of fatty acids and is targeted to the peroxisomal matrix via the use of different signals in various organisms. In rat, mouse and human, Pox1p contains a canonical peroxisomal targeting signal 1 (PTS1), whereas in the yeasts Candida tropicalis, Saccharomyces cerevisiae, C. maltosa and Yarrowia lipolytica neither a PTS1 nor a PTS2 sequence is present, suggesting that Pox1p might be targeted to the peroxisomes via a third unknown pathway. Alternatively, since proteins lacking a PTS sequence can enter peroxisomes in association with other polypeptides containing a PTS, Pox1p might ‘piggy‐back’ its way into the peroxisomal matrix together with other proteins. To understand the mechanism of peroxisomal targeting of a yeast Pox1p, we cloned the Pichia pastoris POX1 gene to study the pathway of import of PpPox1p into peroxisomes. The gene was cloned by PCR, hybridization and plasmid rescue. The 2157 bp gene encodes a protein with a predicted molecular weight of 80 kDa. Antisera against PpPox1p detected a protein specifically induced on oleate with an apparent molecular weight of 72 kDa. Immunolocalization studies confirmed the peroxisomal localization of PpPox1p. The carboxy‐terminus of PpPox1p ends with a PTS1‐like sequence, APKI. The sequence PKI was necessary for transport of PpPox1p into peroxisomes and interacted with the PTS1 receptor, Pex5p. Furthermore, addition of the sequence APKI to the C‐terminus of the green fluorescent protein directed this fusion protein to the peroxisome. Therefore, PpPox1p uses the PTS1 pathway for its import into peroxisomes. Copyright © 1999 John Wiley & Sons, Ltd.     

Modifying the antigen-immunization schedule improves the variety of monoclonal antibodies obtained from immune-phage antibody libraries against HIV-1 Nef and Vif

Immune phage antibody libraries are an attractive technology for isolating antigen-specific monoclonal antibodies (mAbs). Here we show that the immunization schedule affects the immune phage antibody library properties. We subcutaneously (s.c.) administered HIV-1 Nef and Vif antigens with different schedules (25 μg × 2 s.c. and 10 μg × 3 s.c.). The variety of isolated mAbs in 25 μg × 2 s.c. groups (Nef: 11 clones, Vif: 9 clones) was superior to that in the 10 μg × 3 s.c. groups (Nef: 2 clones, Vif: 1 clone). This finding suggests that it is important to optimize the immunization schedule for isolating a wide variety of mAbs.     

Cloning of a centromere binding factor 3d (CBF3D) gene from Candida glabrata

The gene encoding centromere binding factor 3d (CBF3D) of the human pathogenic yeast Candida glabrata has been isolated by hybridization of Saccharomyces cerevisiae CBF3D (ScCBF3D) DNA to a C. glabrata partial genomic library. Sequence analysis revealed a 540 bp open reading frame encoding a protein of 179 amino acids with a calculated molecular mass of 20·9 kDa. The amino acid sequence is highly homologous (78·6% identity) to ScCbf3d and 48·3% identical to the human homologue p19 (SKP1). Southern blot analysis indicates that CgCbf3d is encoded by an unique nuclear gene. The cloned CgCBF3D gene can functionally substitute the S. cerevisiae homologue in a S. cerevisiae CBF3D‐deletion mutant. The GenBank Accession No. for this gene is AF 072472. Copyright © 1999 John Wiley & Sons, Ltd.     

Electrophoresis to determine the molecular weight distribution in soluble proteins from various foods and their rumen‐resistant residues in cattle

Electrophoresis was used to visually identify and determine the molecular weight (MW) distribution of rumen‐degradable and rumen‐resistant or escape peptides in soluble proteins from 12 agricultural and distillers' food raw materials (RM) and their residues (RU) following 18 h of in sacco rumen incubation (dg₁₈) in cattle. Soluble proteins were extracted by using water, salt, acid and alkali in succession to represent albumins, globulins, glutalin 1 and glutalin 2 respectively. RM and RU differed substantially in the MW range, number and intensity of bands for various soluble proteins. The bands were mostly below the MW range of 66 kDa. Low‐MW (<25 kDa) peptides were greater in number than high‐MW (>25 kDa) peptides in almost all soluble proteins from RM. Individual peptides behaved differently during rumen incubation. Their resistance to or escape from rumen degradation varied with the class of food, type of soluble protein and their MW range. On average, low‐MW albumins in agricultural foods were more resistant to rumen degradation (0.41 RM vs 0.12 RU; 29%) than their high‐MW counterparts (0.12 RM vs 0.02 RU; 21%). In contrast, high‐MW glutalin 1 was more resistant (0.03 RM vs 0.22 RU) than low‐MW glutalin 1 (0.09 RM vs 0.26 RU) in most agricultural foods. Globulins contained the least and glutalins the most resistant peptides in distillers' foods. While this study reveals an association between dg₁₈ and protein type, structure and size, we do not recommend the immediate use of electrophoresis for routine food evaluation unless more studies are undertaken. It may, however, be suitable for further characterisation of the degradation of specific, selected peptides by specific micro‐organisms. © 2001 Society of Chemical Industry     

VII. Yeast sequencing reports. The sequence of a 27 kb segment on the right arm of chromosome VII from Saccharomyces cerevisiae reveals MOL1, NAT2, RPL30B,RSR1, CYS4, PEM1/CHO2, NSR1 genes and ten new open reading frames

The DNA sequence of a 26 677 bp fragment from the right arm of chromosome VII from Saccharomyces cerevisiae reveals 18 open reading frames (ORFs) longer than 300 bp. Eight ORFs correspond to previously characterized genes. G6620 is the 3′ end of the MOL1 gene coding for a polypeptide similar to stress-inducible proteins from Fusarium; G6630 is the NAT2 gene which encodes a methionine N-acetyltransferase; G6635 is the RPL30B gene coding for the ribosomal protein L30; G6658 is RSR1 encoding a ras-related protein; G6667 is CYS4, the gene for cystathionine β-synthase; G6670 is identical to ORF2 located close to CYS4; G6673 is PEM1/CHO2 encoding a phosphatidylethanolamine methyltransferase; G7001 is the NSR1 gene coding for a nuclear signal recognition protein. G6664 shares significant homology with the ORF YKR076w from chromosome XI. The other nine ORFs show no significant homology to any protein sequence presently available in the public data bases. The sequence has been deposited in the EMBL data library under Accession Number X85807.