22 results on '"Ronald A. Hitzeman"'
Search Results
2. Vanadate-resistant yeast mutants are defective in protein glycosylation
- Author
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Lun Ballou, Clinton E. Ballou, Mark Lewis, and Ronald A. Hitzeman
- Subjects
Glycosylation ,Mutant ,Saccharomyces cerevisiae ,Biology ,medicine.disease_cause ,chemistry.chemical_compound ,medicine ,Glycoproteins ,chemistry.chemical_classification ,Mutation ,Multidisciplinary ,Genetic Complementation Test ,Drug Resistance, Microbial ,biology.organism_classification ,Phenotype ,Molecular biology ,carbohydrates (lipids) ,Complementation ,chemistry ,Biochemistry ,Cinnamates ,Hygromycin B ,Vanadates ,Glycoprotein ,Protein Processing, Post-Translational ,Research Article - Abstract
Spontaneous recessive orthovanadate-resistant mutants of Saccharomyces cerevisiae were obtained in five complementation groups, and all show defects in protein glycosylation that mimic the previously isolated mnn mutants. Three of the groups are allelic to the known mnn8, mnn9, and mnn10 mutants, whereas the other two groups show other glycosylation defects. The vanadate-resistant phenotype was associated with enhanced hygromycin B sensitivity. The glycosylation phenotypes of the mutants are all reflections of defects in glycoprotein trafficking, and the easy isolation of vanadate-resistant or hygromycin B-sensitive mutants should facilitate the study of this process.
- Published
- 1991
3. Automatic Eukaryotic Artificial Chromosomes
- Author
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Carole Weaver, Bryant E. Fong, Nathan C. Hitzeman, Ronald A. Hitzeman, Chin Y. Loh, Meghan E. Bowser, George E. Chisholm, and Lynne M. Giere
- Subjects
Yeast artificial chromosome ,Genetics ,Eukaryotic chromosome fine structure ,C-value ,Chromosome ,Human artificial chromosome ,Computational biology ,Bacterial genome size ,Biology ,Genome ,Eukaryotic chromosome structure - Abstract
Publisher Summary This chapter discusses the automatic eukaryotic artificial chromosomes (AEAC). Evolutionary theory proposes that mitochondria, plastids, and chloroplasts originated by the engulfment or cell fusion of prokaryotes by eukaryotes. As this endo-symbiotic relationship evolved, the size of the bacterial DNA genome decreased and the functions of genes lost from the bacterial genome were assumed by the eukaryotic chromosome. These experiments demonstrate a new technology that could be used for transferring an entire prokaryotic genome or other large DNA molecules, in circular form, into a eukaryotic cell, where the circular DNA is automatically converted into an artificial linear chromosome in vivo. The production of AEAC-bacterial genomes for animals, human cells (AHACs), and plants preferably require different telomeres, centromeres, and selectable markers that function in these cells to make automatic chromosomes. AHACs carrying genomic or cDNAs for the necessary genes to be transferred would be ideal because of their formation of automatic functional chromosomes upon reaching the nuclei of the human cells.
- Published
- 2002
4. Production of human type I collagen in yeast reveals unexpected new insights into the molecular assembly of collagen trimers
- Author
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Winson Ong, P. David Toman, George E. Chisholm, David R. Olsen, Robert C. Chang, Scott D. Leigh, Ronald A. Hitzeman, Ernest Tai, David E. Birk, Hugh McMullin, and Richard A. Berg
- Subjects
Protein Folding ,Saccharomyces cerevisiae ,Biochemistry ,law.invention ,Protein structure ,law ,Endopeptidases ,Humans ,Collagenases ,Protein precursor ,Protein Structure, Quaternary ,Molecular Biology ,Sequence Deletion ,Chemistry ,Circular Dichroism ,Cell Biology ,Cell biology ,Collagen, type I, alpha 1 ,Procollagen peptidase ,Microscopy, Electron ,Recombinant DNA ,Protein folding ,Collagen ,Type I collagen ,Procollagen ,Triple helix - Abstract
Substantial evidence supports the role of the procollagen C-propeptide in the initial association of procollagen polypeptides and for triple helix formation. To evaluate the role of the propeptide domains on triple helix formation, human recombinant type I procollagen, pN-collagen (procollagen without the C-propeptides), pC-collagen (procollagen without the N-propeptides), and collagen (minus both propeptide domains) heterotrimers were expressed in Saccharomyces cerevisiae. Deletion of the N- or C-propeptide, or both propeptide domains, from both proalpha-chains resulted in correctly aligned triple helical type I collagen. Protease digestion assays demonstrated folding of the triple helix in the absence of the N- and C-propeptides from both proalpha-chains. This result suggests that sequences required for folding of the triple helix are located in the helical/telopeptide domains of the collagen molecule. Using a strain that does not contain prolyl hydroxylase, the same folding mechanism was shown to be operative in the absence of prolyl hydroxylase. Normal collagen fibrils were generated showing the characteristic banding pattern using this recombinant collagen. This system offers new opportunities for the study of collagen expression and maturation.
- Published
- 2001
5. Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae
- Author
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Robert C. Chang, Lynne M. Giere, Scott D. Leigh, David R. Olsen, George E. Chisholm, Ronald A. Hitzeman, Robert J. Kovach, Richard A. Berg, P. David Toman, Bryant E. Fong, Gregory A. Daniels, and Hugh McMullin
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integumentary system ,biology ,Saccharomyces cerevisiae ,macromolecular substances ,Cell Biology ,biology.organism_classification ,Biochemistry ,Saccharomyces ,Recombinant Proteins ,law.invention ,Culture Media ,Procollagen peptidase ,Biopolymers ,law ,Heterotrimeric G protein ,Gene expression ,Recombinant DNA ,Humans ,Protein precursor ,Molecular Biology ,Gene ,Procollagen - Abstract
The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.
- Published
- 2000
6. Aspergillus Niger var. Awamori as a Host for the Expression of Heterologous Genes
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Kirk J. Hayenga, Randy M. Berka, Daniel Cullen, Michael W. Rey, Nigel Dunn-Coleman, Michael H. Lamsa, Ronald A. Hitzeman, Lori J. Wilson, Frank T. Bayliss, Katherine H. Kodama, Peggy Bloebaum, Melinda M. Przetak, and Michael Ward
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Genetics ,biology ,Saccharomyces cerevisiae ,Heterologous ,biology.organism_classification ,medicine.disease_cause ,Yeast ,Biochemistry ,Aspergillus nidulans ,Gene expression ,medicine ,Escherichia coli ,Gene ,Aspergillus awamori - Abstract
Among the diversity of cellular systems that have been developed for the expression of heterologous gene products, certain species of filamentous fungi possess features which make them exceptionally attractive for this purpose. These include (a) the ability to produce high levels (>25 grams per liter) of secreted protein in submerged culture, (b) a long history of safe use in the production of enzymes, antibiotics, and biochemicals which are used for human consumption, and (c) established fermentation processes which are inexpensive by comparison with animal cell culture processes done on a similar scale. These attributes have prompted several biotechnology companies to explore the use of filamentous fungi as hosts for the expression and secretion of foreign proteins. Some of the heterologous gene products which have been made using fungal expression systems are shown in Table 1. Compared to highly refined expression systems such as Escherichia coli or Saccharomyces cerevisiae, the evolution of filamentous fungi as hosts has barely begun, and many fundamental aspects of cell biology and biochemistry in fungi have not been studied. Fortunately, many of the molecular details and principles which have been elucidated in yeast and in mammalian cell systems appear to be applicable to the study of heterologous gene expression and protein secretion in filamentous fungi as well.
- Published
- 1991
7. [35] Use of heterologous and homologous signal sequences for secretion of heterologous proteins from yeast
- Author
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Roger Pai, Donald Dowbenko, Chung Liu, Mark Renz, Arjun Singh, Nancy J. Simpson, Chang Cn, Robert Hamilton, Vanessa Chisholm, Christina Y. Chen, William J. Kohr, and Ronald A. Hitzeman
- Subjects
Signal peptide ,Plasmid ,biology ,Biochemistry ,Saccharomyces cerevisiae ,Heterologous ,Secretion ,Heterologous expression ,biology.organism_classification ,Molecular biology ,Peptide sequence ,Yeast - Published
- 1990
8. [37] Molecular and genetic approach to enhancing protein secretion
- Author
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Ronald A. Hitzeman, Christina Y. Chen, Nancy J. Simpson, and Vanessa Chisholm
- Subjects
Genetics ,Mutation ,Secretory protein ,Gene expression ,Saccharomyces cerevisiae ,medicine ,Secretion ,Biology ,medicine.disease_cause ,biology.organism_classification ,Cell biology - Published
- 1990
9. Expression of hepatitis B virus surface antigen in yeast
- Author
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Jeffrey V. Miller, Hermann Oppermann, Chung-Cheng Liu, Avima Yaffe, Ronald A. Hitzeman, David A. Estell, Eric J. Patzer, Dennis G. Kleid, Christina Y. Chen, Arthur D. Levinson, and Frank E. Hagie
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HBsAg ,Hepatitis B Surface Antigens ,Hepatitis B virus DNA polymerase ,Autonomously replicating sequence ,Structural gene ,Saccharomyces cerevisiae ,Biology ,biology.organism_classification ,Molecular biology ,Yeast ,Molecular Weight ,Methionine ,Plasmid ,Gene Expression Regulation ,Genetics ,Cysteine ,Gene - Abstract
The structural gene of Hepatitis B virus surface protein (HBsAg) was introduced into a plasmid capable of autonomous replication and selection in both the yeast Saccharomyces cerevisiae and E. coli. In this plasmid transcription of the HBsAg is initiated by the 5'-flanking sequence of the yeast 3-phosphoglycerate kinase (PGK) gene and terminated by the 3'-flanking region of the yeast TRP1 gene. Yeast cells containing this plasmid produce a new major species of mRNA of 1200 nucleotides in length coding for HBsAg. Viral surface antigen is made in nonglycosylated form at a level of about 1-2 percent of total yeast protein. A small fraction of this polypeptide (2-5 percent) is found in aggregated form upon yeast cell disruption by glass beads. This material is similar in size, density, and shape to the 22nm particle, isolated from the plasma of human hepatitis carriers, and induced comparable levels of HBsAg antibodies in mice when compared with the natural particle.
- Published
- 1983
10. Secretion of Human Interferons by Yeast
- Author
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Howard L. Levine, David W. Leung, Ronald A. Hitzeman, L. J. Perry, William J. Kohr, and David V. Goeddel
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Signal peptide ,Multidisciplinary ,biology ,Cell growth ,Saccharomyces cerevisiae ,Protein Sorting Signals ,biology.organism_classification ,Yeast ,Microbiology ,Cell biology ,Plasmid ,Gene Expression Regulation ,Humans ,Eukaryote ,Secretion ,Interferons ,Cloning, Molecular ,RNA Processing, Post-Transcriptional ,Peptides ,Protein Processing, Post-Translational ,Gene ,Plasmids - Abstract
Plasmids were constructed to direct synthesis of the human interferons IFN-alpha 1, IFN-alpha 2, and IFN-gamma in the yeast Saccharomyces cerevisiae. Expression of IFN genes containing coding sequences for secretion signals resulted in the secretion of IFN activity. A large proportion of the IFN-alpha 1 and IFN-alpha 2 isolated from the yeast cell growth media had the same amino termini as the natural mature interferons, suggesting a removal of the signal sequences identical to that of human cells. These results show that a lower eukaryote, such as yeast, can utilize and process a human signal sequence.
- Published
- 1983
11. Human, yeast and hybrid 3-phosphoglycerate kinase gene expression in yeast
- Author
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Christina Y. Chen and Ronald A. Hitzeman
- Subjects
Phosphoglycerate kinase ,Protein Conformation ,Genes, Fungal ,Structural gene ,Saccharomyces cerevisiae ,DNA Restriction Enzymes ,Biology ,biology.organism_classification ,Molecular biology ,Gene dosage ,Yeast ,Phosphoglycerate Kinase ,Genes ,Biochemistry ,Sequence Homology, Nucleic Acid ,Complementary DNA ,Gene expression ,Genetics ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Gene ,Plasmids - Abstract
When the gene for yeast 3-phosphoglycerate kinase (PGK) is present on a high copy number plasmid in Saccharomyces cerevisiae, 30-40 percent of yeast protein is produced as PGK. However, when the structural part of this gene is replaced by as many as twenty different heterologous genes, production of gene products is greatly reduced--usually by more than 20 fold. This decrease in protein production is accompanied by large decreases in the steady-state levels of mRNA. However, in contrast to these coding sequences, replacement of the yeast PGK structural gene with a human PGK cDNA has little effect on the steady-state mRNA level in yeast. PGK is a two-domain enzyme and its 3-dimensional structure is highly conserved among species. These observations and others have led us to propose that the PGK protein itself might influence its own mRNA levels (Chen et al., Nucleic Acids Res. 12, pp. 8951-8969, 1984). In addition, data is presented here which suggest that the human PGK mRNA is less efficiently translated than the yeast PGK mRNA. Two different mechanisms of controlling gene expression are indicated. Both mechanisms appear to be independent of gene copy number.
- Published
- 1987
12. Saccharomyces cerevisiae Secretes and Correctly Processes Human Interferon Hybrid Proteins Containing Yeast Invertase Signal Peptides
- Author
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J. J. Wulf, L. J. Perry, M. Matteucci, Chung Nan Chang, Ronald A. Hitzeman, and Christina Y. Chen
- Subjects
Signal peptide ,Signal peptidase ,Signal recognition particle ,Expression vector ,Glycoside Hydrolases ,beta-Fructofuranosidase ,biology ,Saccharomyces cerevisiae ,DNA Restriction Enzymes ,Cell Biology ,Protein Sorting Signals ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Yeast ,Invertase ,Biochemistry ,Interferon Type I ,Escherichia coli ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Research Article - Abstract
Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.
- Published
- 1986
13. Active Human-Yeast Chimeric Phosphoglycerate Kinases Engineered by Domain Interchange
- Author
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Maria T. Mas, Arthur D. Riggs, Christina Y. Chen, and Ronald A. Hitzeman
- Subjects
Models, Molecular ,Conformational change ,Protein Conformation ,Genes, Fungal ,Saccharomyces cerevisiae ,Protein structure ,Humans ,Amino Acid Sequence ,Binding site ,Peptide sequence ,chemistry.chemical_classification ,Phosphoglycerate kinase ,Binding Sites ,Multidisciplinary ,biology ,Chimera ,Active site ,Yeast ,Kinetics ,Phosphoglycerate Kinase ,Enzyme ,Genes ,Biochemistry ,chemistry ,biology.protein ,Protein Multimerization ,Genetic Engineering ,Plasmids - Abstract
Phosphoglycerate kinase (PGK) is a monomeric protein composed of two domains of approximately equal size, connected by a hinge. Substrate-induced conformational change results in the closure of the active site cleft, which is situated between these two domains. In a study of the relations between structure and function of this enzyme, two interspecies hybrids were constructed, each composed of one domain from the human enzyme and one domain from the yeast enzyme. Despite a 35% difference in the amino acid composition between human and yeast PGK, catalytic properties of the hybrid enzymes are very similar to those of the parental proteins. This result demonstrates that the evolutionary substitutions within these two distantly related molecules do not significantly affect formation of the active site cleft, mechanism of domain closure, or enzyme activity itself.
- Published
- 1986
14. Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone
- Author
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Ronald A. Hitzeman, Chung N. Chang, Peter H. Seeburg, June M. Lugovoy, Ellson Y. Chen, and Arjun Singh
- Subjects
Genetics ,chemistry.chemical_classification ,biology ,Base Sequence ,Saccharomyces cerevisiae ,Nucleic acid sequence ,Nucleic Acid Hybridization ,DNA Restriction Enzymes ,biology.organism_classification ,Pheromones ,Amino acid ,Nucleic acid thermodynamics ,chemistry ,Biochemistry ,Coding region ,Animals ,Genomic library ,Amino Acid Sequence ,Cloning, Molecular ,Gene ,Peptide sequence ,Plasmids - Abstract
Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.
- Published
- 1983
15. SYNTHESIS OF HUMAN INTERFERONS AND ANALOGS IN HETEROLOGOUS CELLS
- Author
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Diane Pennica, Richard M. Lawn, David V. Goeddel, Frank E. Hagie, H. M. Shepard, A D Levinson, Patrick W. Gray, Rik Derynck, Richard Najarian, David W. Leung, Elizabeth Mcleod Yelverton, Ronald A. Hitzeman, Axel Ullrich, and Pamela J. Sherwood
- Subjects
Antigenicity ,Biological studies ,Heterologous ,Biology ,Molecular biology ,law.invention ,genomic DNA ,medicine.anatomical_structure ,Immune system ,law ,Complementary DNA ,Recombinant DNA ,medicine ,Fibroblast - Abstract
Publisher Summary Human interferons (IFNs) are classified in three groups based on antigenicity: the acid-stable α-(leukocyte)-IFN, the acid-stable β-(fibroblast)-IFN, and the acid-labile γ-(immune)-IFN. The application of the recombinant DNA technology has caused a major breakthrough in the study of these IFNs and has led to the establishment of their structure. cDNA clones for different human IFNs have been isolated and characterized. High-level bacterial expression of these has been obtained, thus allowing structural and biological studies with the purified proteins. Hybridization studies with total genomic DNA and with a cloned human chromosomal DNA library have revealed the existence of a single gene corresponding to the human β-IFN cDNA. The bacterial synthesis of mature human fibroblast IFN has been achieved in E. coli using either the lac- or the trp-promoter. cDNA clones for human α-IFN have also been isolated using the same approach as for β-IFN.
- Published
- 1982
16. Expression of Human Interferon-γ in Heterologous Systems Genentech, Inc. Contribution no. 134
- Author
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Ronald A. Hitzeman, Patrick W. Gray, David V. Goeddel, and Rik Derynck
- Subjects
chemistry.chemical_classification ,Messenger RNA ,biology ,Heterologous ,RNA ,Molecular biology ,Amino acid ,Immune system ,chemistry ,Interferon ,Complementary DNA ,medicine ,biology.protein ,Antibody ,medicine.drug - Abstract
Publisher Summary This chapter discusses the expression of human interferon-γ (IFN-γ) in heterologous systems. On induction with various mitogens, mammalian T lymphocytes from peripheral blood lymphocytes, tonsils, or other sources are stimulated to synthesize and secrete “immune” interferon or interferon-γ. This interferon is distinct from the leukocyte interferons (IFNs-α) or fibroblast interferon (IFN-β), not only by its cellular origin but also on the basis of neutralization studies with antibodies raised against different species. Its induction differs from IFN-α and IFN -β in that neither viral infection nor double-stranded RNA is sufficient for inducing its synthesis. Interferon-γ is also distinct in its biological activities from IFN-α and IFN-β. A cloned cDNA of human IFN-γ has been isolated, and synthesized using mRNA from induced peripheral blood lymphocytes. Nucleotide-sequence analysis of the full-size cDNA insert of 1200 bp revealed the presence of a reading frame coding for 166 amino acids. The N-terminal 20 amino acids constitute the putative signal sequence, cleaved off during the secretion from the cell.
- Published
- 1983
17. Deoxyribonucleoside Triphosphates and DNA Polymerase in Bacteriophage PBS1-Infected Bacillus Subtilis
- Author
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Ronald A. Hitzeman, Henrik Møllgaard, Jan Neuhard, and Alan R. Price
- Subjects
chemistry.chemical_classification ,Deoxyribonucleoside triphosphate ,biology ,DNA polymerase ,Uracil ,Bacillus subtilis ,biology.organism_classification ,Thymine ,Deoxyribonucleoside ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biochemistry ,biology.protein ,DNA - Abstract
Bacteriophage PBS1 and its clear-plaque variant PBS2 are unique viruses in that their DNA contains uracil instead of thymine (1,2). One of the changes affecting deoxyribonucleotide metabolism in Bacillus subtilis following PBS1 or PBS2 infection (see Fig. 1) is an increase in the level of DNA polymerase activity. We predicted that the phage-induced DNA polymerase would be a new enzyme, whose substrate in vivo would be dUTP for the synthesis of uracil-DNA. This report describes the discovery of dUTP in vivo, evidence for its biosynthetic origin, and characteristics of the purified phage DNA polymerase.
- Published
- 1978
18. The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase
- Author
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Frank E. Hagie, Ronald A. Hitzeman, Joei S. Hayflick, Peter H. Seeburg, Rik Derynck, and Christina Y. Chen
- Subjects
Genetics ,Phosphoglycerate kinase ,Base Sequence ,Transcription, Genetic ,Nucleic acid sequence ,Protein primary structure ,DNA Restriction Enzymes ,Saccharomyces cerevisiae ,Biology ,Phosphoglycerate Kinase ,Open reading frame ,Genes ,Biochemistry ,Consensus sequence ,Coding region ,Amino Acid Sequence ,RNA, Messenger ,Gene ,Peptide sequence ,Research Article ,Plasmids - Abstract
The DNA sequence of the gene for the yeast glycolytic enzyme, 3-phosphoglycerate kinase (PGK), has been obtained by sequencing part of a 3.1 kbp HindIII fragment obtained from the yeast genome. The structural gene sequence corresponds to a reading frame of 1251 bp coding for 416 amino acids with no intervening DNA sequences. The amino acid sequence is approximately 65 percent homologous with human and horse PGK protein sequences and is in general agreement with the published protein sequence for yeast PGK. As for other highly expressed structural genes in yeast, the coding sequence is highly codon biased with 95 percent of the amino acids coded for by a select 25 codons (out of 61 possible). Besides structural DNA sequence, 291 bp of 5'-flanking sequence and 286 bp of 3'-flanking sequence were determined. Transcription starts 36 nucleotides upstream from the translational start and stops 86-93 nucleotides downstream from the translational stop. These results suggest a non-polyadenylated mRNA length of 1373 to 1380 nucleotides, which is consistent with the observed length of 1500 nucleotides for polyadenylated PGK mRNA. A sequence TATATATAAA is found at 145 nucleotides upstream from the translational start. This sequence resembles the TATAAA box that is possibly associated with RNA polymerase II binding.
- Published
- 1982
19. Contributors
- Author
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MERVYN J. BIBB, JAMES R. BROACH, J. PAUL BURNETT, KEITH F. CHATER, JACK COLEMAN, RIK DERYNCK, DAVID DUBNAU, MARY-JANE GETHING, ELI GILBOA, DAVID V. GOEDDEL, MAX E. GOTTESMAN, PATRICK W. GRAY, DEAN H. HAMER, PATRICK HEARING, RONALD A HITZEMAN, YEN-SEN HO, DAVID A. HOPWOOD, BRUCE H. HOWARD, MASAYORI INOUYE, SUMIKO INOUYE, MAKKUNI JAYARAM, JOHN D. KEMP, YU-YANG LI, YOSHIHIRO MASUI, RICHARD C. MULLIGAN, MARTIN ROSENBERG, JOE SAMBROOK, ALLAN SHATZMAN, THOMAS SHENK, GEORGE P. VLASUK, LING-CHUAN CHEN WU, and THOMAS YEE
- Published
- 1983
20. Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae
- Author
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Ronald A. Hitzeman, Christina Y. Chen, and Hermann Oppermann
- Subjects
Transcription, Genetic ,Saccharomyces cerevisiae ,Genes, Fungal ,Genetic Vectors ,Heterologous ,Biology ,medicine.disease_cause ,Chromosomes ,Plasmid ,Genetics ,medicine ,Escherichia coli ,Animals ,RNA, Messenger ,Gene ,Mutation ,Phosphoglycerate kinase ,Nucleic Acid Hybridization ,DNA Restriction Enzymes ,biology.organism_classification ,Molecular biology ,Yeast ,Phosphoglycerate Kinase ,Genes ,Cattle ,Heterologous expression ,Plasmids - Abstract
DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels.
- Published
- 1984
21. Expression of a human gene for interferon in yeast
- Author
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Ronald A. Hitzeman, David V. Goeddel, Frank E. Hagie, Gustav Ammerer, Howard L. Levine, and Benjamin D. Hall
- Subjects
Transcription, Genetic ,Autonomously replicating sequence ,Genetic Linkage ,Saccharomyces cerevisiae ,DNA, Recombinant ,Biology ,law.invention ,chemistry.chemical_compound ,Plasmid ,Transformation, Genetic ,law ,Operon ,Humans ,Genomic library ,Cloning, Molecular ,Gene ,Multidisciplinary ,biology.organism_classification ,Molecular biology ,Yeast ,Alcohol Oxidoreductases ,chemistry ,Gene Expression Regulation ,Genes ,Protein Biosynthesis ,Recombinant DNA ,Interferons ,DNA ,Plasmids - Abstract
A DNA sequence coding for mature human leukocyte interferon D (LeIF-D) was linked with DNA fragments of the 5'-flanking sequences of the Saccharomyces cerevisiae (yeast) alcohol dehydrogenase I gene in a plasmid capable of autonomous replication and selection in both yeast and Escherichia coli. Yeast cells transformed by these plasmids synthesize up to 1 x 10(6) molecules of biologically active LeIF-D per cell.
- Published
- 1981
22. Protein products from yeast
- Author
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Chang Cn, Chen Cy, Chisholm, Donald Dowbenko, Arjun Singh, Etcheverry T, Ronald A. Hitzeman, Vehar G, Mark Renz, and William J. Kohr
- Subjects
Glycosylation ,Text mining ,Gene Expression Regulation ,Chemistry ,business.industry ,Retroviridae Proteins ,HIV ,Saccharomyces cerevisiae ,Computational biology ,HIV Envelope Protein gp120 ,business ,Biochemistry ,Yeast - Published
- 1988
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