Yeast Genomic DNA Library Research Articles

Screening for novel genes ofSaccharomyces cerevisiaeinvolved in recombinant antibody production

Cost-effective manufacturing of biopharmaceuticals in non-mammalian hosts still requires tremendous efforts in strain development. In order to expedite identification of novel leads for strain engineering, we used a transposon-mutagenized yeast genomic DNA library to create a collection of Saccharomyces cerevisiae deletion strains expressing a full-length IgG antibody. Using a high-throughput screening, transformants with either significantly higher or lower IgG expression were selected. The integration site of the transposon in three of the selected strains was located by DNA sequencing. The inserted DNA lay within the VPS30 and TAR1 open reading frame, and upstream of the HEM13 open reading frame. The complete coding sequence of these genes was deleted in the wild-type strain background to confirm the IgG expression phenotypes. Production of recombinant antibody was increased 2-fold in the Δvps30 strain, but only mildly affected secretion levels in the Δtar1 strain. Remarkably, expression of endogenous yeast acid phosphatase was increased 1.7- and 2.4-fold in Δvps30 and Δtar1 strains. The study confirmed the power of genome-wide high-throughput screens for strain development and highlights the importance of using the target molecule during the screening process.

Multipurpose Transposon-Insertion Libraries in Yeast.

Libraries of transposon-insertion alleles constitute powerful and versatile tools for large-scale analysis of yeast gene function. Transposon-insertion libraries are constructed most simply through mutagenesis of a plasmid-based genomic DNA library; modification of the mutagenizing transposon by incorporation of yeast selectable markers, recombination sites, and an epitope tag enables the application of insertion alleles for phenotypic screening and protein localization. In particular, yeast genomic DNA libraries have been mutagenized with modified bacterial transposons carrying the URA3 marker, lox recombination sites, and sequence encoding multiple copies of the hemagglutinin (HA) epitope. Mutagenesis with these transposons has yielded a large resource of insertion alleles affecting nearly 4000 yeast genes in total. Through well-established protocols, these insertion libraries can be introduced into the desired strain backgrounds and the resulting insertional mutants can be screened or systematically analyzed. Relative to alternative methods of UV irradiation or chemical mutagenesis, transposon-insertion alleles can be easily identified by PCR-based approaches or high-throughput sequencing. Transposon-insertion libraries also provide a cost-effective alternative to targeted deletion approaches, although, in contrast to start-codon to stop-codon deletions, insertion alleles might not represent true null-mutants. For protein-localization studies, transposon-insertion alleles can provide encoded epitope tags in-frame with internal codons; in many cases, these transposon-encoded epitope tags can provide a more accurate localization for proteins in which terminal sequences are crucial for intracellular targeting. Thus, overall, transposon-insertion libraries can be used quickly and economically and have a particular utility in screening for desired phenotypes and localization patterns in nonstandard genetic backgrounds.

Using Yeast Transposon-Insertion Libraries for Phenotypic Screening and Protein Localization.

This protocol details how to use a transposon-insertion library for phenotypic screening and protein localization. The insertion library was generated by mutagenesis of a plasmid-based yeast genomic DNA library by using a multipurpose transposon; the transposon produces gene disruptions, and, by Cre-mediated recombination at lox sites incorporated within the transposon, alleles with an in-frame insertion can be truncated to a residual transposon encoding multiple copies of the hemagglutinin epitope. Insertions are generated in yeast by shuttle mutagenesis. Yeast genomic DNA containing a transposon insertion is released from the library, and the mutagenized DNA sequences are introduced into a desired strain of yeast, where the insertion alleles replace native loci by homologous recombination. The insertion mutants can be screened for phenotypes, and the site of transposon insertion can subsequently be identified in selected mutants by inverse polymerase chain reaction (PCR). In-frame insertions within genes of interest can be truncated to an epitope-tagged allele by Cre-lox recombination, and the subcellular localization of the encoded protein product can be identified by standard methods of indirect immunofluorescence. In summary, the transposon-insertion libraries represent an informative resource for large-scale mutagenesis, presenting a straightforward alternative to labor-intensive targeted approaches for the construction of deletion alleles and fluorescent protein fusions.

Overexpression of ESBP6 improves lactic acid resistance and production in Saccharomyces cerevisiae

Polylactic acid plastics are receiving increasing attention for the control of atmospheric CO2 emissions. Lactic acid, the building block for polylactic acid, is produced by fermentation technology from renewable carbon sources. The yeast Saccharomyces cerevisiae, harboring the lactate dehydrogenases gene (LDH), produces lactic acid at a large scale due to its strong acid resistance, to its simple nutritional requirements and to its ease of genetic engineering. Since improvement of lactic acid resistance is correlated with an increase of lactic acid production under non-neutralizing condition, we isolated a novel gene that enhances lactic acid resistance using a multi-copy yeast genomic DNA library. In this study, we identified the ESBP6 gene, which increases lactic acid resistance when overexpressed and which encodes a protein with similarity to monocarboxylate permeases. Although ESBP6 was not induced in response to lactic acid stress, it caused weak but reproducible sensitivity to lactic acid when disrupted. Furthermore, intracellular pH in the ESBP6 overexpressing strain was higher than that in the wild-type strain under lactic acid stressed condition, suggesting that Esbp6 plays some roles in lactic acid adaptation response. The ESBP6 overexpressing strain carrying the LDH gene induced 20% increase in lactic acid production compared with the wild-type strain carrying the LDH gene under non-neutralizing conditions. These results indicate that overexpression of ESBP6 provides a novel and useful tool to improve lactic acid resistance and lactic acid production in yeast.

Multicopy suppression of oxidant-sensitive eos1 mutation by IZH2 in Saccharomyces cerevisiae and the involvement of Eos1 in zinc homeostasis

EOS1 is required for tolerance to oxidative stress in Saccharomyces cerevisiae; mutants are defective in the gene sensitive to hydrogen peroxide and tolerant to tunicamycin. To clarify the function of Eos1, we screened yeast genomic DNA libraries for heterologous genes that, when overexpressed from a plasmid, can suppress the hydrogen peroxide-sensitive eos1 mutation. We identified one such gene, IZH2, which has previously been reported to be a Zap1-regulated gene. However, the EOS1 and IZH2 genes do not themselves appear to be functionally interchangeable. Double disruption of the EOS1 and IZH2 genes yielded a slow-growth phenotype, suggesting that the two proteins are involved in related cellular processes. DNA microarray analysis revealed decreased expression of Zap1-regulated genes in the eos1-deletion mutant (Deltaeos1). Thus, it is likely that Eos1 is involved in zinc homeostasis.

Identifying Novel Factors Involved In Yeast Mitochondrial Protein Synthesis

The goal of our work is to identify protein factors involved in the initiation of mitochondrial translation using yeast genetics. FMT1 encodes the S. cerevisiae methionyl‐tRNA formyltransferase, which catalyzes the formylation of mitochondrial initiator Met‐tRNA. A genetic screen was performed with an fmt1 deletion strain to generate synthetic petite strains – strains containing mutations that in combination with the fmt1 deletion lead to the loss of respiratory growth. One mutant strain, s.p. 72–1, was transformed with a yeast genomic DNA library to identify open reading frames that complement the respiratory‐deficient phenotype. A region of chromosome XVI, including the LPE10 gene, was found to rescue growth on non‐fermentable carbon sources. Sequencing of DNA from the mutant strain revealed that the LPE10 gene, encoding a mitochondrial inner membrane magnesium transporter, contained a point mutation leading to a premature stop codon. This mutation likely causes the synthetic petite phenotype with the deletion of fmt1. Indeed, an lpe10 fmt1 double mutant has a more severe respiratory growth defect than the single lpe10 deletion strain. Deletion of the MIS1 gene, also required for the formylation of mitochondrial initiator tRNA, also leads to the petite phenotype in combination with the lpe10 deletion. This work is supported by HHMI and NSF grants to the Freshman Research Initiative at UT‐Austin.

Identifying the Cox24 Protein as a Factor Involved in Mitochondrial Protein Synthesis

The current understanding of mitochondrial protein synthesis is far from complete; it is likely that many accessory factors required for this process have not yet been identified. The FMT1 gene encodes the S. cerevisiae methionyl‐tRNA formyltransferase, which formylates the mitochondrial initiator Met‐tRNA. A genetic screen was designed to identify accessory factors involved in mitochondrial translation, particularly in the absence of formylated Met‐tRNA. This screen yielded yeast mutants that in combination with the fmt1 deletion, cause dysfunctional mitochondrial respiration. One mutant strain, s.p. 106–1, was transformed with a yeast genomic DNA library to identify open reading frames that complement the synthetic petite phenotype. Library plasmids that complemented the mutation were sequenced and resulting open reading frames were subcloned and expressed in the mutant strain. Expression of the COX24 gene rescued respiratory growth, and sequencing of s.p. 106–1 yeast genomic DNA revealed a T‐to‐C mutation in COX24 that leads to a leucine to proline substitution at amino acid 93. Studies indicate that Cox24p is required for proper splicing of mitochondrial COX1 pre‐mRNA. Our results indicate that Cox24p may also play a role in the initiation of mitochondrial translation, particularly when formylated Met‐tRNA is not available. Work supported by HHMI and NSF grants to the Freshman Research Initiative.

Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Delta mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Delta cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Delta cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.

Low‐copy episomal vector pFY20 and high‐saturation coverage genomic libraries for the fission yeast Schizosaccharomyces pombe

In fission yeast, as in many organisms, episomally replicating plasmid DNA molecules can be used for a wide variety of applications. However, replicating plasmids described previously are each propagated at a high copy number per cell. Plasmid fission yeast twenty (pFY20) contains the ura4(+) gene for positive and negative selection, an origin of replication (ars1) and a stability element (stb). Although this plasmid does not have a centromere, it is propagated with a copy number of about two plasmids per haploid genome equivalent and it is transmitted with relatively high fidelity in mitosis and meiosis. This low-copy vector is useful for screens and mutational studies where overexpression (e.g. from high copy plasmids) is undesirable. We therefore constructed multiple partial-digest, size-fractionated, fission yeast genomic DNA libraries in pFY20 and in the cloning vector pBluescript KS(+). These libraries have sufficient complexity (average of 2100 genome equivalents each) for saturation screening by complementation, plasmid shuffle or hybridization.

A novel yeast genomic DNA library on a geneticin-resistance vector

We have constructed a Saccharomyces cerevisiae genomic library utilizing a novel centromere-containing vector bearing the TRP1 and KANMX4 G418-resistance selectable markers. Random Sau3AI genomic DNA fragments with an average size of 6.4 kb were introduced into a unique BamHI site in the vector's polylinker. Six independent library pools were generated and the characteristics of these pools were determined. This library can be used to introduce low-copy genomic DNA fragments into any strain that is not G418-resistant, making it useful for low-copy overexpression analysis or complementation of mutant phenotypes in strains that lack a suitable auxotrophy marker or only exhibit phenotypes on rich, undefined, growth media.

Large-scale mutagenesis of the yeast genome using a Tn7-derived multipurpose transposon.

We present here an unbiased and extremely versatile insertional library of yeast genomic DNA generated by in vitro mutagenesis with a multipurpose element derived from the bacterial transposon Tn7. This mini-Tn7 element has been engineered such that a single insertion can be used to generate a lacZ fusion, gene disruption, and epitope-tagged gene product. Using this transposon, we generated a plasmid-based library of approximately 300,000 mutant alleles; by high-throughput screening in yeast, we identified and sequenced 9032 insertions affecting 2613 genes (45% of the genome). From analysis of 7176 insertions, we found little bias in Tn7 target-site selection in vitro. In contrast, we also sequenced 10,174 Tn3 insertions and found a markedly stronger preference for an AT-rich 5-base pair target sequence. We further screened 1327 insertion alleles in yeast for hypersensitivity to the chemotherapeutic cisplatin. Fifty-one genes were identified, including four functionally uncharacterized genes and 25 genes involved in DNA repair, replication, transcription, and chromatin structure. In total, the collection reported here constitutes the largest plasmid-based set of sequenced yeast mutant alleles to date and, as such, should be singularly useful for gene and genome-wide functional analysis.

Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase alpha.

Eukaryotic DNA polymerase (pol) alpha is a complex of four subunits. The subunits in the yeast Saccharomyces cerevisiae are: 167, 79, 62 and 48 kDa polypeptides. The p79 subunit has no known enzymatic functions, but it is essential for growth and chromosomal DNA replication. We have analyzed the interaction between the subunits of yeast pol alpha, particularly p167 and p79, using a yeast two-hybrid screen and deletion analysis. We have identified the interaction sites in each of these two subunits leading to p167.p79 complex formation, and correlated our results with the available genetic data. A detailed two-hybrid analysis, using the p79 gene as the bait and a yeast genomic DNA library, identified two independent groups of positive clones. One group that displayed strong positive interaction (delta1) with p79 represented a fusion of the p167 open reading frame at 3502 bp (Ile1168), and the second group, displaying weak positive interaction (delta2) with p79, had a fusion at 3697 bp (Asn1233) with the DNA-binding domain of the yeast Gal4 transcription factor. A detailed deletion analysis of the downstream region indicated the existence of two subdomains that interact with p79. Subdomain I encompasses a 65 amino acid segment between Ile1168 and Phe1232, and subdomain II is a 25 amino acid segment between Glu1259 and Leu1283. Deletion and two-hybrid interaction analysis of the p79 subunit of pol alpha revealed a complementary region with two subdomains: a 67 amino acid segment between Asn189 and Gln255 (I) and a 68 amino acid segment between Glu256 and Met323 (II). The p79 subdomains I and II appeared to interact with the p167 subdomains I and II, respectively. Analysis of interaction between p62 and various deletion clones of p167 did not result in an unambiguous and stable positive interaction in the two-hybrid screen between these two subunits. A strong interaction between p167 and p62 would probably require the presence of either p79 or p48 in the complex.

Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector.

Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.

A ubiquitin-proteasome system is responsible for the protection of yeast and human cells against methylmercury.

The mechanism responsible for the toxic effects of methylmercury (MeHg), an important environmental pollutant, is poorly understood. We have identified a gene, CDC34, that confers resistance to MeHg in Saccharomyces cerevisiae by screening a yeast genomic DNA library. CDC34 encodes a ubiquitin-conjugating enzyme, Cdc34, which is involved in ubiquitin-dependent proteolysis. Overexpression of Cdc34 results in significant resistance to MeHg both in yeast and human cells, and it increases the cellular level of ubiquitinated proteins. The ubiquitin-conjugating activity of Cdc34 is essential for the Cdc34-mediated resistance to MeHg, and the protective effect of the overexpression of Cdc34 is depressed by inhibition of proteasome activity. Our results support the hypothesis that MeHg induces the cellular accumulation of a certain protein(s) that causes cell damage and that this protein(s) is degraded after its ubiquitination in proteasomes.

Overexpression of L-glutamine: D-fructose-6-phosphate amidotransferase provides resistance to methylmercury in Saccharomyces cerevisiae

To identify novel genes that confer resistance to methylmercury (MeHg), a yeast genomic DNA library was transfected into Saccharomyces cerevisiae. Two functional plasmids were isolated from transfected yeast clones D1 and H5 that exhibited resistance to MeHg. The yeast transfected with plasmid isolated from clone H5 was several-fold more resistant than yeast transfected with plasmid from clone D1. Functional characterization of the genomic DNA fragment obtained from clone H5 determined that the GFA1 gene conferred resistance to MeHg. GFA1 was reported to encode L-glutamine: D-fructose-6-phosphate amidotransferase (GFAT) which catalyzes the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate. Accumulation of mercury in yeast clone W303B/pGFA1, which contains the transfected GFA1 gene, did not differ from that in control yeast clone W303B/pYES2. The W303B/pGFA1 strain did not show resistance to mercuric chloride, zinc chloride, cadmium chloride or copper chloride, suggesting that the resistance acquired by GFA1 gene transfection might be specific to MeHg. This is the first report of a gene involved in MeHg resistance in eukaryotic cells identified by screening a DNA library.

The yeast NOP4 gene product is an essential nucleolar protein required for pre-rRNA processing and accumulation of 60S ribosomal subunits.

The Saccharomyces cerevisiae NOP4 gene was isolated by screening a lambda gt11 yeast genomic DNA library with a monoclonal antibody against a yeast nucleolar protein. NOP4 encodes a 78 kDa protein that contains two prototypical RNA recognition motifs (RRMs) flanking an imperfect RRM lacking characteristic RNP1 and RNP2 motifs. In addition, there is a fourth incomplete RRM. NOP4 is a single copy essential gene present on chromosome XVI, between RAD1 and PEP4. To examine the function of Nop4p, we constructed a conditional null allele of NOP4 by placing this gene under the control of the glucose-repressible GAL1 promoter. When cells are shifted from galactose-containing medium to glucose-containing medium, NOP4 transcription is terminated, Nop4 protein is depleted and cell growth is impaired. Nop4 protein depletion results in diminished accumulation of 60S ribosomal subunits, assignable to a defect in ribosome biogenesis arising from a lack of production of mature 25S rRNA from 27S precursor rRNA.

A multitude of suppressors of group II intron-splicing defects in yeast.

Disruption of the nuclear MRS2 gene (mrs2-1 mutation) causes a strong pet- phenotype in strains with mitochondrial group II introns, and a leaky pet- phenotype in strains without group II introns. MRS3 and MRS4, the genes for two mitochondrial-solute carrier proteins, can suppress both phenotypes when present in high-copy-number plasmids. In order to search for further multicopy suppressors of the mrs2-1 mutant phenotype, an yeast genomic DNA library, MW90, was constructed in YEp351 from a strain deleted for the MRS2, MRS3 and MRS4 genes. Ten different Sau3A DNA fragments that act as multicopy suppressors of the mrs2-1 respiratory-deficient phenotype were isolated from this library. Some of the newly isolated genes suppress the pet- phenotypes of mrs2-1 cells in strains with and without mitochondrial group II introns. Other genes, however, are suppressors only for the mitochondrial intron-less strains. This supports the notion that the MRS2 gene product is bifunctional i.e., it is essential for the splicing of group II introns and is also involved in processes of mitochondrial biogenesis other than RNA splicing.

Molecular cloning and analysis of the nuclear gene MRP-L6 coding for a putative mitochondrial ribosomal protein from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae nuclear gene MRP-L6 was cloned by complementation of the respiratory-deficient mutant pet-ts 2523 with a library of wild-type yeast genomic DNA. The isolated gene was part of a 3.8-kb sequenced DNA fragment containing, in addition to MRP-L6, two unassigned reading frames, ORF1 and ORF2. MRP-L6 codes for a basic protein of 205 amino acids and a molecular mass of 22.8 kDa. The protein exhibits significant sequence similarity to the ribosomal protein L6 of bacteria and chloroplasts. Unlike the corresponding bacterial proteins, however, the MRP-L6 protein (MRP-L6p) contains at its N-terminus a 16 amino-acid leader sequence exhibiting the known characteristics of mitochondrial import signals. Disruption of MRP-L6 leads to the phenotype of a mitochondrial translation-defective, rho-negative yeast mutant. The results are consistent with MRP-L6p representing an essential component of yeast mitochondrial ribosomes. Expression of MRP-L6 was examined, under conditions of glucose repression and derepression, in wild-type cells and in a series of catabolite repression-defective yeast mutants. In most cases, a distinct though small influence of the carbon source on the expression of an MRP-L6/lacZ reported construct was observed.

Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae.

The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1 beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed. Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1 beta subunits. The DNA fragments were subcloned and sequenced. The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486. Yeast and human E1 beta exhibit 62% sequence identity. The size of the mRNA is approximately 1.5 kilobases. Hybridization analysis showed that the E1 beta gene (PDH beta 1) is localized to chromosome II. Disruption of PDH beta 1 is not lethal under vegetative growth conditions. The null mutant transformed with PDH beta 1 on a unit-copy plasmid produced mature E1 beta and a functional PDH complex.

Molecular genetics of the yeast vacuolar H(+)-ATPase.

The yeast vacuolar proton-translocating ATPase was discovered in 1981 as the first member of the V-ATPases, which are now known to be ubiquitously distributed in eukaryotic vacuo-lysosomal organelles and archaebacteria. Nine VMA genes that are indispensable for expression of vacuolar ATPase activity have been identified in the yeast Saccharomyces cerevisiae. VMA1, VMA2, VMA3, VMA5 and VMA6 were cloned and characterized on the basis of partial amino acid sequences determined with the purified subunits. Genetic and biochemical studies of the yeast Pet-cls mutants have demonstrated that they are related to vma defects. Based on this evidence, VMA11 (CLS9), VMA12 (CLS10) and VMA13 (CLS11) were isolated from a yeast genomic DNA library by complementation of the vma11, vma12 and vma13 mutations, respectively. This article summarizes currently available information on the VMA genes and the molecular biological functions of the VMA gene products.