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1
Erickson, J. R., and M. Johnston. "Direct cloning of yeast genes from an ordered set of lambda clones in Saccharomyces cerevisiae by recombination in vivo." Genetics 134, no. 1 (May 1, 1993): 151–57. http://dx.doi.org/10.1093/genetics/134.1.151.
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Abstract We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Escherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura+ transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.
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Zhang, Jiantao, Zsigmond Benko, Chenyu Zhang, and Richard Y. Zhao. "Advanced Protocol for Molecular Characterization of Viral Genome in Fission Yeast (Schizosaccharomyces pombe)." Pathogens 13, no. 7 (July 4, 2024): 566. http://dx.doi.org/10.3390/pathogens13070566.
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Fission yeast, a single-cell eukaryotic organism, shares many fundamental cellular processes with higher eukaryotes, including gene transcription and regulation, cell cycle regulation, vesicular transport and membrane trafficking, and cell death resulting from the cellular stress response. As a result, fission yeast has proven to be a versatile model organism for studying human physiology and diseases such as cell cycle dysregulation and cancer, as well as autophagy and neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Huntington’s diseases. Given that viruses are obligate intracellular parasites that rely on host cellular machinery to replicate and produce, fission yeast could serve as a surrogate to identify viral proteins that affect host cellular processes. This approach could facilitate the study of virus–host interactions and help identify potential viral targets for antiviral therapy. Using fission yeast for functional characterization of viral genomes offers several advantages, including a well-characterized and haploid genome, robustness, cost-effectiveness, ease of maintenance, and rapid doubling time. Therefore, fission yeast emerges as a valuable surrogate system for rapid and comprehensive functional characterization of viral proteins, aiding in the identification of therapeutic antiviral targets or viral proteins that impact highly conserved host cellular functions with significant virologic implications. Importantly, this approach has a proven track record of success in studying various human and plant viruses. In this protocol, we present a streamlined and scalable molecular cloning strategy tailored for genome-wide and comprehensive functional characterization of viral proteins in fission yeast.
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Sclafani, Robert A., and Walton L. Fangman. "THYMIDINE UTILIZATION BY tut MUTANTS AND FACILE CLONING OF MUTANT ALLELES BY PLASMID CONVERSION IN S. CEREVISIAE." Genetics 114, no. 3 (November 1, 1986): 753–67. http://dx.doi.org/10.1093/genetics/114.3.753.
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ABSTRACT Plasmid pJM81 contains a Herpes simplex virus thymidine kinase (TK) gene that is expressed in yeast. Cells containing the plasmid utilize thymidine (TdR) and the analogue 5-bromodeoxyuridine (BUdR) for specific incorporation into DNA. TdR auxotrophs, harboring plasmid pJM81 and a mutation in the yeast gene TMP1 require high concentrations of TdR (300 μg/ml) to support normal growth rates and the wild-type mitochondrial genome (ρ+) cannot be maintained. We have identified a yeast gene, TUT1, in which recessive mutations allow efficient utilization of lower concentrations of TdR. Strains containing the mutations tmp1 and tut1, as well as plasmid pJM81, form colonies at 2 μg/ml TdR, grow at nearly normal rates and maintain the ρ+ genome at 50 μg/ml TdR. These strains can be used to radiolabel DNA specifically and to synchronize DNA replication by TdR starvation. In addition, the substitution of BUdR for TdR allows the selective killing of DNA-synthesizing cells by 310-nm irradiation and allows the separation of replicated and unreplicated forms of DNA by CsCl equilibrium density banding. We also describe a unique, generally applicable system for cloning mutant alleles that exploits the fact that Tk+ yeast cells are sensitive to 5-fluorodeoxyuridine (FUdR) and that gene conversions can occur between a yeast chromosome and a TK-containing plasmid.
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Hiltunen, J. K., F. Okubo, V. A. S. Kursu, K. J. Autio, and A. J. Kastaniotis. "Mitochondrial fatty acid synthesis and maintenance of respiratory competent mitochondria in yeast." Biochemical Society Transactions 33, no. 5 (October 26, 2005): 1162–65. http://dx.doi.org/10.1042/bst0331162.
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Mitochondrial FAS (fatty acid synthesis) of type II is a widely conserved process in eukaryotic organisms, with particular importance for respiratory competence and mitochondrial morphology maintenance in Saccharomyces cerevisiae. The recent characterization of three missing enzymes completes the pathway. Etr1p (enoyl thioester reductase) was identified via purification of the protein followed by molecular cloning. To study the link between FAS and cell respiration further, we also created a yeast strain that has FabI enoyl-ACP (acyl-carrier protein) reductase gene from Escherichia coli engineered to carry a mitochondrial targeting sequence in the genome, replacing the endogenous ETR1 gene. This strain is respiratory competent, but unlike the ETR1 wild-type strain, it is sensitive to triclosan on media containing only non-fermentable carbon source. A colony-colour-sectoring screen was applied for cloning of YHR067w/RMD12, the gene encoding mitochondrial 3-hydroxyacyl-ACP dehydratase (Htd2/Yhr067p), the last missing component of the mitochondrial FAS. Finally, Hfa1p was shown to be the mitochondrial acetyl-CoA carboxylase.
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Zhang, Xiao-Ran, Jia-Bei He, Yi-Zheng Wang, and Li-Lin Du. "A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast." G3: Genes|Genomes|Genetics 8, no. 6 (April 27, 2018): 2067–77. http://dx.doi.org/10.1534/g3.118.200164.
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Leppert, G., R. McDevitt, S. C. Falco, T. K. Van Dyk, M. B. Ficke, and J. Golin. "Cloning by gene amplification of two loci conferring multiple drug resistance in Saccharomyces." Genetics 125, no. 1 (May 1, 1990): 13–20. http://dx.doi.org/10.1093/genetics/125.1.13.
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Abstract Yeast DNA fragments that confer multiple drug resistance when amplified were isolated. Cells containing a yeast genomic library cloned in the high copy autonomously replicating vector, YEp24, were plated on medium containing cycloheximide. Five out of 100 cycloheximide-resistant colonies were cross-resistant to the unrelated inhibitor, sulfometuron methyl, due to a plasmid-borne resistance determinant. The plasmids isolated from these resistant clones contained two nonoverlapping regions in the yeast genome now designated PDR4 and PDR5 (for pleiotropic drug resistant). PDR4 was mapped to chromosome XIII, 31.5 cM from LYS7 and 9 cM from the centromere. PDR4 was mapped to chromosome XV between ADE2 and H1S3. Genetic analysis demonstrated that at least three tightly linked genes (PDR5, PDR2 and SMR3) that mediate resistance to inhibitors are located in this region. Insertion mutations in the either PDR4 or PDR5 genes are not lethal, but the insertion in PDR5 results in a drug-hypersensitive phenotype.
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Mülleder, Michael, Kate Campbell, Olga Matsarskaia, Florian Eckerstorfer, and Markus Ralser. "Saccharomyces cerevisiae single-copy plasmids for auxotrophy compensation, multiple marker selection, and for designing metabolically cooperating communities." F1000Research 5 (September 20, 2016): 2351. http://dx.doi.org/10.12688/f1000research.9606.1.
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Auxotrophic markers are useful tools in cloning and genome editing, enable a large spectrum of genetic techniques, as well as facilitate the study of metabolite exchange interactions in microbial communities. If unused background auxotrophies are left uncomplemented however, yeast cells need to be grown in nutrient supplemented or rich growth media compositions, which precludes the analysis of biosynthetic metabolism, and which leads to a profound impact on physiology and gene expression. Here we present a series of 23 centromeric plasmids designed to restore prototrophy in typicalSaccharomyces cerevisiaelaboratory strains. The 23 single-copy plasmids complement for deficiencies inHIS3, LEU2, URA3, MET17 or LYS2genes and in their combinations, to match the auxotrophic background of the popular functional-genomic yeast libraries that are based on the S288c strain. The plasmids are further suitable for designing self-establishing metabolically cooperating (SeMeCo) communities, and possess a uniform multiple cloning site to exploit multiple parallel selection markers in protein expression experiments.
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Kuspa, A., D. Vollrath, Y. Cheng, and D. Kaiser. "Physical mapping of the Myxococcus xanthus genome by random cloning in yeast artificial chromosomes." Proceedings of the National Academy of Sciences 86, no. 22 (November 1, 1989): 8917–21. http://dx.doi.org/10.1073/pnas.86.22.8917.
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Hanekamp, Theodor, Mary K. Thorsness, Indrani Rebbapragada, Elizabeth M. Fisher, Corrine Seebart, Monica R. Darland, Jennifer A. Coxbill, Dustin L. Updike, and Peter E. Thorsness. "Maintenance of Mitochondrial Morphology Is Linked to Maintenance of the Mitochondrial Genome in Saccharomyces cerevisiae." Genetics 162, no. 3 (November 1, 2002): 1147–56. http://dx.doi.org/10.1093/genetics/162.3.1147.
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Abstract In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss. While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype. This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology. Yeast strains bearing null mutations of MMM1 have altered mitochondrial morphology and a slow growth rate on all carbon sources and quantitatively lack mitochondrial DNA. Extragenic suppressors of MMM1 deletion mutants partially restore mitochondrial morphology to the wild-type state and have a corresponding increase in growth rate and mitochondrial DNA stability. A dominant suppressor also suppresses the phenotypes caused by a point mutation in MMM1, as well as by specific mutations in YME4 and MDM10.
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Andleeb, S., F. Latif, S. Afzal, Z. Mukhtar, S. Mansoor, and I. Rajoka. "CLONING AND EXPRESSION OF CHAETOMIUM THERMOPHILUM XYLANASE 11-A GENE IN PICHIA PASTORIS." Nucleus 45, no. 1-2 (July 1, 2020): 75–81. https://doi.org/10.71330/nucleus.45.01-2.1001.
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The various thermophilic fungi like Chaetomium thermophile has potential to secrete xylanase and cellulase enzymes. Inthe present study eukaryotic expression system of Pichia pastoris (yeast) was used to express xylanase gene. Thexylanase (Xyn 11-A) gene was isolated from C. thermophile strain NIBGE-1. Primers were designed to amplify the gene,ligated into P. pastoris pPIC3.5K vector, the resultant recombinant clone pSSZ810 was transformed into the genome ofP. pastoris GS115 strain through electroporation. Transformants were selected on yeast peptone dextrose medium(YPD) plates containing antibiotic geneticin (100 mg/mL) upto final concentration of 0.75 mg/mL. The maximum activityof xylanase 2.04 U/mL after incubation of 2 hrs at 50ºC was observed in the presence of 100% methanol inducer uptofinal concentration of 30μL (0.5%) as compared to control. HPLC analysis represented high peak of xylose as comparedto control. SDS-PAGE indicated approx. 28 kDa protein of expressed xylanase gene.
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Queiroz, Sabrina R. A., Andréa N. M. R. Silva, Jefferson J. S. Santos, Ernesto T. A. Marques Jr, Giovani R. Bertani, and Laura H. V. G. Gil. "Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique." Anais da Academia Brasileira de Ciências 85, no. 1 (March 1, 2013): 159–68. http://dx.doi.org/10.1590/s0001-37652013005000008.
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RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.
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Prince, James P., and Steven D. Tanksley. "Restriction fragment length polymorphisms in plant breeding and genetics." Proceedings of the Royal Society of Edinburgh. Section B. Biological Sciences 99, no. 3-4 (1992): 23–29. http://dx.doi.org/10.1017/s0269727000005479.
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SynopsisThe usefulness of restriction fragment length polymorphisms (RFLPs) in plant breeding and genetics is discussed, with particular emphasis on tagging genes, map-based cloning, the assessment of genetic variability and distances, and comparative genome mapping.The Department of Plant Breeding and Biometry has currently established tight linkages between RFLPs and more than 20 genes of economic importance. Approximately half of these genes confer resistance to major pathogens including nematodes, bacteria, fungi, and viruses. Other genes tagged are involved in various aspects of crop quality.Locating genes with respect to DNA markers on an RFLP map provides a starting point for cloning the genes by chromosome walking. This strategy is currently being pursued for three disease-resistance genes that have been placed on the tomato RFLP map; Pto (resistance to Pseudomonas syringae), Mi (resistance to root-knot nemotodes) and Tm2a (resistance to tobacco mosaic virus). Further discussion will include the construction of a yeast artificial chromosome library and the collection of additional DNA markers in the regions of interest through RAPD analysis of nearly isogenic lines.The assessment of genetic variability and fingerprinting varieties based on RFLP data will be briefly discussed.Comparative genome mapping in the family Solanaceae has allowed the relationships among tomato, potato, and pepper to be unravelled. Tomato and potato share a near perfect conservation of gene order throughout their genomes. In contrast, while pepper shares most of its single copy DNA with tomato and potato, the order of these markers is highly rearranged compared with the other two species.
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Carrano, A. V., P. J. de Jong, E. Branscomb, T. Slezak, and B. W. Watkins. "Constructing chromosome- and region-specific cosmid maps of the human genome." Genome 31, no. 2 (January 15, 1989): 1059–65. http://dx.doi.org/10.1139/g89-182.
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A chromosome-specific ordered set of cosmids would be a significant contribution toward understanding human chromosome structure and function. We are developing two parallel approaches for creating an ordered cosmid library of human chromosome 19 and other selected subregions of the human genome. The "bottom up" approach is used to establish sets of overlapping cosmids as islands or "contigs" along the chromosome, while the "top down" approach, using pulsed-field gel electrophoresis and yeast cloning, will establish a large-fragment map and close the inevitable gaps remaining from the "bottom up" approach. Source DNA consists of a single homolog of chromosome 19 from a hamster–human hybrid cell and human fragments cloned in yeast artificial chromosomes. We have constructed cosmid libraries in a vector that facilitates cloning small amounts of DNA, allows transcription of the insert termini, and contains unique sites for partial-digest mapping. Computer simulations of cosmid contig building suggest that near-optimal efficiency can be achieved with high-density restriction fragment digest schemes that can detect 20–30% overlap between cosmids. We developed the chemistry and data analysis tools to compare the ordering efficiencies of several cosmid restriction digest fingerprinting strategies. Restriction fragments from a four-cutter digest are labeled with a fluorochrome, separated by polyacrylamide gel electrophoresis, and detected after laser excitation as they traverse a fixed point in the gel. We have also developed the software to rapidly process the output signal to define and analyze the fragment peaks. Up to three cosmids (or three different digests of the same cosmid) plus a size standard are analyzed simultaneously in a single gel lane. In two pilot experiments, cosmid contig maps from a small region of chromosome 19 and a 600-kilobase region of chromosome 14q21 have been constructed. The resulting maps, when coupled to the known genetic map, provide a collection of cloned cosmids for further study.Key words: human, genome, cosmid, fingerprinting, DNA, electrophoresis, contig, automation, restriction enzyme.
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Aguilar, Rhiannon R., Zih-Jie Shen, and Jessica K. Tyler. "A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9." Methods and Protocols 5, no. 5 (October 4, 2022): 79. http://dx.doi.org/10.3390/mps5050079.
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Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome.
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Altmann, M., C. Handschin, and H. Trachsel. "mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae." Molecular and Cellular Biology 7, no. 3 (March 1987): 998–1003. http://dx.doi.org/10.1128/mcb.7.3.998-1003.1987.
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We have isolated genomic and cDNA clones encoding protein synthesis initiation factor eIF-4E (mRNA cap-binding protein) of the yeast Saccharomyces cerevisiae. Their identity was established by expression of a cDNA in Escherichia coli. This cDNA encodes a protein indistinguishable from purified eIF-4E in terms of molecular weight, binding to and elution from m7GDP-agarose affinity columns, and proteolytic peptide pattern. The eIF-4E gene was isolated by hybridization of cDNA to clones of a yeast genomic library. The gene lacks introns, is present in one copy per haploid genome, and encodes a protein of 213 amino acid residues. Gene disruption experiments showed that the gene is essential for growth.
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Altmann, M., C. Handschin, and H. Trachsel. "mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae." Molecular and Cellular Biology 7, no. 3 (March 1987): 998–1003. http://dx.doi.org/10.1128/mcb.7.3.998.
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We have isolated genomic and cDNA clones encoding protein synthesis initiation factor eIF-4E (mRNA cap-binding protein) of the yeast Saccharomyces cerevisiae. Their identity was established by expression of a cDNA in Escherichia coli. This cDNA encodes a protein indistinguishable from purified eIF-4E in terms of molecular weight, binding to and elution from m7GDP-agarose affinity columns, and proteolytic peptide pattern. The eIF-4E gene was isolated by hybridization of cDNA to clones of a yeast genomic library. The gene lacks introns, is present in one copy per haploid genome, and encodes a protein of 213 amino acid residues. Gene disruption experiments showed that the gene is essential for growth.
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Heinisch, Jürgen J., Andrea Murra, Kai Jürgens, and Hans-Peter Schmitz. "A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum." International Journal of Molecular Sciences 24, no. 3 (January 17, 2023): 1859. http://dx.doi.org/10.3390/ijms24031859.
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Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.
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Saji, Shoko, Yosuke Umehara, Baltazar A. Antonio, Hiroko Yamane, Hiroshi Tanoue, Tomoya Baba, Hiroyoshi Aoki, et al. "A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes." Genome 44, no. 1 (February 1, 2001): 32–37. http://dx.doi.org/10.1139/g00-076.
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A new YAC (yeast artificial chromosome) physical map of the 12 rice chromosomes was constructed utilizing the latest molecular linkage map. The 1439 DNA markers on the rice genetic map selected a total of 1892 YACs from a YAC library. A total of 675 distinct YACs were assigned to specific chromosomal locations. In all chromosomes, 297 YAC contigs and 142 YAC islands were formed. The total physical length of these contigs and islands was estimated to 270 Mb which corresponds to approximately 63% of the entire rice genome (430 Mb). Because the physical length of each YAC contig has been measured, we could then estimate the physical distance between genetic markers more precisely than previously. In the course of constructing the new physical map, the DNA markers mapped at 0.0-cM intervals were ordered accurately and the presence of potentially duplicated regions among the chromosomes was detected. The physical map combined with the genetic map will form the basis for elucidation of the rice genome structure, map-based cloning of agronomically important genes, and genome sequencing.Key words: physical mapping, YAC contig, rice genome, rice chromosomes.
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Weller, S. K., A. Spadaro, J. E. Schaffer, A. W. Murray, A. M. Maxam, and P. A. Schaffer. "Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis." Molecular and Cellular Biology 5, no. 5 (May 1985): 930–42. http://dx.doi.org/10.1128/mcb.5.5.930-942.1985.
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The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.
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Weller, S. K., A. Spadaro, J. E. Schaffer, A. W. Murray, A. M. Maxam, and P. A. Schaffer. "Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis." Molecular and Cellular Biology 5, no. 5 (May 1985): 930–42. http://dx.doi.org/10.1128/mcb.5.5.930.
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The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.
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Maceluch, J., M. Kmieciak, Z. Szweykowska-Kulińska, and A. Jarmołowski. "Cloning and characterization of Arabidopsis thaliana AtNAP57--a homologue of yeast pseudouridine synthase Cbf5p." Acta Biochimica Polonica 48, no. 3 (September 30, 2001): 699–709. http://dx.doi.org/10.18388/abp.2001_3904.
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Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (Arabidopsis thaliana homologue of NAP57). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.
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Larin, Zoia, and Hans Lehrach. "Yeast artificial chromosomes: an alternative approach to the molecular analysis of mouse developmental mutations." Genetics Research 56, no. 2-3 (October 1990): 203–8. http://dx.doi.org/10.1017/s0016672300035308.
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SummaryMammalian genetics now allows a molecular study of genomic regions previously analysed by genetic and embryological techniques. To simplify such an analysis, we have established a number of libraries of mouse DNA in Yeast Artificial Chromosome (YAC) vectors, constructed either by partial digestion with EcoRI, or by complete digestion with enzymes which cut rarely in the mammalian genome. In this paper we report the construction of complete digest libraries prepared from mouse genomic DNA using the rare cutter enzymes NoiI and BssHII, and the detection of gene loci from the H-2 complex, the t–complex, and other loci from the mouse genome. Due to their large insert size, YAC clones simplify the cloning of extended regions of the mouse genome surrounding known developmental mutations and should, after introduction into the germ line, offer a high probability of correct expression of the genes contained within the cloned region. We hope that this will allow the use of YAC clones to scan regions of interest such as the t–complex for specific genes by testing DNA introduced into transgenic mice for the ability to complement mutations localised to this region.
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Liu, Lina, Shicui Zhang, Zhenhui Liu, Hongyan Li, Mei Liu, Yongjun Wang, and Lifang Ma. "Ribosomal proteins L34 and S29 of amphioxus Branchiostoma belcheri tsingtauense: cDNAs cloning and gene copy number." Acta Biochimica Polonica 52, no. 4 (July 11, 2005): 857–62. http://dx.doi.org/10.18388/abp.2005_3398.
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The complete cDNA and deduced amino-acid sequences of ribosomal proteins L34 (AmphiL34) and S29 (AmphiS29) from the amphioxus Branchiostoma belcheri tsingtauense were identified in this study. The AmphiL34 cDNA is 435 nucleotides in length and encodes a 118 amino-acid protein with calculated molecular mass of 13.6 kDa. It shares 53.6-67.5% amino-acid sequence identity with its eukaryotic counterparts including human, mouse, rat, pig, frog, catfish, fruit fly, mosquito, armyworm, nematode and yeast. The AmphiS29 cDNA comprises 453 nucleotides and codes for a 56 amino-acid protein with a calculated molecular mass of 6.6 kDa. It shows 66.1-78.6% amino-acid sequence identity to eukaryotic S29 proteins from human, mouse, rat, pig, zebrafish, seahorse, fruit fly, nematode, sea hare and yeast. AmphiL34 contains a putative nucleolar localization signal, while AmphiS29 has a zinc finger-like domain. A phylogenetic tree deduced from the conserved sequences of AmphiL34 and AmphiS29 and other known counterparts indicates that the positions of AmphiL34/AmphiS29 are intermediate between the vertebrate and invertebrate L34/S29. Southern blot analysis demonstrates the presence of one copy of the L34 gene and 2-3 copies of the S29 gene in the genome of the amphioxus B. belcheri tsingtauense. This is in sharp contrast to the existence of 7-9 copies of the L34 gene and 14-17 copies of the S29 gene in the rat genome. These date suggest that housekeeping genes like AmphiL34 and AmphiS29 have undergone large-scale duplication in the chordate lineage.
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Andreotti, Patrícia Ferrari, Juliana Leal Monteiro da Silva, Elaine Cristina Teixeira, Maria Célia Bertolini, Christiane Pienna Soares, Gil Benard, and Maria José Soares Mendes-Giannini. "Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis." Journal of Medical Microbiology 56, no. 7 (July 1, 2007): 884–87. http://dx.doi.org/10.1099/jmm.0.47127-0.
Full textAbstract:
Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pb18) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pb18a by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein β-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.
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Song, Junqi, Fenggao Dong, Jason W. Lilly, Robert M. Stupar, and Jiming Jiang. "Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences." Genome 44, no. 3 (June 1, 2001): 463–69. http://dx.doi.org/10.1139/g01-029.
Full textAbstract:
The cloning and propagation of large DNA fragments as bacterial artificial chromosomes (BACs) has become a valuable technique in genome research. BAC clones are highly stable in the host, Escherichia coli, a major advantage over yeast artificial chromosomes (YACs) in which recombination-induced instability is a major drawback. Here we report that BAC clones containing tandemly repeated DNA elements are not stable and can undergo drastic deletions during routine library maintenance and DNA preparation. Instability was observed in three BAC clones from sorghum, rice, and potato, each containing distinct tandem repeats. As many as 46% and 74% of the single colonies derived from a rice BAC clone containing 5S ribosomal RNA genes had insert deletions after 24 and 120 h of growth, respectively. We also demonstrated that BAC insert rearrangement can occur in the early stage of library construction and duplication. Thus, a minimum growth approach may not avoid the instability problem of such clones. The impact of BAC instability on genome research is discussed.Key words: repetitive DNA, large insert DNA library, genome research.
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Cardoso, J. C. R., M. S. Clark, F. A. Viera, P. D. Bridge, A. Gilles, and D. M. Power. "The secretin G-protein-coupled receptor family: teleost receptors." Journal of Molecular Endocrinology 34, no. 3 (June 2005): 753–65. http://dx.doi.org/10.1677/jme.1.01730.
Full textAbstract:
Twenty-one members of the secretin family (family 2) of G-protein-coupled receptors (GPCRs) were identified via directed cloning and data-mining of the Fugu Genome Consortium database, representing the most comprehensive description of secretin GPCRs in a teleost fish to date. Duplicated genes were identified for many of the family members, namely the receptors for pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP), calcitonin, calcitonin gene-related peptide (CGRP), growth hormone releasing hormone (GHRH), glucagon receptor/glucagon-like peptide (GLP) and parathyroid hormone-related peptide (PTHrP)/PTH. Mining of other teleost genomes (zebrafish and Tetraodon) revealed that the duplicated genes identified in the Takifugu genome were also present in these fish. Additional database searching of the Escherichia coli, yeast, Drosophila, Caenorhabditis elegans and Ciona genomes revealed that the family 2 of GPCRs were only present in the multicellular organisms. Orthologues of all the human secretin receptors were identified with the exception of secretin itself. Additional database searches in the Fugu Genome Consortium database also failed to reveal a secretin ligand and so it is hypothesised that both the receptor and the ligand evolved after the divergence of teleost/tetrapod lineages. Phylogenetic analysis at both the protein and the DNA level provided strong support for each of the individual receptor family groupings, but weak support between groups, making evolutionary inferences difficult. A more critical analysis of the PACAP/VIP receptor family confirmed previous hypotheses that the vasoactive intestinal peptide receptor (VPAC1R) gene is the ancestral form of the receptor.
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Raymond, Christopher K., Elizabeth H. Sims, Arnold Kas, David H. Spencer, Tanya V. Kutyavin, Richard G. Ivey, Yang Zhou, Rajinder Kaul, James B. Clendenning, and Maynard V. Olson. "Genetic Variation at the O-Antigen Biosynthetic Locus in Pseudomonas aeruginosa." Journal of Bacteriology 184, no. 13 (July 1, 2002): 3614–22. http://dx.doi.org/10.1128/jb.184.13.3614-3622.2002.
Full textAbstract:
ABSTRACT The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the P. aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.
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Torres-Garcia, Sito, Lorenza Di Pompeo, Luke Eivers, Baptiste Gaborieau, Sharon A. White, Alison L. Pidoux, Paulina Kanigowska, Imtiyaz Yaseen, Yizhi Cai, and Robin C. Allshire. "SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast." Wellcome Open Research 5 (November 24, 2020): 274. http://dx.doi.org/10.12688/wellcomeopenres.16405.1.
Full textAbstract:
The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15 promoter. The SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3’ end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the ade6+ or ura4+ genes by transformation of cells from asynchronous cultures. SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDIT system displayed reduced toxicity compared to currently available S. pombe editing systems. Thus, SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.
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Hardtke, Christian S., and Thomas Berleth. "Genetic and contig map of a 2200-kb region encompassing 5.5 cM on chromosome 1 of Arabidopsis thaliana." Genome 39, no. 6 (December 1, 1996): 1086–92. http://dx.doi.org/10.1139/g96-136.
Full textAbstract:
In the course of the isolation of the MONOPTEROS (MP) gene, required for primary root formation in Arabidopsis thaliana, a yeast artificial chromosome (YAC) contig encompassing approximately 2200 kilobases corresponding to 5.5 cM on the top arm of chromosome 1 was established. Forty-six YAC clones were characterized and 12 new restriction fragment length polymorphism (RFLP) markers are presented. Three new codominant amplified polymorphic sequence (CAPS) markers were generated that enabled high resolution genetic mapping and correlation of physical and genetic distances along the contig. The map contributes to the completion of a physical map of the Arabidopsis genome and should facilitate positional cloning of other genes in the region as well as studies on genome organization. We also present another set of 11 physically linked probes, as well as mapping data for additional RFLP markers within a broader interval of 10.4 cM. Key words : Arabidopsis, CAPS markers, MONOPTEROS gene, physical map, RFLP markers, YAC contig.
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Kouprina, Natalay, Sun‐Hee Leem, Greg Solomon, Albert Ly, Maxim Koriabine, John Otstot, Eugene Pak, et al. "Segments missing from the draft human genome sequence can be isolated by transformation‐associated recombination cloning in yeast." EMBO reports 4, no. 3 (March 2003): 257–62. http://dx.doi.org/10.1038/sj.embor.embor766.
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Coleman, K. G., H. Y. Steensma, D. B. Kaback, and J. R. Pringle. "Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome." Molecular and Cellular Biology 6, no. 12 (December 1986): 4516–25. http://dx.doi.org/10.1128/mcb.6.12.4516-4525.1986.
Full textAbstract:
Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.
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Coleman, K. G., H. Y. Steensma, D. B. Kaback, and J. R. Pringle. "Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome." Molecular and Cellular Biology 6, no. 12 (December 1986): 4516–25. http://dx.doi.org/10.1128/mcb.6.12.4516.
Full textAbstract:
Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.
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Morroll, Shaun M., and Zoe A. Wilson. "Arabidopsis YAC restriction mapping." Genome 41, no. 6 (December 1, 1998): 806–17. http://dx.doi.org/10.1139/g98-086.
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The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (~27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region. The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used. This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterised genes involved in male gametogenesis (e.g., Ms1 and Ms37). The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterised floral development genes which lie in this region. This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.Key words: yeast artificial chromosome, YAC, Arabidopsis thaliana, partial restriction mapping, floral development.
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Howarth, Deanna L., Sheran H. W. Law, Benjamin Barnes, Julie M. Hall, David E. Hinton, Linda Moore, Jodi M. Maglich, John T. Moore, and Seth W. Kullman. "Paralogous Vitamin D Receptors in Teleosts: Transition of Nuclear Receptor Function." Endocrinology 149, no. 5 (February 7, 2008): 2411–22. http://dx.doi.org/10.1210/en.2007-1256.
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The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we describe the cloning and functional characterization of two novel vitamin D receptor (VDR) paralogs from the freshwater teleost medaka (Oryzias latipes). VDR sequences were identified through mining of the medaka genome database in which gene organization and structure was determined. Two distinct VDR genes were identified in the medaka genome and mapped to defined loci. Each VDR sequence exhibits unique intronic organization and dissimilar 5′ untranslated regions, suggesting they are not isoforms of the same gene locus. Phylogenetic comparison with additional teleosts and mammalian VDR sequences illustrate that two distinct clusters are formed separating aquatic and terrestrial species. Nested within the teleost cluster are two separate clades for VDRα and VDRβ. The topology of teleost VDR sequences is consistent with the notion of paralogous genes arising from a whole genome duplication event prior to teleost radiation. Functional characterization was conducted through the development of VDR expression vectors including Gal4 chimeras containing the yeast Gal4 DNA binding domain fused to the medaka VDR ligand binding domain and full-length protein. The common VDR ligand 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] resulted in significant transactivation activity with both the Gal4 and full-length constructs of medaka (m) VDRβ. Comparatively, transactivation of mVDRα with 1α,25(OH)2D3 was highly attenuated, suggesting a functional divergence between these two nuclear receptor paralogs. We additionally demonstrate through coactivator studies that mVDRα is still functional; however, it exhibits a different sensitivity to 1α,25(OH)2D3, compared with VDRβ. These results suggest that in mVDRα and VDRβ have undergone a functional divergence through a process of sub- and/or neofunctionalization of VDR nuclear receptor gene pairs.
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Pfeifer, Tom A., Dwayne D. Hegedus, and George G. Khachatourians. "The mitochondrial genome of the entomopathogenic fungus Beauveria bassiana: analysis of the ribosomal RNA region." Canadian Journal of Microbiology 39, no. 1 (January 1, 1993): 25–31. http://dx.doi.org/10.1139/m93-004.
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The 28.5-kbp mitochondrial (mt) genome from the entomopathogenic fungus Beauveria bassiana was studied using restriction enzyme analysis, gene probe hybridization, and DNA sequence comparisons. A detailed restriction enzyme map allowed cloning of the entire genome into a number of segments. Hybridization of heterologous gene probes to the mtDNA resulted in the identification of the large ribosomal RNA (lrRNA) and small ribosomal RNA (srRNA) genes. Gene probes derived from several yeasts and fungi failed to identify any additional genes. However, partial DNA sequence analysis revealed the lrRNA and srRNA genes as well as four protein-encoding genes: the NADH dehydrogenase subunit 1 (NAD1), NADH dehydrogenase subunit 6 (NAD6), cytochrome oxidase subunit 3 (C03), and ATPase subunit 6 (ATP6) genes. The ATPase subunit 9 (ATP9) gene was not identified by hybridization to mtDNA, but could be detected by hybridization to total cellular DNA. The portions of the genes sequenced were homologous to the equivalent genes from yeast and other filamentous fungi, most notably Aspergillus nidulans. No introns were identified in these regions. The organization of the sequenced region of the B. bassiana mt genome more closely resembled that of A. nidulans than that of Podospora anserina or Neurospora crassa.Key words: Beauveria bassiana, mtDNA, gene mapping, mitochondrial rRNA, mitochondrial organization.
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Chibana, Hiroji, Elizabeth L. Heinecke, Janna L. Beckerman, and Paul T. Magee. "A system of rapid isolation of end-DNA from a small amount of fosmid DNA, with vector-based PCR for chromosome walking." Genome 44, no. 2 (April 1, 2001): 305–8. http://dx.doi.org/10.1139/g00-116.
Full textAbstract:
The pBAC 108L and pFos 1 vectors were developed as stable propagation vectors which, due to their extremely low copy number, facilitate the cloning of a large-sized insert containing repeated DNA. However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome walking. Here we describe efficient methods for end-DNA isolation. The entire process, including small-scale DNA preparation, restriction digestion, self-ligation, and PCR with vector-based primers, is carried out in 96-well formats. Using a Fosmid library of genomic DNA of Candida albicans, PCR products ranging in size from 0.1 to 8 kbp were generated from 118 end sequences in 140 reactions from 70 Fosmid clones. A single or a prominent band was found in 101 of these reactions. Twenty-six of these bands were tested for walking and all of them proved to be specific. Thus, the system overcomes the disadvantage caused by low copy number. This system allows rapid physical mapping of genomes, and is adaptable for several other vectors including BAC (bacterial artificial chromosome), PAC (P1-derived artificial chromosome) and YAC (yeast artificial chromosome).Key words: IPCR, LM-PCR, chromosome walk, genome project, contig map.
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Chapple, RM, AM Chaudhury, KC Blomer, LB Farrell, and ES Dennis. "Construction of a YAC Contig of 2 Megabases Around the MS1 Gene in Arabidopsis thaliana." Functional Plant Biology 23, no. 4 (1996): 453. http://dx.doi.org/10.1071/pp9960453.
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The ms1 mutation of Arabidopsis thaliana causes male sterility by preventing the development of normal microspores in the developing anther. The gene is located on a region of chromosome 5 containing the RFLP markers g4111, g4560 and g21503. Using yeast artificial chromosome (YAC) libraries, we have constructed a contig of 38 YACs spanning approximately 2.1 megabases (approximately 2% of the genome) around MS1 and redefined the order of these RFLP markers. Chimeric YACs and repetitive DNA caused problems in chromosome 'walking'. A method for cloning YAC right ends by plasmid rescue was applied to arabidopsis. One YAC end contained a portion of the A. thaliana sucrose synthase gene ASUSI, hence locating this gene on chromosome 5 near MS1. Using recombinant plant populations containing ms1 and flanking markers, MS1 was localised to a 200 kb region within the YAC contig. In this contig the relationship between physical and genetic distance varied from less than 100 kb to 720 kb per centimorgan.
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TABUCHI, Mitsuaki, Tsutomu YOSHIDA, Kaoru TAKEGAWA, and Fumio KISHI. "Functional analysis of the human NRAMP family expressed in fission yeast." Biochemical Journal 344, no. 1 (November 8, 1999): 211–19. http://dx.doi.org/10.1042/bj3440211.
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The Bcg/Ity/Lsh locus in the mouse genome regulates macrophage activation for antimicrobial activity against intracellular pathogens, and the positional cloning of this locus identified the Nramp1 (natural resistance-associated macrophage protein) gene. Nramp2 was initially isolated as a homologue of Nramp1. Recently, the rat divalent metal transporter DMT1 was identified electrophysiologically, and was found to be an isoform of Nramp2, a mutation which was subsequently identified in rats suffering from hereditary iron-deficiency anaemia. Despite the 64% amino acid sequence identity of Nramp1 and Nramp2, no divalent metal transport activity has yet been detected from Nramp1, and the function of Nramp1 on the molecular level is still unclear. To investigate the divalent metal transport activity of NRAMP molecules, we constructed four chimeric NRAMP genes by swapping the domains of human NRAMP1 and NRAMP2 with each other. The functional characteristics of wild-type NRAMP1, NRAMP2 and their chimeras were determined by expression in the divalent metal transporter-disrupted strain of fission yeast, pdt1δ, and we analysed the divalent metal transport activity by complementation of the EGTA- and pH-sensitive phenotype of pdt1δ. Replacement of the N-terminal cytoplasmic domain of NRAMP2 with the NRAMP1 counterpart resulted in inactive chimeras, indicating that the functional difference between NRAMP1 and NRAMP2 is located in this region. However, results obtained with the reverse construct and other chimeras indicated that these regions are not solely responsible for the differences in EGTA- and pH-sensitivity of NRAMP1 and NRAMP2. These findings indicate that NRAMP1 itself cannot represent the divalent metal transport activity in S. pombe and the additional protein segments of the molecules located elsewhere in NRAMP1 are also functionally distinct from their NRAMP2 counterparts.
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Hansen, Bjarne G., Bo Salomonsen, Morten T. Nielsen, Jakob B. Nielsen, Niels B. Hansen, Kristian F. Nielsen, Torsten B. Regueira, Jens Nielsen, Kiran R. Patil, and Uffe H. Mortensen. "Versatile Enzyme Expression and Characterization System for Aspergillus nidulans, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case." Applied and Environmental Microbiology 77, no. 9 (March 11, 2011): 3044–51. http://dx.doi.org/10.1128/aem.01768-10.
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ABSTRACTAssigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location ofAspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp largempaCgene fromPenicillium brevicompactumtoA. nidulans. In parallel, we produced defined mutant derivatives ofmpaC, and the combined analysis ofA. nidulansstrains expressingmpaCor mutatedmpaCgenes unequivocally demonstrated thatmpaCindeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.
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Liszewska, Frantz, Dali Gaganidze, and Agnieszka Sirko. "Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits." Acta Biochimica Polonica 52, no. 1 (March 31, 2005): 117–28. http://dx.doi.org/10.18388/abp.2005_3496.
Full textAbstract:
We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.
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Tokai, Masaya, Hideki Kawasaki, Yasuhiro Kikuchi, and Kozo Ouchi. "Cloning and Characterization of the CSF1 Gene ofSaccharomyces cerevisiae, Which Is Required for Nutrient Uptake at Low Temperature." Journal of Bacteriology 182, no. 10 (May 15, 2000): 2865–68. http://dx.doi.org/10.1128/jb.182.10.2865-2868.2000.
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ABSTRACT We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial baker's yeast that have practically no fermentation capacity at 5°C and return to their normal capacity at 25 to 40°C.CSF1 was cloned by functional complementation of the Csf phenotype. CSF1 contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids. The nucleotide sequence was identical to that of the YLR087C gene in theSaccharomyces genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein. Gene disruption shows that CSF1 is required for growth and fermentation only at low temperatures. Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10°C. The disruptant cells had the same glucose uptake rates as the parental cells at 30°C, but three- to fivefold-lower rates than the parental cells at 10°C. These findings suggest that CSF1 associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.
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Wu, Youting, Pingzhi Wu, Shaoming Xu, Yaping Chen, Meiru Li, Guojiang Wu, and Huawu Jiang. "Genome-Wide Identification, Expression Patterns and Sugar Transport of the Physic Nut SWEET Gene Family and a Functional Analysis of JcSWEET16 in Arabidopsis." International Journal of Molecular Sciences 23, no. 10 (May 12, 2022): 5391. http://dx.doi.org/10.3390/ijms23105391.
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The Sugars Will Eventually be Exported Transporters (SWEET) family is a class of sugar transporters that play key roles in phloem loading, seed filling, pollen development and the stress response in plants. Here, a total of 18 JcSWEET genes were identified in physic nut (Jatropha curcas L.) and classified into four clades by phylogenetic analysis. These JcSWEET genes share similar gene structures, and alternative splicing of messenger RNAs was observed for five of the JcSWEET genes. Three (JcSWEET1/4/5) of the JcSWEETs were found to possess transport activity for hexose molecules in yeast. Real-time quantitative PCR analysis of JcSWEETs in different tissues under normal growth conditions and abiotic stresses revealed that most are tissue-specifically expressed, and 12 JcSWEETs responded to either drought or salinity. The JcSWEET16 gene responded to drought and salinity stress in leaves, and the protein it encodes is localized in both the plasma membrane and the vacuolar membrane. The overexpression of JcSWEET16 in Arabidopsis thaliana modified the flowering time and saline tolerance levels but not the drought tolerance of the transgenic plants. Together, these results provide insights into the characteristics of SWEET genes in physic nut and could serve as a basis for cloning and further functional analysis of these genes.
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Khatri, Praveen, Ling Chen, Istvan Rajcan, and Sangeeta Dhaubhadel. "Functional characterization of Cinnamate 4-hydroxylase gene family in soybean (Glycine max)." PLOS ONE 18, no. 5 (May 15, 2023): e0285698. http://dx.doi.org/10.1371/journal.pone.0285698.
Full textAbstract:
Cinnamate 4-hydroxylase (C4H) is the first key cytochrome P450 monooxygenase (P450) enzyme in the phenylpropanoid pathway. It belongs to the CYP73 family of P450 superfamily, and catalyzes the conversion of trans-cinnamic acid to p-coumaric acid. Since p-coumaric acid serves as the precursor for the synthesis of a wide variety of metabolites involved in plant development and stress resistance, alteration in the expression of soybean C4H genes is expected to affect the downstream metabolite levels, and its ability to respond to stress. In this study, we identified four C4H genes in the soybean genome that are distributed into both class I and class II CYP73 family. GmC4H2, GmC4H14 and GmC4H20 displayed tissue- and developmental stage-specific gene expression patterns with their transcript accumulation at the highest level in root tissues. GmC4H10 appears to be a pseudogene as its transcript was not detected in any soybean tissues. Furthermore, protein homology modelling revealed substrate docking only for GmC4H2, GmC4H14 and GmC4H20. To demonstrate the function of GmC4Hs, we modified a cloning vector for the heterologous expression of P450s in yeast, and used it for microsomal protein production and enzyme assay. Our results confirmed that GmC4H2, GmC4H14 and GmC4H20 contain the ability to hydroxylate trans-cinnamic acid with varying efficiencies.
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Pan, Feng, Min Wu, Wenfang Hu, Rui Liu, Hanwei Yan, and Yan Xiang. "Genome-Wide Identification and Expression Analyses of the bZIP Transcription Factor Genes in moso bamboo (Phyllostachys edulis)." International Journal of Molecular Sciences 20, no. 9 (May 5, 2019): 2203. http://dx.doi.org/10.3390/ijms20092203.
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The basic leucine zipper (bZIP) transcription factor (TF) family is one of the largest gene families, and play crucial roles in many processes, including stress responses, hormone effects. The TF family also participates in plant growth and development. However, limited information is available for these genes in moso bamboo (Phyllostachys edulis), one of the most important non-timber forest products in the world. In the present study, 154 putative PhebZIP genes were identified in the moso bamboo genome. The phylogenetic analyses indicate that the PhebZIP gene proteins classify into 9 subfamilies and the gene structures and conserved motifs that analyses identified among all PhebZIP proteins suggested a high group-specificity. Microsynteny and evolutionary patterns analyses of the non-synonymous (Ka) and synonymous (Ks) substitution rates and their ratios indicated that paralogous pairs of PhebZIP genes in moso bamboo underwent a large-scale genome duplication event that occurred 7–15 million years ago (MYA). According to promoter sequence analysis, we further selected 18 genes which contain the higher number of cis-regulatory elements for expression analysis. The result showed that these genes are extensively involved in GA-, ABA- and MeJA-responses, with possibly different mechanisms. The tissue-specific expression profiles of PhebZIP genes in five plant tissues/organs/developmental stages suggested that these genes are involved in moso bamboo organ development, especially seed development. Subcellular localization and transactivation activity analysis showed that PhebZIP47 and PhebZIP126 were localized in the nucleus and PhebZIP47 with no transcriptional activation in yeast. Our research provides a comprehensive understanding of PhebZIP genes and may aid in the selection of appropriate candidate genes for further cloning and functional analysis in moso bamboo growth and development, and improve their resistance to stress during their life.
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Beklemishev, A. B., M. B. Pykhtina, Ya M. Kulikov, T. N. Goryachkovskaya, D. V. Bochkov, S. V. Sergeeva, A. R. Vasileva, V. P. Romanov, D. S. Novikova, and S. E. Peltek. "Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album." Vavilov Journal of Genetics and Breeding 25, no. 8 (January 1, 2022): 882–88. http://dx.doi.org/10.18699/vj21.102.
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The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.
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Lee, Jung-Kul, Bong-Seong Koo, and Sang-Yong Kim. "Cloning and Characterization of the xyl1 Gene, Encoding an NADH-Preferring Xylose Reductase from Candida parapsilosis, and Its Functional Expression in Candida tropicalis." Applied and Environmental Microbiology 69, no. 10 (October 2003): 6179–88. http://dx.doi.org/10.1128/aem.69.10.6179-6188.2003.
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ABSTRACT Xylose reductase (XR) is a key enzyme in d-xylose metabolism, catalyzing the reduction of d-xylose to xylitol. An NADH-preferring XR was purified to homogeneity from Candida parapsilosis KFCC-10875, and the xyl1 gene encoding a 324-amino-acid polypeptide with a molecular mass of 36,629 Da was subsequently isolated using internal amino acid sequences and 5′ and 3′ rapid amplification of cDNA ends. The C. parapsilosis XR showed high catalytic efficiency (k cat/Km = 1.46 s−1 mM−1) for d-xylose and showed unusual coenzyme specificity, with greater catalytic efficiency with NADH (k cat/Km = 1.39 × 104 s−1 mM−1) than with NADPH (k cat/Km = 1.27 × 102 s−1 mM−1), unlike all other aldose reductases characterized. Studies of initial velocity and product inhibition suggest that the reaction proceeds via a sequentially ordered Bi Bi mechanism, which is typical of XRs. Candida tropicalis KFCC-10960 has been reported to have the highest xylitol production yield and rate. It has been suggested, however, that NADPH-dependent XRs, including the XR of C. tropicalis, are limited by the coenzyme availability and thus limit the production of xylitol. The C. parapsilosis xyl1 gene was placed under the control of an alcohol dehydrogenase promoter and integrated into the genome of C. tropicalis. The resulting recombinant yeast, C. tropicalis BN-1, showed higher yield and productivity (by 5 and 25%, respectively) than the wild strain and lower production of by-products, thus facilitating the purification process. The XRs partially purified from C. tropicalis BN-1 exhibited dual coenzyme specificity for both NADH and NADPH, indicating the functional expression of the C. parapsilosis xyl1 gene in C. tropicalis BN-1. This is the first report of the cloning of an xyl1 gene encoding an NADH-preferring XR and its functional expression in C. tropicalis, a yeast currently used for industrial production of xylitol.
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Szuplewski, Sébastien, and Régine Terracol. "The cyclope Gene of Drosophila Encodes a Cytochrome c Oxidase Subunit VIc Homolog." Genetics 158, no. 4 (August 1, 2001): 1629–43. http://dx.doi.org/10.1093/genetics/158.4.1629.
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Abstract Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7–10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits.
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House, Brent L., Michael W. Mortimer, and Michael L. Kahn. "New Recombination Methods for Sinorhizobium meliloti Genetics." Applied and Environmental Microbiology 70, no. 5 (May 2004): 2806–15. http://dx.doi.org/10.1128/aem.70.5.2806-2815.2004.
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ABSTRACT The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative α-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.
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Kimura, Makoto, Takeshi Tokai, Gentaro Matsumoto, Makoto Fujimura, Hiroshi Hamamoto, Katsuyoshi Yoneyama, Takehiko Shibata, and Isamu Yamaguchi. "Trichothecene Nonproducer Gibberella Species Have Both Functional and Nonfunctional 3-O-Acetyltransferase Genes." Genetics 163, no. 2 (February 1, 2003): 677–84. http://dx.doi.org/10.1093/genetics/163.2.677.
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Abstract The trichothecene 3-O-acetyltransferase gene (FgTri101) required for trichothecene production by Fusarium graminearum is located between the phosphate permease gene (pho5) and the UTP-ammonia ligase gene (ura7). We have cloned and sequenced the pho5-to-ura7 regions from three trichothecene nonproducing Fusarium (i.e., F. oxysporum, F. moniliforme, and Fusarium species IFO 7772) that belong to the teleomorph genus Gibberella. BLASTX analysis of these sequences revealed portions of predicted polypeptides with high similarities to the TRI101 polypeptide. While FspTri101 (Fusarium species Tri101) coded for a functional 3-O-acetyltransferase, FoTri101 (F. oxysporum Tri101) and FmTri101 (F. moniliforme Tri101) were pseudogenes. Nevertheless, F. oxysporum and F. moniliforme were able to acetylate C-3 of trichothecenes, indicating that these nonproducers possess another as yet unidentified 3-O-acetyltransferase gene. By means of cDNA expression cloning using fission yeast, we isolated the responsible FoTri201 gene from F. oxysporum; on the basis of this sequence, FmTri201 has been cloned from F. moniliforme by PCR techniques. Both Tri201 showed only a limited level of nucleotide sequence similarity to FgTri101 and FspTri101. The existence of Tri101 in a trichothecene nonproducer suggests that this gene existed in the fungal genome before the divergence of producers from nonproducers in the evolution of Fusarium species.
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PRICE, Nigel T., Scot R. KIMBALL, Leonard S. JEFFERSON, and Christopher G. PROUD. "Cloning of cDNA for the γ-subunit of mammalian translation initiation factor 2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2." Biochemical Journal 318, no. 2 (September 1, 1996): 631–36. http://dx.doi.org/10.1042/bj3180631.
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Peptide sequence data were obtained from rabbit protein synthesis initiation factor subunit eIF2Bγ. Searching the database of expressed sequence tags (dbEST) revealed nucleotide sequences potentially encoding human eIF2Bγ that contained peptides corresponding to those from the rabbit subunit. PCR primers were derived from these sequences and used to generate a probe. This was used to screen a rat skeletal muscle cDNA library, and a clone encoding rat eIF2Bγ was isolated. This cDNA gave a product in coupled transcription/translation that co-migrated with the γ-subunit of purified eIF2B under SDS/PAGE. The sequence of this rat eIF2Bγ cDNA is reported. The protein sequence shows homology with that of yeast eIF2Bγ (the GCD1 gene product). We have also identified an open reading frame from the Caenorhabditis elegans genome project that probably encodes the γ-subunit of C. elegans eIF2B. All these sequences show similarity to nucleotidyl- and acyltransferases, as previously reported for GCD1 [Koonin (1995) Protein Sci. 4, 1608–1617], and contain conserved motifs potentially involved in nucleotide binding. They also contain ‘I-patch’ motifs: isoleucine-rich hexamer repeats that have been associated with the binding of acyl groups in bacterial acyltransferases. The roles of these motifs are discussed in relation to the known properties of eIF2B.