scholarly journals Tetrad analysis without tetrad dissection: Meiotic recombination and genomic diversity in the yeast Komagataella phaffii (Pichia pastoris)

2019 ◽  
Author(s):  
Stephanie Braun-Galleani ◽  
Julie A. Dias ◽  
Aisling Y. Coughlan ◽  
Adam P. Ryan ◽  
Kevin P. Byrne ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Diversity ◽  
Pichia Pastoris ◽  
Meiotic Recombination ◽  
Genomic Diversity ◽  
Tetrad Analysis ◽  
Komagataella Phaffii ◽  
Rich Media ◽  
Natural Isolates ◽  
Almost All

AbstractKomagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were formerly called Pichia pastoris. However, almost all laboratory work on K. phaffii has been done on strains derived from a single natural isolate, CBS7435. There is little information about the genetic properties of K. phaffii or its sequence diversity. Genetic analysis is difficult because, although K. phaffii makes asci with four spores, the spores are small and tend to clump together, making the asci hard to dissect. Here, we sequenced the genomes of all the known isolates of this species, and find that K. phaffii has only been isolated from nature four times. We analyzed the meiotic recombination landscape in a cross between auxotrophically marked strains derived from two isolates that differ at 44,000 single nucleotide polymorphism sites. We conducted tetrad analysis by making use of the property that haploids of this species do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetratype ascus germinates. We found that approximately 25 crossovers occur per meiosis, which is 3.5 times fewer than in Saccharomyces cerevisiae. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our method of tetrad analysis without tetrad dissection will be applicable to other species whose spores do not mate spontaneously after germination.Author summaryTo better understand the basic genetics of the budding yeast Komagataella phaffii, which has many applications in biotechnology, we investigated its genetic diversity and its meiotic recombination landscape. We made a genetic cross between strains derived from two natural isolates, and developed a method for characterizing the genomes of the four spores resulting from meiosis, which were previously impossible to isolate. We found that K. phaffii has a lower recombination rate than Saccharomyces cerevisiae. It shows a large zone of suppressed recombination around its centromeres, which may be due to the structural differences between centromeres in K. phaffii and S. cerevisiae.

scholarly journals Genomic diversity and meiotic recombination among isolates of the biotech yeast Komagataella phaffii (Pichia pastoris)

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Stephanie Braun-Galleani ◽  
Julie A. Dias ◽  
Aisling Y. Coughlan ◽  
Adam P. Ryan ◽  
Kevin P. Byrne ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Meiotic Recombination ◽  
Genomic Diversity ◽  
Yeast Culture ◽  
Culture Collections ◽  
Komagataella Phaffii ◽  
Rich Media ◽  
Natural Isolates ◽  
Trait Locus ◽  
Almost All

Abstract Background Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were previously called Pichia pastoris. However, almost all laboratory work on K. phaffii has utilized strains derived from a single natural isolate, CBS7435. There is little information about the sequence diversity of K. phaffii or the genetic properties of this species. Results We sequenced the genomes of all the known isolates of K. phaffii. We made a genetic cross between derivatives of two isolates that differ at 44,000 single nucleotide polymorphism sites, and used this cross to analyze the rate and landscape of meiotic recombination. We conducted tetrad analysis by making use of the property that K. phaffii haploids do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetra-type ascus germinates. Conclusions We found that only four distinct natural isolates of K. phaffii exist in public yeast culture collections. The meiotic recombination rate in K. phaffii is approximately 3.5 times lower than in Saccharomyces cerevisiae, with an average of 25 crossovers per meiosis. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our work lays a foundation for future quantitative trait locus analysis in K. phaffii.

scholarly journals Crossover Interference in Saccharomyces cerevisiae Requires a TID1/RDH54- and DMC1-Dependent Pathway

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1273-1286 ◽  
Author(s):  
Miki Shinohara ◽  
Kazuko Sakai ◽  
Akira Shinohara ◽  
Douglas K Bishop
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Tetrad Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Crossover Interference ◽  
Meiotic Progression ◽  
Invasion Stage ◽  
Strand Invasion ◽  
Dependent Pathway

Abstract Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Δ mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Δ cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.

scholarly journals Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression inPichia pastoris

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Thomas Vogl ◽  
Leigh Gebbie ◽  
Robin W. Palfreyman ◽  
Robert Speight
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Protein Production ◽  
Eukaryotic Expression ◽  
Gene Copy ◽  
Clonal Variation ◽  
Reporter Protein ◽  
Content Type ◽  
Komagataella Phaffii ◽  
Highly Uniform

ABSTRACTPichia pastoris(syn.Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast toSaccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient inP. pastorisand ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted inP. pastoris. Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs).IMPORTANCEYeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeastSaccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such asPichia pastoris(syn.Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation inP. pastorisand, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.

scholarly journals Genome Duplication Increases Meiotic Recombination Frequency: A Saccharomyces cerevisiae Model

2020 ◽  
Author(s):  
Ou Fang ◽  
Lin Wang ◽  
Yuxin Zhang ◽  
Jixuan Yang ◽  
Qin Tao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Genome Stability ◽  
Genome Duplication ◽  
Genetic Recombination ◽  
Recombination Frequency ◽  
Abiotic Factors ◽  
Strand Break ◽  
Homologous Chromosomes ◽  
Almost All

Abstract Genetic recombination characterized by reciprocal exchange of genes on paired homologous chromosomes is the most prominent event in meiosis of almost all sexually reproductive organisms. It contributes to genome stability by ensuring the balanced segregation of paired homologs in meiosis, and it is also the major driving factor in generating genetic variation for natural and artificial selection. Meiotic recombination is subjected to the control of a highly stringent and complex regulating process and meiotic recombination frequency (MRF) may be affected by biological and abiotic factors such as sex, gene density, nucleotide content, and chemical/temperature treatments, having motivated tremendous researches for artificially manipulating MRF. Whether genome polyploidization would lead to a significant change in MRF has attracted both historical and recent research interests; however, tackling this fundamental question is methodologically challenging due to the lack of appropriate methods for tetrasomic genetic analysis, thus has led to controversial conclusions in the literature. This article presents a comprehensive and rigorous survey of genome duplication-mediated change in MRF using Saccharomyces cerevisiae as a eukaryotic model. It demonstrates that genome duplication can lead to consistently significant increase in MRF and rate of crossovers across all 16 chromosomes of S. cerevisiae, including both cold and hot spots of MRF. This ploidy-driven change in MRF is associated with weakened recombination interference, enhanced double-strand break density, and loosened chromatin histone occupation. The study illuminates a significant evolutionary feature of genome duplication and opens an opportunity to accelerate response to artificial and natural selection through polyploidization.

scholarly journals Poorly Repaired Mismatches in Heteroduplex DNA are Hyper-Recombinagenic in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 407-416 ◽  
Author(s):  
P Manivasakam ◽  
Susan M Rosenberg ◽  
P J Hastings
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Genetic Markers ◽  
Meiotic Recombination ◽  
Repair System ◽  
Normal Allele ◽  
Heteroduplex Dna ◽  
Mismatch Repair System ◽  
Postmeiotic Segregation ◽  
System Operating

Abstract In yeast meiotic recombination, alleles used as genetic markers fall into two classes as regards their fate when incorporated into heteroduplex DNA. Normal alleles are those that form heteroduplexes that are nearly always recognized and corrected by the mismatch repair system operating in meiosis. High PMS (postmeiotic segregation) alleles form heteroduplexes that are inefficiently mismatch repaired. We report that placing any of several high PMS alleles very close to normal alleles causes hyperrecombination between these markers. We propose that this hyperrecombination is caused by the high PMS allele blocking a mismatch repair tract initiated from the normal allele, thus preventing corepair of the two alleles, which would prevent formation of recombinants. The results of three point crosses involving two PMS alleles and a normal allele suggest that high PMS alleles placed between two alleles that are normally corepaired block that corepair.

scholarly journals Phylogeographic Genetic Diversity in the White Sucker Hepatitis B Virus across the Great Lakes Region and Alberta, Canada

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 285
Author(s):  
Cynthia R. Adams ◽  
Vicki S. Blazer ◽  
Jim Sherry ◽  
Robert Scott Cornman ◽  
Luke R. Iwanowicz
Keyword(s):  
Genetic Diversity ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Lake Michigan ◽  
Illumina Miseq ◽  
Genomic Variation ◽  
Genomic Diversity ◽  
White Sucker ◽  
Fish Health ◽  
B Virus

Hepatitis B viruses belong to a family of circular, double-stranded DNA viruses that infect a range of organisms, with host responses that vary from mild infection to chronic infection and cancer. The white sucker hepatitis B virus (WSHBV) was first described in the white sucker (Catostomus commersonii), a freshwater teleost, and belongs to the genus Parahepadnavirus. At present, the host range of WSHBV and its impact on fish health are unknown, and neither genetic diversity nor association with fish health have been studied in any parahepadnavirus. Given the relevance of genomic diversity to disease outcome for the orthohepadnaviruses, we sought to characterize genomic variation in WSHBV and determine how it is structured among watersheds. We identified WSHBV-positive white sucker inhabiting tributaries of Lake Michigan, Lake Superior, Lake Erie (USA), and Lake Athabasca (Canada). Copy number in plasma and in liver tissue was estimated via qPCR. Templates from 27 virus-positive fish were amplified and sequenced using a primer-specific, circular long-range amplification method coupled with amplicon sequencing on the Illumina MiSeq. Phylogenetic analysis of the WSHBV genome identified phylogeographical clustering reminiscent of that observed with human hepatitis B virus genotypes. Notably, most non-synonymous substitutions were found to cluster in the pre-S/spacer overlap region, which is relevant for both viral entry and replication. The observed predominance of p1/s3 mutations in this region is indicative of adaptive change in the polymerase open reading frame (ORF), while, at the same time, the surface ORF is under purifying selection. Although the levels of variation we observed do not meet the criteria used to define sub/genotypes of human and avian hepadnaviruses, we identified geographically associated genome variation in the pre-S and spacer domain sufficient to define five WSHBV haplotypes. This study of WSHBV genetic diversity should facilitate the development of molecular markers for future identification of genotypes and provide evidence in future investigations of possible differential disease outcomes.

scholarly journals Unveiling a unique genetic diversity of cultivated Coffea arabica L. in its main domestication center: Yemen

2021 ◽  
Author(s):  
C. Montagnon ◽  
A. Mahyoub ◽  
W. Solano ◽  
F. Sheibani
Keyword(s):  
Genetic Diversity ◽  
Genetic Distance ◽  
Coffea Arabica ◽  
Genetic Improvement ◽  
The World ◽  
Genetic Clusters ◽  
Domestication Process ◽  
Almost All ◽  
Coffea Arabica L ◽  
Cup Quality

AbstractWhilst it is established that almost all cultivated coffee (Coffea arabica L.) varieties originated in Yemen after some coffee seeds were introduced into Yemen from neighboring Ethiopia, the actual coffee genetic diversity in Yemen and its significance to the coffee world had never been explored. We observed five genetic clusters. The first cluster, which we named the Ethiopian-Only (EO) cluster, was made up exclusively of the Ethiopian accessions. This cluster was clearly separated from the Yemen and cultivated varieties clusters, hence confirming the genetic distance between wild Ethiopian accessions and coffee cultivated varieties around the world. The second cluster, which we named the SL-17 cluster, was a small cluster of cultivated worldwide varieties and included no Yemen samples. Two other clusters were made up of worldwide varieties and Yemen samples. We named these the Yemen Typica-Bourbon cluster and the Yemen SL-34 cluster. Finally, we observed one cluster that was unique to Yemen and was not related to any known cultivated varieties and not even to any known Ethiopian accession: we name this cluster the New-Yemen cluster. We discuss the consequences of these findings and their potential to pave the way for further comprehensive genetic improvement projects for the identification of major resilience/adaptation and cup quality genes that have been shaped through the domestication process of C. arabica.

scholarly journals Suppressor Analysis of the Saccharomyces cerevisiae Gene REC104 Reveals a Genetic Interaction With REC102

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1261-1272 ◽  
Author(s):  
Laura Salem ◽  
Natalie Walter ◽  
Robert Malone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Null Allele ◽  
Genetic Interaction ◽  
In Vitro Mutagenesis ◽  
Conditional Mutations ◽  
Suppressor Analysis

Abstract REC104 is a gene required for the initiation of meiotic recombination in Saccharomyces cerevisiae. To better understand the role of REC104 in meiosis, we used an in vitro mutagenesis technique to create a set of temperature-conditional mutations in REC104 and used one ts allele (rec104-8) in a screen for highcopy suppressors. An increased dosage of the early exchange gene REC102 was found to suppress the conditional recombinational reduction in rec104-8 as well as in several other conditional rec104 alleles. However, no suppression was observed for a null allele of REC104, indicating that the suppression by REC102 is not “bypass” suppression. Overexpression of the early meiotic genes REC114, RAD50, HOP1, and RED1 fails to suppress any of the rec104 conditional alleles, indicating that the suppression might be specific to REC102.

scholarly journals Competition Between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces cerevisiae

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 661-670 ◽  
Author(s):  
Qing-Qing Fan ◽  
Fei Xu ◽  
Michael A White ◽  
Thomas D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Telomeric Sequence ◽  
Coding Region ◽  
Dna Breaks ◽  
Local Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Recombination Hotspots ◽  
Double Strand Dna Breaks ◽  
His4 Gene

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.

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