Compacting a synthetic yeast chromosome arm

Abstract Background Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction. Results We develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified. Conclusions We develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.

Download Full-text

DNA replication, transcription, and H3K56 acetylation regulate copy number and stability at tandem repeats

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab082 ◽

2021 ◽

Cited By ~ 1

Author(s):

Devika Salim ◽

William D Bradford ◽

Boris Rubinstein ◽

Jennifer L Gerton

Keyword(s):

Dna Replication ◽

Copy Number Variation ◽

Histone Acetylation ◽

Copy Number ◽

Tandem Repeats ◽

Gene Array ◽

Rdna Locus ◽

Yeast Genome ◽

Number Variation ◽

H3k56 Acetylation

AbstractTandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, we used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. We developed powerful, highly sensitive, and quantitative assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of both arrays, we demonstrate that instability can be induced by DNA replication stress and transcription. Importantly, while changes in stability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection over time. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and restricts transcription-induced amplification. Our work suggests that the modulation of replication and transcription is a direct, reversible strategy to alter stability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the omnipresence of DNA replication, transcription, and chromatin marks like histone acetylation, the fundamental mechanisms we have uncovered significantly advance our understanding of the plasticity of tandem repeats more generally.

Download Full-text

Construction and Characterization of a Fusion Protein of Single-Chain Anti-CD20 Antibody and Human β-Glucuronidase for Antibody-Directed Enzyme Prodrug Therapy

Blood ◽

10.1182/blood.v92.1.184.413k26_184_190 ◽

1998 ◽

Vol 92 (1) ◽

pp. 184-190 ◽

Cited By ~ 34

Author(s):

Hidde J. Haisma ◽

M. Fleur Sernee ◽

Erik Hooijberg ◽

Ruud H. Brakenhoff ◽

Ida H. v.d. Meulen-Muileman ◽

...

Keyword(s):

Fusion Protein ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Tumor Site ◽

Single Chain ◽

Enzyme Prodrug Therapy ◽

Synthetic Sequence ◽

Cd20 Antibody ◽

Sequence Encoding ◽

Anti Cd20

The CD20 antigen is an attractive target for specific treatment of B-cell lymphoma. Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a nontoxic prodrug at the tumor site by an enzyme targeted by a tumor-specific antibody such as anti-CD20. We constructed a fusion protein of the single-chain Fv anti-CD20 mouse monoclonal antibody (MoAb) 1H4 and human β-glucuronidase for the activation of the nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-β-glucuronyl carbamate to doxorubicin at the tumor site. The cDNAs encoding the light- and heavy-chain variable regions of 1H4 were cloned, joined by a synthetic sequence encoding a 15-amino acid linker and fused to human β-glucuronidase by a synthetic sequence encoding a 6-amino acid linker. An antibody-enzyme fusion protein-producing cell line was established by transfection of the construct into human embryonic kidney 293/EBNA cells. The yield of active fusion protein was 100 ng/mL transfectoma supernatant. Antibody affinity, antibody specificity, and enzyme activity were fully retained by the fusion protein. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the fusion protein has a relative molecular weight (Mw) of 100 kD under denaturing conditions. Gel filtration analysis indicated that the enzymatically active form of the fusion protein is a tetramer with an Mw of approximately 400 kD. The nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-β-glucuronyl carbamate was hydrolyzed by the fusion protein at a hydrolysis rate similar to that of human β-glucuronidase. When the fusion protein was specifically bound to Daudi lymphoma cells, the prodrug induced similar antiproliferative effects as doxorubicin. Thus, it is feasible to construct a eukaryotic fusion protein consisting of a single-chain anti-CD20 antibody and human β-glucuronidase for future use in the activation of anticancer prodrugs in B-cell lymphoma.

Download Full-text

Efficacy of Allicin against Plant Pathogenic Fungi and Unveiling the Underlying Mode of Action Employing Yeast Based Chemogenetic Profiling Approach

Applied Sciences ◽

10.3390/app10072563 ◽

2020 ◽

Vol 10 (7) ◽

pp. 2563 ◽

Cited By ~ 1

Author(s):

Muhammad Sarfraz ◽

Muhammad Jawad Nasim ◽

Claus Jacob ◽

Martin C. H. Gruhlke

Keyword(s):

Mode Of Action ◽

Plant Pathogens ◽

Essential Gene ◽

Pathogenic Fungi ◽

Yeast Cells ◽

Yeast Genome ◽

Plant Pathogenic Fungi ◽

Mitochondrial Translation ◽

Antifungal Activities ◽

Wide Range

Allicin (diallylthiosulfinate) is the principal organosulfur compound present in freshly damaged garlic tissue which exhibits a wide range of biological actions including antibacterial, antifungal, antiviral and anticancer properties. The antifungal activities of allicin were investigated against plant pathogenic fungi of agriculture importance. Furthermore, a yeast genome haploinsufficiency screening was also employed to decipher the antifungal mode of action of allicin. Wildtype and 1152 yeast mutant strains (each deprived of one specific allele of an essential gene in a diploid strain) were screened against allicin. Allicin exhibited promising antifungal properties against all the tested plant pathogens. Haploinsufficiency screening revealed three hypersensitive yeast mutants with gene deletions coding for proteins involved in DNA replication, mitochondrial translation and chromatids cohesion. These processes play a vital role in the cell cycle, growth and viability of yeast cells. Taken together, the results of the present study unravel the excellent antifungal activities and mechanisms and modes of action of allicin. These findings also indicate the potential use of allicin as an alternative “green” fungicide (fumigant) in agriculture.

Download Full-text

A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.3081-3087.1989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 3081-3087

Author(s):

M Foiani ◽

C Santocanale ◽

P Plevani ◽

G Lucchini

Keyword(s):

Dna Polymerase ◽

Essential Gene ◽

Large Subunit ◽

Mitotic Cell ◽

Yeast Genome ◽

Gene Encoding ◽

Dna Primase ◽

Dna Library ◽

Calculated Molecular Weight ◽

Primase Complex

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.

Download Full-text

Conditional mutants of RPC160, the gene encoding the largest subunit of RNA polymerase C in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/119.3.517 ◽

1988 ◽

Vol 119 (3) ◽

pp. 517-526

Author(s):

R Gudenus ◽

S Mariotte ◽

A Moenne ◽

A Ruet ◽

S Memet ◽

...

Keyword(s):

Rna Polymerase ◽

Essential Gene ◽

Mutant Cell ◽

Yeast Genome ◽

Temperature Sensitive ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Gene Encoding

Abstract A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO3 and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA. An unlinked mutation (pcil-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation.

Download Full-text

Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations

10.1101/2021.07.26.453910 ◽

2021 ◽

Author(s):

Bin Jia ◽

Jin Jin ◽

Mingzhe Han ◽

Ying-Jin Yuan

Keyword(s):

Genome Evolution ◽

Rational Design ◽

Chromosome Rearrangement ◽

Genomic Variation ◽

Yeast Genome ◽

Structural Variations ◽

Heterologous Biosynthesis ◽

Naturally Occurring ◽

3D Architecture ◽

Random Screen

Background: Naturally occurring structural variations (SVs) are a considerable source of genomic variation and can reshape chromosomes 3D architecture. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been proved to generate random SVs to impact phenotypes and thus constitutes powerful drivers of directed genome evolution. However, controllable methods to introduce complex SVs and revealing the related molecular mechanism has so far remained challenging. Results: We develop a SV-prone yeast strain by using SCRaMbLE with two synthetic chromosomes, synV and synX. An heterologous biosynthesis pathway allowing a high throughput screen for increased yield of astaxanthin is used as readout and a proof of concept for the application of SV in industry. We report here that complex SVs, including a pericentric inversion and a trans-chromosomes translocation between synV and synX, result in two neochromosomes and a 2.7-fold yield of astaxanthin. We mapped genetic targets contributing to higher astaxanthin yield and demonstrated the SVs can led to large reorganization of the genetic information along the chromosomes. We also used the model learned from the aforementioned random screen and successfully harnessed the precise introduction of trans-chromosomes translocation and pericentric inversions by rational design. Conclusions: Our work provides an effective tool to not only accelerate the directed genome evolution but also reveal mechanistic insight of complex SVs for altering phenotypes.

Download Full-text

A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.3081 ◽

1989 ◽

Vol 9 (7) ◽

pp. 3081-3087 ◽

Cited By ~ 39

Author(s):

M Foiani ◽

C Santocanale ◽

P Plevani ◽

G Lucchini

Keyword(s):

Dna Polymerase ◽

Essential Gene ◽

Large Subunit ◽

Mitotic Cell ◽

Yeast Genome ◽

Gene Encoding ◽

Dna Primase ◽

Dna Library ◽

Calculated Molecular Weight ◽

Primase Complex

Download Full-text

Factors that affect the location and frequency of meiosis-induced double-strand breaks in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/140.1.55 ◽

1995 ◽

Vol 140 (1) ◽

pp. 55-66 ◽

Cited By ~ 6

Author(s):

T C Wu ◽

M Lichten

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromatin Structure ◽

Meiotic Recombination ◽

Double Strand Breaks ◽

Yeast Genome ◽

Dna Breaks ◽

Strand Breaks ◽

Double Strand Dna Breaks ◽

A Site ◽

Chromosomal Sequences

Abstract Double-strand DNA breaks (DSBs) initiate meiotic recombination in Saccharomyces cerevisiae. DSBs occur at sites that are hypersensitive in nuclease digests of chromatin, suggesting a role for chromatin structure in determining DSB location. We show here that the frequency of DSBs at a site is not determined simply by DNA sequence or by features of chromatin structure. An arg4-containing plasmid was inserted at several different locations in the yeast genome. Meiosis-induced DSBs occurred at similar sites in pBR322-derived portions of the construct at all insert loci, and the frequency of these breaks varied in a manner that mirrored the frequency of meiotic recombination in the arg4 portion of the insert. However, DSBs did not occur in the insert-borne arg4 gene at a site that is frequently broken at the normal ARG4 locus, even though the insert-borne arg4 gene and the normal ARG4 locus displayed similar DNase I hypersensitivity patterns. Deletions that removed active DSB sites from an insert at HIS4 restored breaks to the insert-borne arg4 gene and to a DSB site in flanking chromosomal sequences. We conclude that the frequency of DSB at a site can be affected by sequences several thousand nucleotides away and suggest that this is because of competition between DSB sites for locally limited factors.

Download Full-text