scholarly journals Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

2018 ◽  
Author(s):  
Václav Vopálenský ◽  
Michal Sýkora ◽  
Tomáš Mašek ◽  
Martin Pospíšek
Keyword(s):  
Yeast Species ◽  
Kluyveromyces Lactis ◽  
Messenger Rnas ◽  
Alternative Promoters ◽  
Linear Plasmids ◽  
Promoter Regions ◽  
Translation Factors ◽  
Mrna Capping ◽  
Close Relationship ◽  
Race Analysis

AbstractLinear plasmids with almost identical compact genetic organization have been found in the cytoplasm of yeast species from nine genera. We employed pGKL1,2 plasmids fromKluyveromyces lactisas a model to investigate the previously unstudied transcriptome of yeast cytoplasmic linear plasmids. We performed 5’ and 3’ RACE analysis of all the pGKL1,2 mRNAs and found them not 3’ polyadenylated and containing mostly uncapped 5’ poly(A) leaders that are not complementary to the plasmid DNA. The degree of 5’ capping and/or 5’ polyadenylation is specific to each gene and is controlled by the corresponding promoter regions. We refined the description of the pGKL1,2 promoters and found new alternative promoters of several genes. We also provide evidence thatK2ORF3encodes an mRNA cap guanine-N7-methyltransferase and that 5’ capped pGKL1,2 transcripts contain N7-methylated caps. Translation of pGKL1,2 transcripts is enhanced inIsm1Δandpab1Δstrains and is independent of eIF4E and Pab1 translation factors. We suggested a model of a primitive regulation of pGKL1,2 plasmids gene expression where degree of 5’ mRNA capping, degree of 5’ non-template polyadenylation and presence of negative regulators as PAB1 and Lsm1 play an important role. Our data also suggest a close relationship between linear plasmids and poxviruses.

scholarly journals Conservation of the Prion Properties of Ure2p through Evolution

2003 ◽  
Vol 14 (8) ◽  
pp. 3449-3458 ◽  
Author(s):  
Agnès Baudin-Baillieu ◽  
Eric Fernandez-Bellot ◽  
Fabienne Reine ◽  
Eric Coissac ◽  
Christophe Cullin
Keyword(s):  
Functional Analysis ◽  
Candida Albicans ◽  
Gene Deletion ◽  
Yeast Species ◽  
Kluyveromyces Lactis ◽  
In Silico Analysis ◽  
Functional Domain ◽  
Extreme Situation ◽  
Homologous Genes ◽  
Silico Analysis

The yeast inheritable [URE3] element corresponds to a prion form of the nitrogen catabolism regulator Ure2p. We have isolated several orthologous URE2 genes in different yeast species: Saccharomyces paradoxus, S. uvarum, Kluyveromyces lactis, Candida albicans, and Schizosaccharomyces pombe. We show here by in silico analysis that the GST-like functional domain and the prion domain of the Ure2 proteins have diverged separately, the functional domain being more conserved through the evolution. The more extreme situation is found in the two S. pombe genes, in which the prion domain is absent. The functional analysis demonstrates that all the homologous genes except for the two S. pombe genes are able to complement the URE2 gene deletion in a S. cerevisiae strain. We show that in the two most closely related yeast species to S. cerevisiae, i.e., S. paradoxus and S. uvarum, the prion domains of the proteins have retained the capability to induce [URE3] in a S. cerevisiae strain. However, only the S. uvarum full-length Ure2p is able to behave as a prion. We also show that the prion inactivation mechanisms can be cross-transmitted between the S. cerevisiae and S. uvarum prions.

scholarly journals Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters

2002 ◽  
Vol 184 (2) ◽  
pp. 427-432 ◽  
Author(s):  
Paola Goffrini ◽  
Iliana Ferrero ◽  
Claudia Donnini
Keyword(s):  
Yeast Species ◽  
Kluyveromyces Lactis ◽  
Sugar Uptake ◽  
Sugar Transporters ◽  
High Substrate ◽  
Fermentative Growth ◽  
Major Bottleneck ◽  
Respiratory Conditions

ABSTRACT In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained K. lactis strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for K. lactis cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K. lactis and probably in many other yeasts.

scholarly journals Structure–function analysis of the nsp14 N7–guanine methyltransferase reveals an essential role in Betacoronavirus replication

2021 ◽  
Vol 118 (49) ◽  
pp. e2108709118
Author(s):  
Natacha S. Ogando ◽  
Priscila El Kazzi ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Brenda W. Bontes ◽  
Alice Decombe ◽  
...  
Keyword(s):  
Function Analysis ◽  
Nonstructural Protein ◽  
Site Directed Mutagenesis ◽  
Messenger Rnas ◽  
Mtase Activity ◽  
Mrna Capping ◽  
Activity Spectrum ◽  
Immune Sensing ◽  
Key Residues

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5′ exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3′-to-5′ ExoN domain and a C-terminal (N7-guanine)–methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14’s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)–CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

CRABTREE POZİTİF VE CRABTREE NEGATİF MAYA TÜRLERİNDE GCR1 GENİNİN IN SILICO ANALİZİ

2021 ◽  
Vol 8 (17) ◽  
pp. 207-216
Author(s):  
Tülay TURGUT GENÇ ◽  
Ataberk ÇAKAN ◽  
Melih GÜNAY
Keyword(s):  
In Silico ◽  
Protein Production ◽  
Heterologous Protein ◽  
Yeast Species ◽  
Yeast Cells ◽  
Crabtree Effect ◽  
Promoter Regions ◽  
Yeast Strains ◽  
Genes Encoding ◽  
Chromatin Folding

The use of fermentation in the presence of oxygen and at high glucose concentrations is referred to as the Crabtree effect. Yeast species that have the Crabtree effect are called Crabtree positive, and yeast species that do not have the Crabtree effect are called Crabtree negative. While Crabtree negative yeast strains are mostly used for heterologous protein production in the industrial field, Crabtree positive yeast strains are used to understand metabolic events in cancer cells. The genes encoding the enzymes involved in the glycolytic pathway in S. cerevisiae yeast cells are controlled by Gcr1p. Gcr1p binds to CT elements located in the promoter regions of glycolytic genes and activates their transcription. In our study, Crabtree positive and negative yeast strains containing Sc-Gcr1p similar proteins were determined, and protein similarity analyzes and promoter analyzes of genes encoding the relevant proteins in these yeast strains were compared in silico using different databases and analysis programs. For this purpose, SGD, UNIPROT, NCBI-Genome and Yeastract databases and BLASTp-NCBI, MEGA-X and Chromatin Folding V2 programs were used. Using the SGD database, 32 different yeast strains were identified that matched with Sc-Gcr1p. Five different Crabtree positive and 5 different Crabtree negative yeast strains were selected from these yeast strains and in silico analyzes were performed using these yeast strains. After protein analysis and promoter analysis, it was determined that the similarities and differences between yeast species were not specific for Crabtree positive and Crabtree negative yeast species, but varied between species.

scholarly journals Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function inCandida albicans

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Swati Bijlani ◽  
Mathuravani A. Thevandavakkam ◽  
Hung-Ji Tsai ◽  
Judith Berman
Keyword(s):  
Candida Albicans ◽  
Colony Size ◽  
Transformation Efficiency ◽  
Kluyveromyces Lactis ◽  
Baker’S Yeast ◽  
Dependent Manner ◽  
Baker's Yeast ◽  
Linear Plasmids ◽  
Content Type ◽  
Bona Fide

ABSTRACTThe ability to generate autonomously replicating plasmids has been elusive inCandida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants (tiny, medium, and large, respectively).Candida albicansURA3and a heterologous marker,ARG4,yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologousLEU2marker was highly dependent upon the addition of abona fideorigin sequence. Severalbona fidechromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin,panARS, fromKluyveromyces lactis, drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus,C. albicansmaintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner.IMPORTANCECircular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, inCandida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate inC. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of aC. albicansorigin and an origin sequence from a distantly related yeast can both function as origins inC. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ betweenC. albicansand baker’s yeast.

scholarly journals Kazachstania siamensis sp. nov., an ascomycetous yeast species from forest soil in Thailand

2007 ◽  
Vol 57 (2) ◽  
pp. 419-422 ◽  
Author(s):  
Savitree Limtong ◽  
Wichien Yongmanitchai ◽  
Moe Moe Tun ◽  
Hiroko Kawasaki ◽  
Tatsuji Seki
Keyword(s):  
Deciduous Forest ◽  
Yeast Species ◽  
Large Subunit ◽  
The Novel ◽  
Phenotypic Characteristics ◽  
Nucleotide Substitutions ◽  
Ascomycetous Yeast ◽  
Rdna Sequences ◽  
Close Relationship ◽  
Large Subunit Rdna

Two strains (S-34T and S-35) of a novel ascomycetous yeast species belonging to the genus Kazachstania were isolated from soil from a mixed deciduous forest in Amphoe Wang Nam Khiao, Nakhon Ratchasima Province, Thailand. The D1/D2 domains of the large-subunit rDNA sequences of the two strains were identical and also indicated a close relationship with respect to Kazachstania aquatica, Kazachstania unispora, Kazachstania aerobia, Kazachstania servazzii and Kazachstania solicola. The most closely related species, K. aquatica, has 14 nucleotide substitutions and three gaps in 566 nt. The phenotypic characteristics of the two strains were typical of those of members of the genus Kazachstania. These characteristics include the formation of a single globose ascospore in an unconjugated and persistent ascus, multilateral budding, the absence of arthrospores and ballistospores, the fermentation of glucose, the inability to assimilate nitrate, negative diazonium blue B and urease reactions, and the presence of ubiquinone Q-6. The novel strains can be distinguished from K. aquatica on the basis of a number of phenotypic characteristics and represent a novel species in the genus Kazachstania, for which the name Kazachstania siamensis sp. nov. is proposed. The type strain is S-34T (=CBS 10361T=NBRC 101968T=BCC 21230T).

scholarly journals The NAD+-dependent deacetylase Sir2 enables evolution of new traits by regulating distinct gene sets in two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis

2019 ◽  
Author(s):  
Kristen M. Humphrey ◽  
Lisha Zhu ◽  
Meleah A. Hickman ◽  
Shirin Hasan ◽  
Haniam Maria ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Networks ◽  
Environmental Changes ◽  
Yeast Species ◽  
Kluyveromyces Lactis ◽  
Carbon Sources ◽  
Adaptive Responses ◽  
Gene Sets ◽  
Conserved Genes ◽  
Heavy Metal Efflux

ABSTRACTEvolutionary adaptation increases the fitness of an organism in its environment. It can occur through rewiring of gene regulatory networks, such that an organism responds appropriately to environmental changes. We investigated whether sirtuin deacetylases, which repress transcription and require NAD+ for activity, could facilitate the evolution of potentially adaptive responses by serving as transcriptional rewiring points. If so, bringing genes under the control of sirtuins could enable organisms to mount appropriate responses to stresses that decrease NAD+ levels. To explore how the genomic targets of sirtuins shift over evolutionary time, we compared two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis that display differences in cellular metabolism and lifecycle timing in response to nutrient availability. We identified sirtuin-regulated genes through a combination of chromatin immunoprecipitation and RNA expression. In both species, regulated genes were associated with NAD+ homeostasis, mating, and sporulation, but the specific genes differed. In addition, regulated genes in K. lactis were associated with other processes, including utilization of non-glucose carbon sources, heavy metal efflux, DNA synthesis, and production of the siderophore pulcherrimin. Consistent with the species-restricted regulation of these genes, sirtuin deletion impacted relevant phenotypes in K. lactis but not S. cerevisiae. Finally, sirtuin-regulated gene sets were depleted for broadly-conserved genes, consistent with sirtuins regulating processes restricted to a few species. Taken together, these results are consistent with the notion that sirtuins serve as rewiring points that allow species to evolve distinct responses to low NAD+ stress.

scholarly journals Paralogs in the PKA Regulon Traveled Different Evolutionary Routes to Divergent Expression in Budding Yeast

2021 ◽  
Vol 2 ◽  
Author(s):  
Benjamin M. Heineike ◽  
Hana El-Samad
Keyword(s):  
Yeast Species ◽  
Budding Yeast ◽  
Kluyveromyces Lactis ◽  
Functional Divergence ◽  
Promoter Sequence ◽  
Duplicate Genes ◽  
Generating Functional ◽  
Basal Expression ◽  
Genes Expression ◽  
Paralog Gene

Functional divergence of duplicate genes, or paralogs, is an important driver of novelty in evolution. In the model yeast Saccharomyces cerevisiae, there are 547 paralog gene pairs that survive from an interspecies Whole Genome Hybridization (WGH) that occurred ~100MYA. In this work, we report that ~1/6th (110) of these WGH paralogs pairs (or ohnologs) are differentially expressed with a striking pattern upon Protein Kinase A (PKA) inhibition. One member of each pair in this group has low basal expression that increases upon PKA inhibition, while the other has moderate and unchanging expression. For these genes, expression of orthologs upon PKA inhibition in the non-WGH species Kluyveromyces lactis and for PKA-related stresses in other budding yeasts shows unchanging expression, suggesting that lack of responsiveness to PKA was likely the typical ancestral phenotype prior to duplication. Promoter sequence analysis across related budding yeast species further revealed that the subsequent emergence of PKA-dependence took different evolutionary routes. In some examples, regulation by PKA and differential expression appears to have arisen following the WGH, while in others, regulation by PKA appears to have arisen in one of the two parental lineages prior to the WGH. More broadly, our results illustrate the unique opportunities presented by a WGH event for generating functional divergence by bringing together two parental lineages with separately evolved regulation into one species. We propose that functional divergence of two ohnologs can be facilitated through such regulatory divergence.

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