PubMed Health⌕ Search

Biomedical subjects

Mick F Tuite

Publications and source records attributed to Mick F Tuite.

At least 19 recordsLinked to original sources
PubMed2006

Regulated translational bypass of stop codons in yeast.

Stop codons are used to signal the ribosome to terminate the decoding of an mRNA template. Recent studies on translation termination in the yeast Saccharomyces cerevisiae have not only enabled the identification of the key components of the termination machinery, but have also revealed several regulatory mechanisms that might enable the controlled synthesis of C-terminally extended polypeptides via stop-codon readthrough. These include both genetic and epigenetic mechanisms. Rather than being a translation 'error', stop-codon readthrough can have important effects on other cellular processes such as mRNA degradation and, in some cases, can confer a beneficial phenotype to the cell.

Codon, Terminator↗
PubMed2006

The [PSI+] prion of yeast: a problem of inheritance.

The [PSI(+)] prion of the yeast Saccharomyces cerevisiae was first identified by Brian Cox some 40 years ago as a non-Mendelian genetic element that modulated the efficiency of nonsense suppression. Following the suggestion by Reed Wickner in 1994 that such elements might be accounted for by invoking a prion-based model, it was subsequently established that the [PSI(+)] determinant was the prion form of the Sup35p protein. In this article, we review how a combination of classical genetic approaches and modern molecular and biochemical methods has provided conclusive evidence of the prion basis of the [PSI(+)] determinant. In so doing we have tried to provide a historical context, but also describe the results of more recent experiments aimed at elucidating the mechanism by which the [PSI(+)] (and other yeast prions) are efficiently propagated in dividing cells. While understanding of the [PSI(+)] prion and its mode of propagation has, and will continue to have, an impact on mammalian prion biology nevertheless the very existence of a protein-based mechanism that can have a beneficial impact on a cell's fitness provides equally sound justification to fully explore yeast prions.

Amino Acid Sequence↗
PubMed2006

Zeocin resistance as a dominant selective marker for transformation and targeted gene deletions in Candida glabrata.

Many of the genetic tools used to generate gene knockouts in Candida glabrata exploit auxotrophic markers but this is not suitable for use with clinical strains. Antibiotic resistance markers, however, allow one to target genes to be deleted without any prior genetic manipulation of clinical isolates. Such antibiotic selection markers have been widely reported for the manipulation of Saccharomyces cerevisiae. However, very few antibiotic resistance markers have been shown to be useful in C. glabrata. Here, we report the use of Zeocin resistance (ZeoR), encoded by the ble gene from Streptoalloteichus hindustanus, as a new positive selection marker for the genetic manipulation of C. glabrata including clinical strains that we show are significantly more sensitive to Zeocin than to G418. The potential of the ZeoR marker for targeted gene disruption in C. glabrata was confirmed by constructing deletions of the ADE2 in both a laboratory and a clinical strain of C. glabrata, using both short (90 bp) and long (400 bp) homology cassettes.

Bleomycin↗
PubMed2006

The [PSI+] prion of Saccharomyces cerevisiae can be propagated by an Hsp104 orthologue from Candida albicans.

The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI+], [URE3], and [RNQ/PIN+] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI+] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI+] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.

Amino Acid Sequence↗
PubMed2005

Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence.

The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients . Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence . Filamentation is mediated by second messengers such as cyclic adenosine 3',5'-monophosphate (cAMP) synthesized by adenylyl cyclase . The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients . Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity . Here we demonstrate that physiological concentrations of CO2/HCO3- induce filamentation in C. albicans by direct stimulation of cyclase activity. Furthermore, we show that CO2/HCO3- equilibration by carbonic anhydrase is essential for pathogenesis of C. albicans in niches where the available CO2 is limited. We also demonstrate that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3-. These data demonstrate that the link between cAMP signaling and CO2/HCO3- sensing is conserved in fungi and reveal CO2 sensing to be an important mediator of fungal pathogenesis. Novel therapeutic agents could target this pathway at several levels to control fungal infections.

Adenylyl Cyclases↗
PubMed2005

The role of the N-terminal oligopeptide repeats of the yeast Sup35 prion protein in propagation and transmission of prion variants.

The cytoplasmic [PSI+] determinant of Saccharomyces cerevisiae is the prion form of the Sup35 protein. Oligopeptide repeats within the Sup35 N-terminal domain (PrD) presumably are required for the stable [PSI+] inheritance that in turn involves fragmentation of Sup35 polymers by the chaperone Hsp104. The nonsense suppressor [PSI+] phenotype can vary in efficiency probably due to different inheritable Sup35 polymer structures. Here we study the ability of Sup35 mutants with various deletions of the oligopeptide repeats to support [PSI+] propagation. We define the minimal region of the Sup35-PrD necessary to support [PSI+] as amino acids 1-64, which include the first two repeats, although a longer fragment, 1-83, is required to maintain weak [PSI+] variants. Replacement of wild-type Sup35 with deletion mutants decreases the strength of the [PSI+] phenotype. However, with one exception, reintroducing the wild-type Sup35 restores the original phenotype. Thus, the specific prion fold defining the [PSI+] variant can be preserved by the mutant Sup35 protein despite the change of phenotype. Coexpression of wild-type and mutant Sup35 containing three, two, one, or no oligopeptide repeats causes variant-specific [PSI+] elimination. These data suggest that [PSI+] variability is primarily defined by differential folding of the Sup35-PrD oligopeptide-repeat region.

Base Sequence↗
PubMed2005

Chaperoning prions: the cellular machinery for propagating an infectious protein?

Newly made polypeptide chains require the help of molecular chaperones not only to rapidly reach their final three-dimensional forms, but also to unfold and then correctly refold them back to their biologically active form should they misfold. Most prions are an unusual type of protein that can exist in one of two stable conformations, one of which leads to formation of an infectious alternatively folded form. Studies in Baker's yeast (Saccharomyces cerevisiae) have revealed that prions can exploit the molecular chaperone machinery in the cell in order to ensure stable propagation of the infectious, aggregation-prone form. The disaggregation of yeast prion aggregates by molecular chaperones generates forms of the prion protein that can seed the protein polymerisation that underlies the prion propagation cycle. In this article, we review what we have learnt about the role of molecular chaperones in yeast prion propagation, describe a model that can explain the role of various classes of molecular chaperones and their co-chaperones, and speculate on the possible involvement of chaperones in the propagation of mammalian prions.

Cytosol↗
PubMed2004

Propagating prions in fungi and mammals.

Prions constitute a rare class of protein, which can switch to a robust amyloid form and then propagate that form in the absence of a nucleic acid determinant, thereby creating a unique, protein-only infectious agent. Details of the mechanism that drives conversion to the prion form and then subsequent propagation of that form are beginning to emerge using a range of in vivo and in vitro approaches. Recent studies on both mammalian and fungal prions are providing a greater understanding of the structural features that distinguish prions from non-transmissible amyloids.

Amyloid↗
PubMed2004

Dissection and design of yeast prions.

Many proteins can misfold into beta-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.

Amino Acid Motifs↗
PubMed2004

Driving change: the evolution of alternative genetic codes.

Pioneering studies in the 1960s that elucidated the genetic code suggested that all extant forms of life use the same genetic code. This early presumption has subsequently been challenged by the discovery of deviations of the universal genetic code in prokaryotes, eukaryotic nuclear genomes and mitochondrial genomes. These studies have revealed that the genetic code is still evolving despite strong negative forces working against the fixation of mutations that result in codon reassignment. Recent data from in vitro, in vivo and in silico comparative genomics studies are revealing significant, previously overlooked links between modified nucleosides in tRNAs, genetic code ambiguity, genome base composition, codon usage and codon reassignment.

Animals↗
PubMed2003

Prion protein gene polymorphisms in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae genome encodes several proteins that, in laboratory strains, can take up a stable, transmissible prion form. In each case, this requires the Asn/Gln-rich prion-forming domain (PrD) of the protein to be intact. In order to further understand the evolutionary significance of this unusual property, we have examined four different prion genes and their corresponding PrDs, from a number of naturally occurring strains of S. cerevisiae. In 4 of the 16 strains studied we identified a new allele of the SUP35 gene (SUP35delta19) that contains a 19-amino-acid deletion within the N-terminal PrD, a deletion that eliminates the prion property of Sup35p. In these strains a second prion gene, RNQ1, was found to be highly polymorphic, with eight different RNQ1 alleles detected in the six diploid strains studied. In contrast, for one other prion gene (URE2) and the sequence of the NEW1 gene encoding a PrD, no significant degree of DNA polymorphism was detected. Analysis of the naturally occurring alleles of RNQ1 and SUP35 indicated that the various polymorphisms identified were associated with DNA tandem repeats (6, 12, 33, 42 or 57 bp) within the coding sequences. The expansion and contraction of DNA repeats within the RNQ1 gene may provide an evolutionary mechanism that can ensure rapid change between the [PRION+] and [prion-] states.

Alleles↗
PubMed2003

Comparative evolutionary genomics unveils the molecular mechanism of reassignment of the CTG codon in Candida spp.

Using the (near) complete genome sequences of the yeasts Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we address the evolution of a unique genetic code change, which involves decoding of the standard leucine-CTG codon as serine in Candida spp. By using two complementary comparative genomics approaches, we have been able to shed new light on both the origin of the novel Candida spp. Ser-tRNA(CAG), which has mediated CTG reassignment, and on the evolution of the CTG codon in the genomes of C. albicans, S. cerevisiae, and S. pombe. Sequence analyses of newly identified tRNAs from the C. albicans genome demonstrate that the Ser-tRNA(CAG) is derived from a serine and not a leucine tRNA in the ancestor yeast species and that this codon reassignment occurred approximately 170 million years ago, but the origin of the Ser-tRNA(CAG) is more ancient, implying that the ancestral Leu-tRNA that decoded the CTG codon was lost after the appearance of the Ser-tRNA(CAG). Ambiguous CTG decoding by the Ser-tRNA(CAG) combined with biased AT pressure forced the evolution of CTG into TTR codons and have been major forces driving evolution of the CTN codon family in C. albicans. Remarkably, most of the CTG codons present in extant C. albicans genes are encoded by serine and not leucine codons in homologous S. cerevisiae and S. pombe genes, indicating that a significant number of serine TCN and AGY codons evolved into CTG codons either directly by simultaneous double mutations or indirectly through an intermediary codon. In either case, CTG reassignment had a major impact on the evolution of the coding component of the Candida spp. genome.

Amino Acid Sequence↗
PubMed2002

Stop codon decoding in Candida albicans: from non-standard back to standard.

The human pathogen Candida albicans translates the standard leucine-CUG codon as serine. This genetic code change is mediated by a novel ser-tRNA(CAG), which induces aberrant mRNA decoding in vitro, resulting in retardation of the electrophoretic mobility of the polypeptides synthesized in its presence. These non-standard decoding events have been attributed to readthrough of the UAG and UGA stop codons encoded by the Brome Mosaic Virus RNA 4, which codes for the virion coat protein, and the rabbit globin mRNAs, respectively. In order to fully elucidate the behaviour of the C. albicans ser-tRNA(CAG) towards stop codons, we have used other cell-free translation systems and reporter genes. However, the reporter systems used encode several CUG codons, making it impossible to distinguish whether the slow migration of the polypeptides is caused by the replacement of leucines by serines at the CUG codons, readthrough, or a combination of both. Therefore, we have constructed new reporter systems lacking CUG codons and have used them to demonstrate that aberrant mRNA decoding in vitro is not a result from stop codon readthrough or any other non-standard translational event. Our data show that a single leucine to serine replacement at only one of the four CUG codons encoded by the BMV RNA-4 gene is responsible for the aberrant migration of the BMV coat protein on SDS-PAGE, suggesting that this amino acid substitution (ser for leu) significantly alters the structure of the virion coat protein. The data therefore show that the only aberrant event mediated by the ser-tRNA(CAG) is decoding of the leu-CUG codon as serine.

Amino Acid Substitution↗