5,642 research outputs found
Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast
A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT
Metabolic and Chaperone Gene Loss Marks the Origin of Animals: Evidence for Hsp104 and Hsp78 Sharing Mitochondrial Clients
The evolution of animals involved acquisition of an emergent gene repertoire
for gastrulation. Whether loss of genes also co-evolved with this developmental
reprogramming has not yet been addressed. Here, we identify twenty-four genetic
functions that are retained in fungi and choanoflagellates but undetectable in
animals. These lost genes encode: (i) sixteen distinct biosynthetic functions;
(ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which
function in the mitochondria and cytosol, respectively; and (iii) six other
assorted functions. We present computational and experimental data that are
consistent with a joint function for the differentially localized ClpB
disaggregases, and with the possibility of a shared client/chaperone
relationship between the mitochondrial Fe/S homoaconitase encoded by the lost
LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the
evolution of gastrulation-based multicellularity in animals led to efficient
extraction of nutrients from dietary sources, loss of natural selection for
maintenance of energetically expensive biosynthetic pathways, and subsequent
loss of their attendant ClpB chaperones.Comment: This is a reformatted version from the recent official publication in
PLoS ONE (2015). This version differs substantially from first three arXiV
versions. This version uses a fixed-width font for DNA sequences as was done
in the earlier arXiv versions but which is missing in the official PLoS ONE
publication. The title has also been shortened slightly from the official
publicatio
The influence of microgravity on invasive growth in Saccharomyces cerevisiae
This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Sigma 1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Sigma 1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Sigma 1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium
The hypoxic transcription factor KlMga2 mediates the response to oxidative stress and influences longevity in the yeast Kluyveromyces lactis
Hypoxia is defined as the decline of oxygen availability, depending on environmental supply and cellular consumption rate. The decrease in O2 results in reduction of available energy in facultative aerobes. The response and/or adaptation to hypoxia and other changing environmental conditions can influence the properties and functions of membranes by modifying lipid composition. In the yeast Kluyveromyces lactis, the KlMga2 gene is a hypoxic regulatory factor for lipid biosynthesis-fatty acids and sterols-and is also involved in glucose signaling, glucose catabolism and is generally important for cellular fitness. In this work we show that, in addition to the above defects, the absence of the KlMGA2 gene caused increased resistance to oxidative stress and extended lifespan of the yeast, associated with increased expression levels of catalase and SOD genes. We propose that KlMga2 might also act as a mediator of the oxidative stress response/adaptation, thus revealing connections among hypoxia, glucose signaling, fatty acid biosynthesis and ROS metabolism in K. lactis
One Hub-One Process: A Tool Based View on Regulatory Network Topology
The relationship between the regulatory design and the functionality of
molecular networks is a key issue in biology. Modules and motifs have been
associated to various cellular processes, thereby providing anecdotal evidence
for performance based localization on molecular networks. To quantify
structure-function relationship we investigate similarities of proteins which
are close in the regulatory network of the yeast Saccharomyces Cerevisiae. We
find that the topology of the regulatory network show weak remnants of its
history of network reorganizations, but strong features of co-regulated
proteins associated to similar tasks. This suggests that local topological
features of regulatory networks, including broad degree distributions, emerge
as an implicit result of matching a number of needed processes to a finite
toolbox of proteins.Comment: 18 pages, 3 figures, 5 supplementary figure
Molecular and proteomic analyses highlight the importance of ubiquitination for the stress resistance, metabolic adaptation, morphogenetic regulation and virulence of Candida albicans
Peer reviewedPublisher PD
Analysis of the transcriptional program governing meiosis and gametogenesis in yeast and mammals
During meiosis a competent diploid cell replicates its DNA once and then undergoes two consecutive divisions followed by haploid gamete diο¬erentiation. Important aspects of meiotic development that distinguish it from mitotic growth include a highly increased rate of recombination, formation of the synaptonemal complex that aligns the homologous chromosomes, as well as separation of the homologues and sister chromatids during meiosis I and II without an intervening S-phase. Budding yeast is an excellent model organism to study meiosis and gametogenesis and accordingly, to date it belongs to the best studied eukaryotic systems in this context. Knowledge coming from these studies has provided important insights into meiotic development in higher eukaryotes. This was possible because sporulation in yeast and spermatogenesis in higher eukaryotes are analogous developmental pathways that involve conserved genes. For budding yeast a huge amount of data from numerous genome-scale studies on gene expression and deletion phenotypes of meiotic development and sporulation are available. In contrast, mammalian gametogenesis has not been studied on a large-scale until recently. It was unclear if an expression proο¬ling study using germ cells and testicular somatic control cells that underwent lengthy puriο¬cation procedures would yield interpretable results. We have therefore carried out a pioneering expression proο¬ling study of male germ cells from Rattus norvegicus using Aο¬ymetrix U34A and B GeneChips. This work resulted in the ο¬rst comprehensive large-scale expression proο¬ling analysis of mammalian male germ cells undergoing mitotic growth, meiosis and gametogenesis. We have identiο¬ed 1268 diο¬erentially expressed genes in germ cells at diο¬erent developmental stages, which were organized into four distinct expression clusters that reο¬ect somatic, mitotic, meiotic and post-meiotic cell types. This included 293 yet uncharacterized transcripts whose expression pattern suggests that they are involved in spermatogenesis and fertility. A group of 121 transcripts were only expressed in meiotic (spermatocytes) and postmeiotic germ cells (round spermatids) but not in dividing germ cells (spermatogonia),
Sertoli
cells or two somatic control tissues (brain
and skeletal muscle). Functional analysis reveals
that most of the known genes in this
group fulfill essential functions during meiosis,
spermiogenesis (the process of sperm maturation)
and fertility. Therefore it is highly possible
that some of the οΏ½30 uncharacterized transcripts
in this group also contribute to these
processes. A web-accessible database (called
reXbase, which was later on integrated into
GermOnline) has been developed for our expression
profiling study of mammalian male
meiosis, which summarizes annotation information
and shows a graphical display of expression
profiles of every gene covered in our
study.
In the budding yeast Saccharomyces cerevisiae
entry into meiosis and subsequent progression
through sporulation and gametogenesis
are driven by a highly regulated transcriptional
program activated by signal pathways
responding to nutritional and cell-type cues.
Abf1p, which is a general transcription factor,
has previously been demonstrated to participate
in the induction of numerous mitotic as
well as early and middle meiotic genes. In
the current study we have addressed the question
how Abf1p transcriptionally coordinates
mitotic growth and meiotic development on a
genome-wide level. Because ABF1 is an essential
gene we used the temperature-sensitive
allele abf1-1. A phenotypical analysis of mutant
cells revealed that ABF1 plays an important
role in cell separation during mitosis,
meiotic development, and spore formation. In
order to identify genes whose expression depends
on Abf1p in growing and sporulating
cells we have performed expression profiling
experiments using Affymetrix S98 GeneChips
comparing wild-type and abf1-1 mutant cells
at both permissive and restrictive temperature.
We have identified 504 genes whose normal expression
depends on functional ABF1. By combining
the expression profiling data with data
from genome-wide DNA binding assays (ChIPCHIP)
and in silico predictions of potential
Abf1p-binding sites in the yeast genome, we
were able to define direct target genes. Expression
of these genes decreases in the absence
of functional ABF1 and whose promotors are
bound by Abf1p and/or contain a predicted
binding site.
Among 352 such bona fide direct target genes
we found many involved in ribosome biogenesis,
translation, vegetative growth and meiotic
developement and therefore could account for
the observed growth and sporulation defects of
abf1-1 mutant cells. Furthermore, the fact that
two members of the septin family (CDC3 and
CDC10 ) were found to be direct target genes
suggests a novel role for Abf1p in cytokinesis.
This was further substantiated by the observation
that chitin localization and septin ring
formation are perturbed in abf1-1 mutant cells
Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans
Peer reviewedPublisher PD
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