Biomedical subjects

Sanie Mnaimneh

Publications and source records attributed to Sanie Mnaimneh.

10 recordsLinked to original sources

PubMed2006

A survey of essential gene function in the yeast cell division cycle.

Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO(7) promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication.

Alleles↗

PubMed2005

The synthetic genetic interaction spectrum of essential genes.

The nature of synthetic genetic interactions involving essential genes (those required for viability) has not been previously examined in a broad and unbiased manner. We crossed yeast strains carrying promoter-replacement alleles for more than half of all essential yeast genes to a panel of 30 different mutants with defects in diverse cellular processes. The resulting genetic network is biased toward interactions between functionally related genes, enabling identification of a previously uncharacterized essential gene (PGA1) required for specific functions of the endoplasmic reticulum. But there are also many interactions between genes with dissimilar functions, suggesting that individual essential genes are required for buffering many cellular processes. The most notable feature of the essential synthetic genetic network is that it has an interaction density five times that of nonessential synthetic genetic networks, indicating that most yeast genetic interactions involve at least one essential gene.

Gene Expression Regulation, Fungal↗

PubMed2005

Genome-wide analysis of mouse transcripts using exon microarrays and factor graphs.

Recent mammalian microarray experiments detected widespread transcription and indicated that there may be many undiscovered multiple-exon protein-coding genes. To explore this possibility, we labeled cDNA from unamplified, polyadenylation-selected RNA samples from 37 mouse tissues to microarrays encompassing 1.14 million exon probes. We analyzed these data using GenRate, a Bayesian algorithm that uses a genome-wide scoring function in a factor graph to infer genes. At a stringent exon false detection rate of 2.7%, GenRate detected 12,145 gene-length transcripts and confirmed 81% of the 10,000 most highly expressed known genes. Notably, our analysis showed that most of the 155,839 exons detected by GenRate were associated with known genes, providing microarray-based evidence that most multiple-exon genes have already been identified. GenRate also detected tens of thousands of potential new exons and reconciled discrepancies in current cDNA databases by 'stitching' new transcribed regions into previously annotated genes.

Algorithms↗

PubMed2004

Exploration of essential gene functions via titratable promoter alleles.

Nearly 20% of yeast genes are required for viability, hindering genetic analysis with knockouts. We created promoter-shutoff strains for over two-thirds of all essential yeast genes and subjected them to morphological analysis, size profiling, drug sensitivity screening, and microarray expression profiling. We then used this compendium of data to ask which phenotypic features characterized different functional classes and used these to infer potential functions for uncharacterized genes. We identified genes involved in ribosome biogenesis (HAS1, URB1, and URB2), protein secretion (SEC39), mitochondrial import (MIM1), and tRNA charging (GSN1). In addition, apparent negative feedback transcriptional regulation of both ribosome biogenesis and the proteasome was observed. We furthermore show that these strains are compatible with automated genetic analysis. This study underscores the importance of analyzing mutant phenotypes and provides a resource to complement the yeast knockout collection.

Alleles↗

PubMed2004

High-definition macromolecular composition of yeast RNA-processing complexes.

A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins. To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae. We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization. Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules. Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described. Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript. This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.

Amino Acid Sequence↗

PubMed2004

Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors.

Using DNA microarrays, we compared global transcript stability profiles following chemical inhibition of transcription to rpb1-1 (a temperature-sensitive allele of yeast RNA polymerase II). Among the five inhibitors tested, the effects of thiolutin and 1,10-phenanthroline were most similar to rpb1-1. A comparison to various microarray data already in the literature revealed similarity between mRNA stability profiles and the transcriptional response to stresses such as heat shock, consistent with the fact that the general stress response includes a transient shutoff of general mRNA transcription. Genes encoding factors involved in rRNA synthesis and ribosome assembly, which are often observed to be coordinately down-regulated in yeast microarray data, were among the least stable transcripts. We examined the effects of deletions of genes encoding deadenylase components Ccr4p and Pan2p and putative RNA-binding proteins Pub1p and Puf4p on the genome-wide pattern of mRNA stability after inhibition of transcription by chemicals and/or heat stress. This examination showed that Ccr4p, the major yeast mRNA deadenylase, contributes to the degradation of transcripts encoding both ribosomal proteins and rRNA synthesis and ribosome assembly factors and mediates a large part of the transcriptional response to heat stress. Pan2p and Puf4p also contributed to the degradation rate of these mRNAs following transcriptional shutoff, while Pub1p preferentially stabilized transcripts encoding ribosomal proteins. Our results indicate that the abundance of ribosome biogenesis factors is controlled at the level of mRNA stability.

Gene Expression Profiling↗

PubMed2004

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Animals↗

PubMed2003

A panoramic view of yeast noncoding RNA processing.

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.

Cells, Cultured↗

PubMed2002

Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo.

There is growing evidence for the involvement of nitric oxide (NO) -mediated nitrosation in cell signaling and pathology. Although S-nitrosothiols (RSNOs) have been frequently implicated in these processes, it is unclear whether NO forms nitrosyl adducts with moieties other than thiols. A major obstacle in assessing the significance of formation of nitrosated species is the limited reliability of available analytical techniques for measurements in complex biological matrices. Here we report on the presence of nitrosated compounds in plasma and erythrocytes of rats, mice, guinea pigs, and monkeys under basal conditions, in immunologically challenged murine macrophages in vitro and laboratory animals in vivo. Besides RSNOs, all biological samples also contained mercury-stable nitroso species, indicating the additional involvement of amine and heme nitros(yl)ation reactions. Significant differences in the amounts and ratios of RSNOs over N- and heme-nitros(yl)ated compounds were found between species and organs. These observations were made possible by the development of a novel gas-phase chemiluminescence-based technique that allows detection of nitroso species in tissues and biological fluids without prior extraction or deproteinization. The method can quantify as little as 100 fmol bound NO and has been validated extensively for use in different biological matrices. Discrimination between nitrite, RSNOs, and N-nitroso or nitrosylheme compounds is accomplished by use of group-specific reagents. Our findings suggest that NO generation in vivo leads to concomitant formation of RSNOs, nitrosamines, and nitrosylhemes with considerable variation between rodents and primates, highlighting the difficulty in comparing data between different animal models and extrapolating results from experimental animals to human physiology.

Animals↗

PubMedDate not supplied

Circulating antibodies directed against nitrosylated antigens in trypanosome-infected mice.

Nitric oxide has been implicated as an effector cytotoxic molecule in trypanosomiasis. In this work, we investigated the presence of circulating antibodies directed against nitrosylated epitopes as biological indicators for nitric oxide (NO) production in the sera of trypanosome-infected mice. We tested these sera with synthetic antigens, such as S-nitrosated protein or nitrosylated conjugates of amino acids that possess a high affinity to NO, by an immunoenzymatic assay. We detected antibodies directed against nitroso epitopes in the sera of infected mice, as compared to non-infected control mice. The antibody response was linked to the IgM isotype. Our results indicate the production of NO and its derivatives in trypanosomiasis. This production may potentially induce the synthesis of nitroso epitopes in vivo and favor the development of a humoral immune response.

Animals↗