RESUMO
Using a microarray that tiles all known yeast non-coding RNAs, we compared RNA from wild-type cells with RNA from mutants encoding known and putative RNA modifying enzymes. We show that at least five types of RNA modification (dihydrouridine, m1G, m2(2)G, m1A and m6(2)A) catalyzed by 10 different enzymes (Trm1p, Trm5, Trm10p, Dus1p-Dus4p, Dim1p, Gcd10p and Gcd14p) can be detected by virtue of differential hybridization to oligonucleotides on the array that are complementary to the modified sites. Using this approach, we identified a previously undetected m1A modification in GlnCTG tRNA, the formation of which is catalyzed by the Gcd10/Gcd14 complex. complex.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Processamento Pós-Transcricional do RNA , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/metabolismo , Mutação , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , RNA não Traduzido/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates. RESULTS: We measured expression of all known and predicted genes across twenty tissues in chicken, frog and pufferfish. By combining the results with human and mouse data and considering only ten common tissues, we have found evidence of conserved expression for more than a third of unique orthologous genes. We find that, on average, transcription factor gene expression is neither more nor less conserved than that of other genes. Strikingly, conservation of expression correlates poorly with the amount of conserved nonexonic sequence, even using a sequence alignment technique that accounts for non-collinearity in conserved elements. Many genes show conserved human/fish expression despite having almost no nonexonic conserved primary sequence. CONCLUSIONS: There are clearly strong evolutionary constraints on tissue-specific gene expression. A major challenge will be to understand the precise mechanisms by which many gene expression patterns remain similar despite extensive cis-regulatory restructuring.
Assuntos
Regulação da Expressão Gênica , Vertebrados , Animais , Anuros , Sequência de Bases , Galinhas , Sequência Conservada/genética , DNA/análise , DNA/genética , Evolução Molecular , Perfilação da Expressão Gênica , Humanos , Camundongos , Alinhamento de Sequência , Análise de Sequência de DNA , Tetraodontiformes , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Vertebrados/genética , Vertebrados/metabolismoRESUMO
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.