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PubMed2001

Identification of essential genes in cultured mammalian cells using small interfering RNAs.

We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both beta- and gamma-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype. Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.

Animals↗
PubMed2026

Mining of CtOPR2 as an essential gene regulating jasmonic acid mediated insect tolerance in Camellia tachangensis through genome-wide association studies.

Jasmonic acid (JA), a pivotal lipid-derived phytohormone, serves as a critical regulator in plant growth and defense mechanisms. However, the genetic mechanisms of OPR2 gene in JA-dependent biotic defenses of Camellia tachangensis have rarely been investigated. In this study, we performed a genome-wide association study to analyze 100,720 high-quality single nucleotide polymorphisms (SNPs) among 350 tea accessions from Guizhou province to identify genetic variations associated with JA. Analysis showed C. tachangensis displayed higher levels of JA content, further analysis identified 60 high-quality SNPs and nine candidate genes related to JA. Among them, CtOPR2 encoding 12-oxophytodienoic acid reductase 2 is responsible for catalyzing the conversion of 4,5-didehydro-JA (4,5-ddh-JA) to JA. The expression level of CtOPR2 in three tea accessions with different JA content was consistent with the dynamic changes of JA content. The expression level of synthetic (AOS, AOC, and ACX) and responsive (WRKY18 and MYC2) genes were significantly decreased and increased in asODN-CtOPR2-treated shoot tips and transgenic tobacco lines overexpressing CtOPR2, respectively, which were consistent with the JA content. These results further revealed that CtOPR2 gene played essential roles in promoting JA biosynthesis. A significant reduction in insect bite area was observed on transgenic tobacco leaves compared to wild-type leaves in feeding experiments with Spodoptera litura, highlighting that the positive regulatory function of CtOPR2 gene in JA-mediated immune responses. This study provides a robust theoretical foundation for marker-assisted selection breeding in tea, aimed at developing high-JA germplasm with potentially enhanced pest resistance for cultivation in Guizhou.

Journal Article↗
PubMed1994

An essential gene of Saccharomyces cerevisiae coding for an actin-related protein.

Actin filaments provide the internal scaffold of eukaryotic cells; they are involved in maintenance of cell shape, cytokinesis, organelle movement, and cell motility. The major component of these filaments, actin, is one of the most well-conserved eukaryotic proteins. Recently genes more distantly related to the conventional actins were cloned from several organisms. In the budding yeast, Saccharomyces cerevisiae, one conventional actin gene, ACT1 (coding for the filament actin), and a so-called actin-like gene, ACT2 (of unknown function), have so far been identified. We report here the discovery of a third member of the actin gene family from this organism, which we named ACT3. The latter gene is essential for viability and codes for a putative polypeptide, Act3, of 489 amino acids (M(r) = 54,831). The deduced amino acid sequence of Act3 is less related to conventional actins than is the deduced amino acid sequence of Act2, mainly because of three unique hydrophilic [corrected] segments. These segments are found inserted into a part of the sequence corresponding to a surface loop of the known three-dimensional structure of the actin molecule. According to sequence comparison, the basal core structure of conventional actin may well be conserved in Act3. Our findings demonstrate that, unexpectedly, there exist three members of the diverse actin protein family in budding yeast that obviously provide different essential functions for survival.

Actins↗
PubMed2000

Essential genes in autosomal heterochromatin of Drosophila melanogaster.

We are taking two approaches to understanding the structure, function and regulation of essential genes within Drosophila heterochromatin. In the first, we have undertaken a genetic and molecular characterization of essential genes within proximal 3L heterochromatin. The expression of such 'resident' genes within a heterochromatic environment is paradoxical and poorly understood, given that the same environment can inactivate euchromatic sequences (position effect variegation, or PEV). A second approach involves the study of the local chromosomal environment of heterochromatic (het) genes, as assayed both biochemically, and via the effects of genetic modifiers of PEV, the latter being putative components important for het gene expression. Our results to date suggest that the three most proximal genes in 3L heterochromatin have key roles in development, and indicate strong effects of combinations of genetic modifiers of PEV on het gene expression.

Animals↗
PubMed1998

Systematic identification of essential genes by in vitro mariner mutagenesis.

Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens, Haemophilus influenzae and Streptococcus pneumoniae, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.

Base Sequence↗
PubMed1996

The identification of transposon-tagged mutations in essential genes that affect cell morphology in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae reproduces by budding, and many genes are required for proper bud development. Mutations in some of these genes cause cells to die with an unusual terminal morphology-elongated or otherwise aberrantly shaped buds. To gain insight into bud development, we set out to identify novel genes that encode proteins required for proper bud morphogenesis. Previous studies screened collections of conditional mutations to identify genes required for essential functions, including bud formation. However, genes that are not susceptible to the generation of mutations that cause a conditional phenotype will not be identified in such screens. To identify a more comprehensive collection of mutants, we used transposon mutagenesis to generate a large collection of lethal disruption mutations. This collection was used to identify 209 mutants with disruptions that cause an aberrant terminal bud morphology. The disruption mutations in 33 of these mutants identify three previously uncharacterized genes as essential, and the mutant phenotypes suggest roles for their products in bud morphogenesis.

Amino Acid Sequence↗
PubMed2005

Gene essentiality and the topology of protein interaction networks.

The mechanistic bases for gene essentiality and for cell mutational resistance have long been disputed. The recent availability of large protein interaction databases has fuelled the analysis of protein interaction networks and several authors have proposed that gene dispensability could be strongly related to some topological parameters of these networks. However, many results were based on protein interaction data whose biases were not taken into account. In this article, we show that the essentiality of a gene in yeast is poorly related to the number of interactants (or degree) of the corresponding protein and that the physiological consequences of gene deletions are unrelated to several other properties of proteins in the interaction networks, such as the average degrees of their nearest neighbours, their clustering coefficients or their relative distances. We also found that yeast protein interaction networks lack degree correlation, i.e. a propensity for their vertices to associate according to their degrees. Gene essentiality and more generally cell resistance against mutations thus seem largely unrelated to many parameters of protein network topology.

Computational Biology↗
PubMed2006

Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form Trypanosoma brucei.

Glycolysis and gluconeogenesis are, in part, driven by the interconversion of 3- and 2-phosphoglycerate (3-PG and 2-PG) which is performed by phosphoglycerate mutases (PGAMs) which can be cofactor dependant (dPGAM) or cofactor independent (iPGAM). The African trypanosome, Trypanosoma brucei, possesses the iPGAM form which is thought to play an important role in glycolysis. Here, we report on the use of RNA interference to down-regulate the T. brucei iPGAM in procyclic form T. brucei and evaluation of the resulting phenotype. We first demonstrated biochemically that depletion of the steady state levels of iPGM mRNA correlates with a marked reduction of enzyme activity. We further show that iPGAM is required for cell growth in procyclic T. brucei.

Animals↗
PubMed1998

The N-degron protein degradation strategy for investigating the function of essential genes: requirement for replication protein A and proliferating cell nuclear antigen proteins for nucleotide excision repair in yeast extracts.

Nucleotide excision repair (NER) of DNA in the yeast Saccharomyces cerevisiae and in human cells has been shown to be a biochemically complex process involving multiple gene products. In yeast, the involvement of the DNA replication accessory proteins, replication protein A (RPA1) and proliferating cell nuclear antigen (PCNA) in NER has not been demonstrated genetically. In this study we have generated temperature-degradable rfa1 and pcna mutants and show that these mutants are defective in NER in vitro under conditions that promote degradation of the RFA1 and PCNA gene products. We also demonstrate a physical interaction between RPA1 protein and subunits of the RNA polymerase II basal transcription factor IIH (TFIIH).

DNA Repair↗
PubMed2000

The search for essential genes.

The bacterial genomic era began with the publication of the chromosomal sequence of Haemophilus influenzae. As few of the observed genes had been examined experimentally, functional assignments were made by comparative analysis and for many genes no annotation could be made. This mini-review briefly describes the genomic-scale experimental approaches being used to identify genes required for the growth of microorganisms. Identifying 'essential genes', the simplest possible annotation for the unknown open reading frames, is important for antibacterial and antifungal research and is a first step to defining the minimum functional requirement for autonomous growth.

Bacteria↗
PubMed2006

Shuttle mutagenesis and targeted disruption of a telomere-located essential gene of Leishmania.

Leishmania mutants have contributed greatly to extend our knowledge of this parasite's biology. Here we report the use of the mariner in vitro transposition system as a source of reagents for shuttle mutagenesis and targeted disruption of Leishmania genes. The locus-specific integration was achieved by the disruption of the subtelomeric gene encoding a DNA-directed RNA polymerase III subunit (RPC2). Further inactivation of RPC2 alleles required the complementation of the intact gene, which was transfected in an episomal context. However, attempts to generate a RPC2 chromosomal null mutant resulted in genomic rearrangements that maintained copies of the intact locus in the genome. The maintenance of the RPC2 chromosomal locus in complemented mutants was not mediated by an increase in the number of copies and did not involve chromosomal translocations, which are the typical characteristics of the genomic plasticity of this parasite. Unlike the endogenous locus, the selectable marker used to disrupt RPC2 did not display a tendency to remain in its chromosomal location but was targeted into supernumerary episomal molecules.

Animals↗
PubMed1998

Identification of an essential gene encoding a class-V unconventional myosin in Drosophila melanogaster.

Class-V myosins are a unique type of myosin motor with roles in intracellular transport. The mouse dilute gene was the first member of this class to be cloned, with mutations resulting in lightening of the coat colour or neurogenic defects leading to early death. Further examples of class-V myosins have been described in yeast, chicken and rat. Here, we report the cloning of the first class-V myosin from Drosophila. We show that expression of this myosin is predominantly in the adult germ line and early embryo and that the transcript is localised in the oocyte during oogenesis. Genetic and in situ hybridisation experiments have determined that this gene is located in the 43C region. We have evidence that it maps to a mutation in this region with an embryonic lethal phenotype.

Amino Acid Sequence↗
PubMed2006

Rendezvin: An essential gene encoding independent, differentially secreted egg proteins that organize the fertilization envelope proteome after self-association.

Preventing polyspermy during animal fertilization relies on modifications to the egg's extracellular matrix. On fertilization in sea urchins, the contents of cortical granules are secreted and rapidly assemble into the egg's extracellular vitelline layer, forming the fertilization envelope, a proteinaceous structure that protects the zygote from subsequent sperm. Here, we document rendezvin, a gene whose transcript is differentially spliced to yield proteins destined for either cortical granules or the vitelline layer. These distinctly trafficked variants reunite after cortical granule secretion at fertilization. Together, they help coordinate assembly of the functional fertilization envelope, whose proteome is now defined in full.

Alternative Splicing↗
PubMed2002

Identification of in vivo essential genes from Pseudomonas aeruginosa by PCR-based signature-tagged mutagenesis.

We adapted PCR-based signature-tagged mutagenesis (STM) to Pseudomonas aeruginosa. A collection of 1056 mutants was screened in a chronic lung infection rat model. Thirteen mutants were confirmed to be attenuated. Analysis revealed that these STM mutants represented transposon insertions into eight genes previously described in databases, three genes encoding proteins sharing identity with hypothetical proteins and two genes that shared no significant identity with sequences in databases. Five strains mutated in genes involved in protein degradation, stress tolerance, cation transport, ABC transporter, and an unknown protein were shown to be highly attenuated when tested individually in the rat chronic lung infection model.

Cloning, Molecular↗
PubMed1999

Math1: an essential gene for the generation of inner ear hair cells.

The mammalian inner ear contains the cochlea and vestibular organs, which are responsible for hearing and balance, respectively. The epithelia of these sensory organs contain hair cells that function as mechanoreceptors to transduce sound and head motion. The molecular mechanisms underlying hair cell development and differentiation are poorly understood. Math1, a mouse homolog of the Drosophila proneural gene atonal, is expressed in inner ear sensory epithelia. Embryonic Math1-null mice failed to generate cochlear and vestibular hair cells. This gene is thus required for the genesis of hair cells.

Animals↗
PubMed1999

Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria.

We describe a fast and simple method for signature-tagged mutagenesis (STM) using defined oligonucleotides for tag construction into mini-Tn5 and PCR instead of hybridization for rapid screening of bacterial mutants in vivo. A collection of 12 unique 21-mers were synthesized as complementary DNA strands to tag bacterial mutants constructed by insertional mutagenesis using pUTmini-Tn5Km2 plasmids. Tags were tested in a combination of assays by PCR and compared to hybridization for specificity and for large-scale screening. Each defined tag has the same melting temperature, an invariable region to optimize PCRs and a variable region for specific amplification by PCR. A series of "suicide" plasmids carrying mini-Tn5s, each with a specific tag, were transferred into Pseudomonas aeruginosa, giving 12 libraries of mutants; groups of 12 mutants were pooled and arrayed into 96-well microplates, representing approximately one-sixth of the P. aeruginosa 5.9-Mb genome. This simple STM method can be adapted to any bacterial system and used for genome scanning in various growth conditions.

Base Sequence↗