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Results for “conditionally essential genes”

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PubMed2004

From ORFeome to biology: a functional genomics pipeline.

As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

Animals↗

PubMed1997

Cloning and characterization of two immunophilin-like genes, ilpA and fkpA, on a single 3.9-kilobase fragment of Aeromonas hydrophila genomic DNA.

Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a previous study (C. Y. F. Wong, G. Mayrhofer, M. W. Heuzenroeder, H. M. Atkinson, D. M. Quinn, and R. L. P. Flower, FEMS Immunol. Med. Microbiol. 15:233-241, 1996) to protect mice against lethal infection by this organism. In this study, colony blot analysis of an A. hydrophila genomic library using antiserum to A. hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been described previously in aeromonads. The nucleotide sequence of a 3.9-kb fragment derived from this cosmid which expressed the 30-kDa protein revealed two potential open reading frames (ORFs) with homology to known immunophilin proteins. ORF1 encoded a 212-amino-acid protein (molecular mass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of Escherichia coli. ORF1 was subsequently designated ilpA (immunophilin-like protein). ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa. The inferred amino acid sequence showed 46% identity with the sequence of the FkpA protein of E. coli and 40% identity with the sequence of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila. ORF3 was designated fkpA (FK506 binding protein) by analogy with the E. coli FkpA protein. Expression of the FkpA protein was confirmed by Western blot (immunoblot) analysis, which detected a 30-kDa protein, with antiserum to the Mip protein of Legionella longbeachae and a specific antiserum to anA. hydrophila 30-kDa membrane protein. PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested. A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase. This suggests that either the fkpA gene is not essential in the virulence of A. hydrophila under these conditions or there are other genes in A. hydrophila coding for proteins with similar functions.

Aeromonas hydrophila↗

PubMed1986

A bacteriophage RNA polymerase transcribes through a Xenopus 5S RNA gene transcription complex without disrupting it.

Conditions are described for programming with active transcription complexes essentially all the Xenopus 5S RNA genes added to an extract of Xenopus oocyte nuclei. We call this rate enhancement. Transcription by SP6 RNA polymerase of either strand of a Xenopus 5S RNA gene is unimpeded by the presence of a complete transcription complex associated with the internal control region of the gene. Furthermore, the transcription complex remains stably associated with the 5S RNA gene following multiple transits by the prokaryotic polymerase.

Animals↗

PubMed1998

A genome-based approach for the identification of essential bacterial genes.

We have used comparative genomics to identify 26 Escherichia coli open reading frames that are both of unknown function (hypothetical open reading frames or y-genes) and conserved in the compact genome of Mycoplasma genitalium. Not surprisingly, these genes are broadly conserved in the bacterial world. We used a markerless knockout strategy to screen for essential E. coli genes. To verify this phenotype, we constructed conditional mutants in genes for which no null mutants could be obtained. In total we identified six genes that are essential for E. coli (yhbZ, ygjD, ycfB, yfil, yihA, and yjeQ). The respective orthologs of the genes yhbZ, ygjD, ycfB, yjeQ, and yihA are also essential in Bacillus subtilis. This low number of essential genes was unexpected and might be due to a characteristic of the versatile genomes of E. coli and B. subtilis that is comparable to the phenomenon of nonorthologous gene displacement. The gene ygjD, encoding a sialoglycoprotease, was eliminated from a minimal genome computationally derived from a comparison of the Haemophilus influenzae and M. genitalium genomes. We show that ygjD and its ortholog ydiE are essential in E. coli and B. subtilis, respectively. Thus, we include this gene in a minimal genome. This study systematically integrates comparative genomics and targeted gene disruptions to identify broadly conserved bacterial genes of unknown function required for survival on complex media.

Amino Acid Sequence↗

PubMed2003

Diploid-dependent regulation of gene expression: a genetic cause of abnormal development in fish haploid embryos.

A diploid-dependent regulatory mechanism of gene expression for spatial patterning of the eye in vertebrates has been determined by analyzing the phenotypes of haploid goldfish embryos. There are two gene loci in charge of eye spatial patterning during embryonic morphogenesis. The expressional probability for each copy of the two genes in a set of chromosomes is 50%. A pair of genes in two sets of homologous or heterologous chromosomes is 100% and essential for normal gene expression. The haploid condition itself would result in the obstruction of gene expression and abnormal development because the diploid-dependent regulatory apparatus will regulate gene expression in a haploid embryo according to the same rule as in the diploid embryo.

Animals↗

PubMed2005

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

Animals↗

PubMed2003

Fission yeast cdc31p is a component of the half-bridge and controls SPB duplication.

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461-1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Deltacdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.

Amino Acid Sequence↗

PubMed2000

Antisense HEMA1 RNA expression inhibits heme and chlorophyll biosynthesis in arabidopsis.

5-aminolevulinic acid (ALA) is a precursor in the biosynthesis of tetrapyrroles including chlorophylls and heme. The formation of ALA involves two enzymatic steps which take place in the chloroplast in plants. The first enzyme, glutamyl-tRNA reductase, and the second enzyme, glutamate-1-semialdehyde-2,1-aminomutase, are encoded by the nuclear HEMA and GSA genes, respectively. To assess the significance of the HEMA gene for chlorophyll and heme synthesis, transgenic Arabidopsis plants that expressed antisense HEMA1 mRNA from the constitutive cauliflower mosaic virus 35S promoter were generated. These plants exhibited varying degrees of chlorophyll deficiency, ranging from patchy yellow to total yellow. Analysis indicated that these plants had decreased levels of chlorophyll, non-covalently bound hemes, and ALA; their levels were proportional to the level of glutamyl-tRNA reductase expression and were inversely related to the levels of antisense HEMA transcripts. Plants that lacked chlorophyll failed to survive under normal growth conditions, indicating that HEMA gene expression is essential for growth.

Aldehyde Oxidoreductases↗

PubMed2006

High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library.

A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

Chromosome Mapping↗

PubMed2006

[Autophagic-lysosomal system: physiology and pathology].

There is increased evidence for the importance of autophagy as a membrane trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. In this review, we introduce the in vivo role of autophagy in mammals. Recently, we generated conditional-knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 disruption resulted in impairment of starvation-induced protein degradation in the adult liver, and Atg7-null mice died within 1 day after birth, associated with low concentrations of plasma amino acids as well as the other autophagy-essential gene, Atg5. Furthermore, loss of Atg7 led to pleiotropic defects, in particular, accumulation of abnormal organelles and ubiquitin-positive inclusions without obvious failure of proteasome function. These results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

Animals↗

PubMedDate not supplied

Analysis of genes encoding the cell division protein FtsZ and a glutathione synthetase homologue in the cyanobacterium Anabaena sp. PCC 7120.

Heterocysts, cells specialized in nitrogen fixation in Anabaena sp. PCC 7120, lose the potential for cell division once fully differentiated. This suggests that cell division activity is differentially regulated in heterocysts and vegetative cells. FtsZ has been shown to play a crucial role in bacterial cell division. Two degenerate oligonucleotide primers were designed to detect, by polymerase chain reaction (PCR), an ftsZ homologue from the heterocystous cyanobacterium Anabaena sp. PCC 7120. A PCR-amplified DNA fragment was cloned and used as a probe to isolate the entire ftsZ gene of Anabaena sp. PCC 7120. The deduced amino acid sequence shares strong similarities with other FtsZ proteins, suggesting remarkable conservation of the FtsZ protein during evolution. An ORF downstream of ftsZ, which would be transcribed in the opposite direction compared to ftsZ, could encode a polypeptide with significant sequence similarity to the glutathione synthetase from Escherichia coli. Inactivation experiments in vivo for both ftsZ and the glutathione synthetase gene did not yield any double recombinants either in the presence or in the absence of combined nitrogen, suggesting that both genes are essential for cell growth under these conditions.

Amino Acid Sequence↗

PubMed1992

Cloning and characterization of the groESL operon from Bacillus subtilis.

The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated. Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA. The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E. coli GroES. The downstream ORF encodes a 58-kDa protein which is 62% identical to E. coli GroEL. A 2.1-kb groESL mRNA from B. subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe. This demonstrated that groES and groEL are in an operon. The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis. The same promoter is utilized under normal and heat shock conditions. This promoter has the same features as a typical sigma A promoter. A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created. With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions.

Amino Acid Sequence↗

PubMed2001

Cse1l is essential for early embryonic growth and development.

The CSE1L gene, the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation as well as in apoptosis. CSE1 and CSE1L are essential genes in Saccharomyces cerevisiae and mammalian cells, as shown by conditional yeast mutants and mammalian cell culture experiments with antisense-mediated depletion of CSE1L. To analyze whether CSE1L is also essential in vivo and whether its absence can be compensated for by other genes or mechanisms, we have cloned the murine CSE1L gene (Cse1l) and analyzed its tissue- and development-specific expression: Cse1l was detected at embryonic day 7.0 (E7.0), E11.0, E15.0, and E17.0, and in adults, high expression was observed in proliferating tissues. Subsequently, we inactivated the Cse1l gene in embryonic stem cells to generate heterozygous and homozygous knockout mice. Mice heterozygous for Cse1l appear normal and are fertile. However, no homozygous pups were born after interbreeding of heterozygous mice. In 30 heterozygote interbreeding experiments, 50 Cse1l wild-type mice and 100 heterozygotes were born but no animal with both Cse1l alleles deleted was born. Embryo analyses showed that homozygous mutant embryos were already disorganized and degenerated by E5.5. This implicates with high significance (P < 0.0001, Pearson chi-square test) an embryonically lethal phenotype of homozygous murine CSE1 deficiency and suggests that Cse1l plays a critical role in early embryonic development.

Active Transport, Cell Nucleus↗

PubMed1994

Differential effects of iron and iron carrier on hematopoietic cells differentiation and human ADA gene transfer.

The role of iron and heme was examined in bone marrow cells from iron-deficient and chronically iron-overloaded rats. Erythroid colony cultures (CFU-E) demonstrated that iron-overloaded bone marrow cells were poor hemin (iron carrier) and CFU-E responders in vitro, whereas iron-deficient marrows grew exuberant numbers of CFU-E and responded to hemin. Results support the concept that iron, or associated factors, plays an important role in the manipulation of HO activity which modulates the hemopoietic potential of the organism. Using cultures of erythroid (CFU-E, BFU-E) and myeloid (CFU-GM), results demonstrated that exogenous hemin (iron carrier) has a specific beneficial effect on human bone marrow progenitor cells, which is not seen with iron (10(-4)M) or other metalloporphyrins. Higher concentrations of iron (10(-3)M) and ZnPP were in fact inhibitory to bone marrow growth. This inhibition was not reversed with higher concentrations of Epo or GM-CSF. The beneficial effect of iron may be best realized in the bound form such as heme. In addition, the effect of bound and nonbound iron was tested for its effect on gene transfer. By using a murine adherent cell layer (ACL) in a prestimulation phase, followed by human gene transfer of ADA into mouse bone marrow cells and Southern blot analysis, successful gene transfer was accomplished. ADA integration patterns were detected in CFU-S from stem cells of mice 5-11 months after transfer. When 10 microM ferric chloride was included in the ACL prestimulation phase, there was a marked depression in ADA integration into stem cells as compared to heme or non-heme controls. Furthermore, heme-bound iron had no effect and deferoxamine included with iron was able to reverse the inhibitory effect of iron on the gene transfer process. Thus, iron must be non-bound to exert its suppressive effect, and chelation of excess iron under clinical conditions of iron overload may be essential for successive gene transfer.

Animals↗

PubMed1991

ADP/ATP translocator is essential only for anaerobic growth of yeast Saccharomyces cerevisiae.

All three genes (AAC1, AAC2 and AAC3) encoding the mitochondrial ADP/ATP translocator, were inactivated in a haploid yeast strain by a gene disruption technique. The triple mutant was still able to grow on fermentable carbon sources but only in the presence of oxygen. Under aerobic conditions neither translocator-protein nor carrier-mediated transport was detected in all mutants in which the AAC2 gene was disrupted. It was further shown that a functional AAC genes product is essential only for anaerobic growth of Saccharomyces cerevisiae but not for growth under derepressed conditions. Under anaerobic conditions a non-detectable amount of AAC3 gene product is sufficient to ensure the cell growth and multiplication.

Adenosine Diphosphate↗

PubMed1997

The patB gene of Dictyostelium discoideum encodes a P-type H(+)-ATPase isoform essential for growth and development under acidic conditions.

During growth and early development of Dictyostelium discoideum, the amoebae exhibit transient pH changes in their cytosol (pHi) and external medium which correlate with the extrusion of H+ from the cell by a plasma membrane pump. Moreover, the changes in pHi have been postulated to influence early prestalk/prespore differentiation during development. To learn more about the role of H+ fluxes in Dictyostelium, we cloned and analysed cDNAs of the gene patB, which appears to encode a P-type H(+)-ATPase. The patB ORF encodes a protein (termed PAT2) of 1058 amino acids with a calculated molecular mass of 117,460 Da. When aligned with other P-type ion-transport ATPases, PAT2 showed the greatest amino acid sequence identity with plasma membrane H(+)-ATPases of plants and fungi and considerably lower identity with other monovalent cation pumps and with Ca2+ pumps. Northern and Western analyses revealed that patB is expressed at very low levels in cells growing at neutral pH, but it is up-regulated rapidly and dramatically when the cells are shifted to an acidic medium. Immunofluorescence analysis indicated that PAT2 resides on the plasma membrane. When patB was disrupted by homologous recombination, the cells grew and developed normally at neutral and slightly alkaline pHs but they were unable to grow or develop at pH 5.0, and they slowly died. In growth medium at pH 6.8, patB+ and patB cells exhibited similar levels of vanadate-sensitive ATPase activity. However, when the cells were shifted to pH 5.0, this activity rapidly increased about twofold in the control cells but not in the mutant cells. Despite the lower ATPase activity in patB cells, they showed relatively normal H+ fluxes and only a slight decrease in pHi when incubated in acidic medium. Together, these results suggest that patB encodes an acid-inducible P-type H(+)-ATPase which is indispensable for the survival of Dictyostelium cells in moderately acidic external environments.

Amino Acid Sequence↗

PubMed2005

A genomewide screen reveals a role of mitochondria in anaerobic uptake of sterols in yeast.

The mechanisms that govern intracellular transport of sterols in eukaryotic cells are not well understood. Saccharomyces cerevisiae is a facultative anaerobic organism that becomes auxotroph for sterols and unsaturated fatty acids in the absence of oxygen. To identify pathways that are required for uptake and transport of sterols, we performed a systematic screen of the yeast deletion mutant collection for genes that are required for growth under anaerobic conditions. Of the approximately 4800 nonessential genes represented in the deletion collection, 37 were essential for growth under anaerobic conditions. These affect a wide range of cellular functions, including biosynthetic pathways for certain amino acids and cofactors, reprogramming of transcription and translation, mitochondrial function and biogenesis, and membrane trafficking. Thirty-three of these mutants failed to grow on lipid-supplemented media when combined with a mutation in HEM1, which mimics anaerobic conditions in the presence of oxygen. Uptake assays with radio- and fluorescently labeled cholesterol revealed that 17 of the 33 mutants strongly affect uptake and/or esterification of exogenously supplied cholesterol. Examination of the subcellular distribution of sterols in these uptake mutants by cell fractionation and fluorescence microscopy indicates that some of the mutants block incorporation of cholesterol into the plasma membrane, a presumably early step in sterol uptake. Unexpectedly, the largest class of uptake mutants is affected in mitochondrial functions, and many of the uptake mutants show electron-dense mitochondrial inclusions. These results indicate that a hitherto uncharacterized mitochondrial function is required for sterol uptake and/or transport under anaerobic conditions and are discussed in light of the fact that mitochondrial import of cholesterol is required for steroidogenesis in vertebrate cells.

ATP-Binding Cassette Transporters↗

PubMed2002

Molecular approach to common causes of female infertility.

Pivotal genetic information has been derived for a host of rare genetic disorders, but progress has been much slower in relation to the common causes of female infertility. In this chapter, we shall illustrate the approaches being applied in elucidating conditions causing infertility that are inherited in a polygenic/multifactorial fashion. The task is to determine the number of genes responsible and their chromosomal location(s). The first approach is to use genome-wide quantitative linkage analysis, searching throughout the genome with no prior expectation that a given gene or chromosomal region is casually involved. A second approach is to search across the genome for altered gene expression, for example comparing endometriosis and normal (non-endometriosis)cells. The third approach is less indiscriminate and more focused, depending upon identifying specific candidate genes. Aromatase, calhedrin, oestrogen receptor, galactose-1-phosphate uridyl transferase (GALT) and tumour suppressor genes such as p53 are attractive candidate genes for endometriosis. Endometriosis, which has long been suspected to possess a familial tendency, has been subjected to genome-wide linkage analysis in Oxford, UK, where sib-pair analysis uses polymorphic DNA markers and fluorescence-based automated analysis. Several regions of exclusion have been found, but no linkages have so far been reported. A candidate gene approach focuses on the presence of chromosomal aberrations, the assumption being that endometriosis parallels neoplasia. At Baylor College of Medicine, we thus began by showing chromosome alterations involving trisomy 11, monosomy 16 and monosomy 17 in late-stage endometriosis. A loss of only the p53 tumour suppressor gene, rather than a loss (monosomy) of chromosome 17 per se, however, seems to be the pivotal event. A second representative polygenic/multifactorial disorder causing female infertility is polycystic ovarian syndrome. Both quantitative linkage analysis and candidate gene approaches are being pursued. In the far more commonly observed 'idiopathic' variety (non-adrenal polycystic ovarian syndrome and hirsutism), consensus has long existed that one or more dominant genes causes the condition. Although the mode of inheritance in 'essential' polycystic ovarian syndrome remains uncertain, dominant tendencies are clearly more pertinent than recessive ones. Genes for adrenal biosynthetic enzymes, insulin receptors, leptin and leptin receptors, follistatin, activin and inhibins are attractive candidates for polycystic ovarian disease. A linkage to 37 candidate genes was sought using affected sib-pair analysis and transmission/disequilibrium methods.

Endometriosis↗