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PubMedDate not supplied

[Fanconi anemia: genes and function(s) revisited].

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes. Nine of them have been identified. In addition, FANCD1 gene has been shown to be identical to BRCA2, one of the two breast cancer susceptibility genes. Seven of the FANC proteins form a complex, which exists in four different forms depending of its subcellular localisation. Four FANC proteins (D1(BRCA2), D2, I and J) are not associated to the complex. The presence of the nuclear form of the FA core complex is necessary for the mono-ubiquitinylation of FANCD2 protein, a modification required for its re-localization to nuclear foci, likely to be sites of DNA repair. A clue towards understanding the molecular function of the FANC genes comes from the recently identified connection of FANC to the BRCA1, ATM, NBS1 and ATR genes. Two of the FANC proteins (A and D2) directly interact with BRCA1, which in turn interacts with the MRE11/RAD50/NBS1 complex, which is one of the key components in the mechanisms involved in the cellular response to DNA double strand breaks (DSB). Moreover, ATM, a protein kinase that plays a central role in the network of DSB signalling, phosphorylates in vitro and in vivo FANCD2 in response to ionising radiations. Moreover, the NBS1 protein and the monoubiquitinated form of FANCD2 seem to act together in response to DNA crosslinking agents. Taken together with the previously reported impaired DSB and DNA interstrand crosslinks repair in FA cells, the connection of FANC genes to the ATM, ATR, NBS1 and BRCA1 links the FANC genes function to the finely orchestrated network involved in the sensing, signalling and repair of DNA replication-blocking lesions.

Chromosome Aberrations↗
PubMed2001

Rules of nonallelic noncomplementation at the synapse in Caenorhabditis elegans.

Nonallelic noncomplementation occurs when recessive mutations in two different loci fail to complement one another, in other words, the double heterozygote exhibits a phenotype. We observed that mutations in the genes encoding the physically interacting synaptic proteins UNC-13 and syntaxin/UNC-64 failed to complement one another in the nematode Caenorhabditis elegans. Noncomplementation was not observed between null alleles of these genes and thus this genetic interaction does not occur with a simple decrease in dosage at the two loci. However, noncomplementation was observed if at least one gene encoded a partially functional gene product. Thus, this genetic interaction requires a poisonous gene product to sensitize the genetic background. Nonallelic noncomplementation was not limited to interacting proteins: Although the strongest effects were observed between loci encoding gene products that bind to one another, interactions were also observed between proteins that do not directly interact but are members of the same complex. We also observed noncomplementation between genes that function at distant points in the same pathway, implying that physical interactions are not required for nonallelic noncomplementation. Finally, we observed that mutations in genes that function in different processes such as neurotransmitter synthesis or synaptic development complement one another. Thus, this genetic interaction is specific for genes acting in the same pathway, that is, for genes acting in synaptic vesicle trafficking.

Alleles↗
PubMed1987

Isolation and characterization of a processed gene for human ceruloplasmin.

A processed pseudogene for human ceruloplasmin has been isolated that contains DNA corresponding to the functional gene sequence encoding the carboxy-terminal 563 amino acid residues and the 3' untranslated region. The pseudogene appears to have arisen from a processed RNA species, since intervening sequences coincident with those of the functional gene have been removed, with the exception of a short segment of intronic sequence which denotes the 5' boundary of the pseudogene. The nucleotide sequence of the pseudogene is highly homologous (97% sequence identity) with that of the wild-type gene, suggesting that pseudogene formation was a relatively recent evolutionary event. In addition to single base substitutions, there is a large 213 base pair (bp) deletion in the pseudogene sequence which corresponds to the location of an intron-exon junction in the functional gene. A 4 bp duplication that occurs at amino acid residue 683 of the wild-type coding sequence results in a frameshift mutation and introduces a premature translational termination codon at this point. This is concordant with the inability to detect a human liver transcript corresponding to the pseudogene by nuclease S1 mapping analysis. The 3' end of the pseudogene is characterized by a 62 bp segment composed mainly of repeated TC dinucleotides. On the basis of genomic Southern blot analysis performed under high-stringency conditions, the pseudogene that we have identified seems to comprise the only sequence in the human genome that is closely related to the wild-type gene. Using somatic cell hybridization, we have mapped the pseudogene to human chromosome 8.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2006

Expressed sequence tag analysis of Lilium longiflorum generative cells.

The generative cell, the male gametic cell progenitor in flowering plants, undergoes mitotic division to produce two sperm cells. We have examined the gene expression profile of the Lilium longiflorum (lily) generative cell by sequencing expressed sequence tags (ESTs). A total of 886 ESTs derived from the generative cell cDNA library were clustered into 637 unique ESTs comprising 123 cluster ESTs and 514 singleton ESTs. Thirty-nine percent of non-redundant ESTs showing similarity to Arabidopsis genes with known function were thus assigned putative functions. Genes related to the ubiquitin system were abundant, suggesting the key role of ubiquitin-dependent proteolysis in gametogenesis. A total of 168 and 129 non-redundant lily generative cell ESTs showed significant similarity to maize sperm cell ESTs and Arabidopsis male gametophyte-specific transcripts, respectively. Fifty-five ESTs appeared to have significant similarities to both maize sperm cell ESTs and Arabidopsis male gametophyte-specific genes, indicating conservation of male gamete-expressed genes across different plant genera. Thus our data provide a handle to identify Arabidopsis gamete-expressed genes and to investigate their function. Several of these genes are potential candidates for analyzing the molecular basis of fertilization and for investigating mechanisms of gamete-specific transcriptional regulation in Arabidopsis through bioinformatics-based approaches.

Arabidopsis↗
PubMed2006

Gene connectivity, function, and sequence conservation: predictions from modular yeast co-expression networks.

BACKGROUND: Genes and proteins are organized into functional modular networks in which the network context of a gene or protein has implications for cellular function. Highly connected hub proteins, largely responsible for maintaining network connectivity, have been found to be much more likely to be essential for yeast survival. RESULTS: Here we investigate the properties of weighted gene co-expression networks formed from multiple microarray datasets. The constructed networks approximate scale-free topology, but this is not universal across all datasets. We show strong positive correlations between gene connectivity within the whole network and gene essentiality as well as gene sequence conservation. We demonstrate the preservation of a modular structure of the networks formed, and demonstrate that, within some of these modules, it is possible to observe a strong correlation between connectivity and essentiality or between connectivity and conservation within the modules particularly within modules containing larger numbers of essential genes. CONCLUSION: Application of these techniques can allow a finer scale prediction of relative gene importance for a particular process within a group of similarly expressed genes.

Base Sequence↗
PubMed2006

Genomewide gain-of-function genetic screen identifies functionally active genes in mouse embryonic stem cells.

Embryonic stem (ES) cells hold great promise for the future of medicine. To elucidate the molecular mechanisms that control ES cell self-renewal and differentiation, a comprehensive knowledge of the molecules involved in these processes is required. Here we describe an effective approach for genomewide identification of functionally active genes in ES cells. This approach combines genetic screens based on cDNA libraries with microarray detection methods to permit high-throughput functional analyses. We implement this strategy to identify genes whose overexpression can maintain phenotypic properties of undifferentiated mouse ES cells under differentiation-inducing conditions, specifically in the absence of leukemia inhibitory factor. The identified genes encode a variety of regulatory proteins whose function in ES cells was previously unknown. Moreover, our approach is capable of detecting genes whose overexpression promote differentiation or cell death. Overall, our studies establish a methodology for highly sensitive identification of genes that confer particular phenotypes on ES cells.

Animals↗
PubMed2001

Histone H1 diversity: bridging regulatory signals to linker histone function.

Genes encoding linker histone variants have evolved to link their expression to signals controlling the proliferative capacities of cells, i.e. cycling and growth-arrested cells express distinct and specific H1 subtypes. In metazoan, these variants show a tripartite structure, with considerably divergent sequences in their amino and carboxyl terminus domains. The aim of this review is to show how specific regulatory signals control the expression of an individual H1 and to discuss the functional significance of the two variables associated with a linker histone: its primary sequence and the timing of its expression.

Amino Acid Sequence↗
PubMed2000

Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation.

Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that the wboA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760-764, 1999). Disruption of the wboA gene in smooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether the wboA gene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functional wboA gene, and the resulting strain was designated RB51WboA. Colony and Western blot analyses indicated that RB51WboA expressed the O antigen; immunoelectron microscopy revealed that the O antigen was present in the cytoplasm. Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic characteristics similar to those of RB51. Bacterial clearance studies of BALB/c mice indicated no increase in the survival ability of RB51WboA in vivo compared to that of RB51. Vaccination of mice with live RB51WboA induced antibodies to the O antigen which were predominantly of the immunoglobulin G2a (IgG2a) and IgG3 isotypes. After in vitro stimulation of splenocytes with killed bacterial cells, quantitation of gamma interferon in the culture supernatants indicated that RB51WboA immunization induced higher levels of gamma interferon than immunization with RB51. Mice vaccinated with RB51WboA were better protected against a challenge infection with the virulent strain 2308 than those vaccinated with RB51. These studies indicate that in addition to the disruption of the wboA gene there is at least one other mutation in RB51 responsible for its rough phenotype. These studies also suggest that the expressed O antigen in RB51WboA is responsible either directly or indirectly for the observed enhancement in the T-cell response.

Animals↗
PubMed1999

Mouse ENU mutagenesis.

The progress of human genome sequencing is driving genetic approaches to define gene function. Strategies such as gene traps and chemical mutagenesis will soon generate a large mutant mouse resource. Point mutations induced by N -ethyl- N -nitrosourea (ENU) provide a unique mutant resource because they: (i) reflect the consequences of single gene change independent of position effects; (ii) provide a fine-structure dissection of protein function; (iii) display a range of mutant effects from complete or partial loss of function to exaggerated function; and (iv) discover gene functions in an unbiased manner. Phenotype-driven ENU screens in the mouse are emphasizing relevance to human clinical disease by targeting cardiology, physiology, neurology, immunity, hematopoiesis and mammalian development. Such approaches are extremely powerful in understanding complex human diseases and traits: the base-pair changes may accurately model base changes found in human diseases, and subtle mutant alleles in a standard genetic background provide the ability to analyze the consequences of compound genotypes. Ongoing mouse ENU mutagenesis experiments are generating a treasure trove of new mutations to allow an in-depth study of a single gene, a chromosomal region or a biological system.

Animals↗
PubMed2004

Efficient activation of gene expression using a heat-shock inducible Gal4/Vp16-UAS system in medaka.

BACKGROUND: Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand. RESULTS: We have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos. CONCLUSION: The combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products.

Animals↗
PubMedDate not supplied

Glucocerebrosidase recombinant allele: molecular evolution of the glucocerebrosidase gene and pseudogene in primates.

Glucocerebrosidase is a lysosomal enzyme that hydrolyses the beta-glycosidic linkage of glucocerebroside, a ubiquitous sphingolipid present in the plasma membrane of mammalian cells. Deleterious mutations in the glucocerebrosidase gene result in Gaucher disease, the most prevalent lysosomal storage disease. Humans have one glucocerebrosidase functional gene and pseudogene that were located 16 kb apart on chromosome 1q21 and share 96% overall sequence similarity. Recombination between the two genes creates a 'complex allele' that renders glucocerebrosidase non-functional and accounts for >20% of the total Gaucher disease mutations in some population. The glucocerebrosidase pseudogene is absent in all other mammalian species surveyed so far. In order to learn more about the molecular evolution of the glucocerebrosidase functional gene and pseudogene, we have sequenced approximately 1.1 kb of the C-terminal region of these genes that encodes the enzyme catalytic site, from PCR-amplified genomic DNA of gorilla, chimpanzee, orangutan (the great apes), and squirrel monkey (a new-world monkey). In gorilla, chimpanzee, and orangutan, there are two copies of the glucocerebrosidase gene while the squirrel monkey possesses only a single copy. Similar to human, the second copy of glucocerebrosidase gene in gorilla and chimpanzee is non-functional because of a 55-bp deletion in exon 9, while that in orangutan appears to be unaffected and may still be functional. These data suggest that the glucocerebrosidase gene duplication event occurred after squirrel monkey divergence from the great apes, and that the exon 9 deletion that rendered the second copy of the glucocerebrosidase gene non-functional occurred prior to the divergence of gorilla and chimpanzee but after the divergence of orangutan from their common ancestor to human. The two genes in each species are least similar in gorilla and chimpanzee (97.8%) and most similar in orangutan (99.5%). None of the nucleotide variations in the GBA gene among the primates correspond to known mutations in Gaucher disease. Phylogenetic tree analysis using DNAstar and PAUP4.0 software indicates that gene conversion caused the evolution of glucocerebrosidase functional gene and pseudogene to be concerted.

Alleles↗
PubMed2005

Metabolic functions of duplicate genes in Saccharomyces cerevisiae.

The roles of duplicate genes and their contribution to the phenomenon of enzyme dispensability are a central issue in molecular and genome evolution. A comprehensive classification of the mechanisms that may have led to their preservation, however, is currently lacking. In a systems biology approach, we classify here back-up, regulatory, and gene dosage functions for the 105 duplicate gene families of Saccharomyces cerevisiae metabolism. The key tool was the reconciled genome-scale metabolic model iLL672, which was based on the older iFF708. Computational predictions of all metabolic gene knockouts were validated with the experimentally determined phenotypes of the entire singleton yeast library of 4658 mutants under five environmental conditions. iLL672 correctly identified 96%-98% and 73%-80% of the viable and lethal singleton phenotypes, respectively. Functional roles for each duplicate family were identified by integrating the iLL672-predicted in silico duplicate knockout phenotypes, genome-scale carbon-flux distributions, singleton mutant phenotypes, and network topology analysis. The results provide no evidence for a particular dominant function that maintains duplicate genes in the genome. In particular, the back-up function is not favored by evolutionary selection because duplicates do not occur more frequently in essential reactions than singleton genes. Instead of a prevailing role, multigene-encoded enzymes cover different functions. Thus, at least for metabolism, persistence of the paralog fraction in the genome can be better explained with an array of different, often overlapping functional roles.

Gene Duplication↗
PubMed2006

Selective constraint and genetic differentiation in geographically distant barley yellow dwarf virus populations.

Numerous studies have documented molecular variability in plant virus populations, but few have assessed the relative contribution of natural selection and genetic drift in generating the observed pattern of diversity. To this end, gene function, environment and phylogenetic history were examined to observe the effect on genetic diversity and population structure of the PAV and PAS species of Barley yellow dwarf virus (family Luteoviridae). Three functional classes of gene were analysed: transcription-related (RdRp), structural (CP) and movement-related (MP). The results indicate that there were no inherent differences, in terms of total diversity or diversity at synonymous or non-synonymous nucleotide sites, between functional classes of genes or populations. Rather, selective constraints on a gene may be more or less relaxed depending on its function and the phylogenetic history of the population sampled. The CP of the PAS species, but not the PAV species, was differentiated genetically between regions. This is probably due to genetic drift, as there was no evidence that any gene deviated from a neutral model of evolution or is under positive selection. In general, the MP was under considerably less functional constraint than structural or replication-related proteins and four positively selected codon sites were identified. Mutations at these sites differentiate species and geographical subpopulations, so presumably they have aided the virus in adaptation to its host environment and contributed to intra- and interspecies diversification.

Biological Evolution↗
PubMed2002

Influence of functional MDR1 gene polymorphisms on P-glycoprotein activity in CD34+ hematopoietic stem cells.

BACKGROUND AND OBJECTIVES: Expression of the multidrug resistance P-glycoprotein, a transmembrane drug transporter, is influenced by recently described polymorphisms of the human MDR1 gene. Hematopoietic cells, such as lymphocytes, and hematopoietic stem cells, express P-glycoprotein, but the effect of MDR1 gene polymorphisms on P-glycoprotein activity in stem cells is unknown. We investigated whether T-129C, G26677T and C3435T polymorphisms influence P-glycoprotein function in stem cells. DESIGN AND METHODS: P-glycoprotein function was evaluated in immunomagnetically purified bone marrow CD34+ cells from 33 healthy bone marrow donors by the flow cytometric rhodamine 123-efflux assay. For T-129C and C3435T, bone marrow donors were genotyped by polymerase chain reaction amplification followed by MspA1I and DpnII digestion analyses, respectively. For the analysis of C2677T, exon 21 was sequenced. RESULTS: P-glycoprotein function was not different among the C3435T genotypes (CC, 38.2 +/- 3.5%; n=17; CT, 42.2 +/- 3.3%; n=11; and TT, 45.0 +/- 5.3%; n=5) nor was it among the C2677T genotypes (CC, 39.4 +/- 2.4%; n=27; CT, 43.7 +/- 6.4%; n=5; and TT, 54.3%; n=1). Among the 33 subjects, three were heterozygotes for the 129C allele (CT) and no mutant homozygote was identified. P-glycoprotein was similar in heterozygotes (TC, 50.6 +/- 2.9%) and wild-type subjects (TT, 39.5 +/- 2.4%). INTERPRETATION AND CONCLUSIONS: These findings suggest that the known functional MDR1 gene polymorphisms are not major determinants of P-glycoprotein function in hematopoietic stem cells. Other genetic variants might influence P-glycoprotein activity in this cell type.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Pharmacology of antisense oligonucleotide inhibitors of protein expression.

The dramatic increase in recent years of both the amount and rate of accumulation of novel genomic sequence information has generated enormous opportunities for the development of new classes of drugs. For these opportunities to be fully capitalized upon, investigators must choose molecular targets for drug development that are likely to yield attractive therapeutic profiles. This will require rapid and effective determination of gene functions in multiple cellular settings. The development of antisense oligonucleotides as specific inhibitors of gene expression should allow such determination of gene function. In addition, the antisense oligonucleotides themselves will likely prove useful as drugs. In this review, we discuss some of the issues surrounding the use of antisense oligonucleotides as research tools to help elucidate gene function, and highlight some of the approaches that can be taken to generate and use effective antisense reagents.

Forecasting↗
PubMed1992

Yeast casein kinase I homologues: an essential gene pair.

We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues.

Amino Acid Sequence↗
PubMed2000

CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes.

CTXphi is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by Vibrio cholerae. CTX prophages in O1 El Tor and O139 strains of V. cholerae are found within arrays of genetically related elements integrated at a single locus within the V. cholerae large chromosome. The prophages of O1 El Tor and O139 strains generally yield infectious CTXphi. In contrast, O1 classical strains of V. cholerae do not produce CTXphi, although they produce cholera toxin and they contain CTX prophages integrated at two sites. We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally. The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXphi. Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages. RS1, a genetic element that is closely related to CTXphi and is often interspersed with CTX prophages in El Tor strains, was not detected in classical V. cholerae. Our model for CTXphi production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction. Thus, failure of O1 classical strains of V. cholerae to produce CTXphi is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes.

Bacteriophages↗
PubMed2006

In vivo transduction of murine cerebellar Purkinje cells by HIV-derived lentiviral vectors.

Cerebellar Purkinje cells are key elements in motor learning and motor coordination, and therefore, it is important to clarify the mechanisms by which Purkinje cells integrate information and control cerebellar function. Gene transfer into neurons, followed by the assessment of the effects on neural function, is an effective approach for examining gene function. However, this method has not been used fully in the study of the cerebellum because adenovirus vectors, the vectors most commonly used for in vivo gene transfer, have very low affinity for Purkinje cells. In this study, we used a human immunodeficiency virus (HIV)-derived lentiviral vector and examined the transduction profile of the vector in the cerebellum. A lentiviral vector carrying the GFP gene was injected into the cerebellar cortex. Seven days after the injection, Purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. GFP was also expressed, though less efficiently, in other cortical interneurons and Bergmann glias. In contrast to reported findings with other viral vectors, no transduced cells were observed outside of the cerebellar cortex. Thus, when HIV-derived lentiviral vectors were injected into the cerebellar cortex, transduction was limited to the cells in the cerebellar cortex, with the highest tropism for Purkinje cells. These results suggest that HIV-derived lentiviral vectors are useful for the study of gene function in Purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect Purkinje cells.

Animals↗