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PubMed1991

Using antisense RNA to study gene function.

Over the past two years, antisense RNA technology has been developed in plants in several experimental systems. Progress reported in the literature will be reviewed. Special emphasis will be placed on experiments in which antisense RNA has been used to generate novel tomato mutants through the downregulation of polygalacturonase (PG), a major cell wall hydrolase. This work has shown that antisense RNA inhibits PG expression specifically without affecting the expression of other genes. The antisense gene and its phenotypic expression are stably inherited. The inhibitory effect of an antisense gene can be enhanced by increasing the copy number of the gene. The interaction of the antisense gene with its target is only seen when the target gene is expressed. Antisense RNA technology has been used to clarify the biochemical function of polygalacturonase and its role in tomato fruit softening. Recently, this approach has also been applied to elucidate the biochemical role of another gene from tomato, pTOM13, whose function was previously unknown. Thus, antisense RNA technology can now be used to generate novel plant mutants that will make possible the definition of the biochemical and biological role of genes whose functions are otherwise unknown.

Genes, Plant↗
PubMed2006

Discovering functional gene expression patterns in the metabolic network of Escherichia coli with wavelets transforms.

BACKGROUND: Microarray technology produces gene expression data on a genomic scale for an endless variety of organisms and conditions. However, this vast amount of information needs to be extracted in a reasonable way and funneled into manageable and functionally meaningful patterns. Genes may be reasonably combined using knowledge about their interaction behaviour. On a proteomic level, biochemical research has elucidated an increasingly complete image of the metabolic architecture, especially for less complex organisms like the well studied bacterium Escherichia coli. RESULTS: We sought to discover central components of the metabolic network, regulated by the expression of associated genes under changing conditions. We mapped gene expression data from E. coli under aerobic and anaerobic conditions onto the enzymatic reaction nodes of its metabolic network. An adjacency matrix of the metabolites was created from this graph. A consecutive ones clustering method was used to obtain network clusters in the matrix. The wavelet method was applied on the adjacency matrices of these clusters to collect features for the classifier. With a feature extraction method the most discriminating features were selected. We yielded network sub-graphs from these top ranking features representing formate fermentation, in good agreement with the anaerobic response of hetero-fermentative bacteria. Furthermore, we found a switch in the starting point for NAD biosynthesis, and an adaptation of the l-aspartate metabolism, in accordance with its higher abundance under anaerobic conditions. CONCLUSION: We developed and tested a novel method, based on a combination of rationally chosen machine learning methods, to analyse gene expression data on the basis of interaction data, using a metabolic network of enzymes. As a case study, we applied our method to E. coli under oxygen deprived conditions and extracted physiologically relevant patterns that represent an adaptation of the cells to changing environmental conditions. In general, our concept may be transferred to network analyses on biological interaction data, when data for two comparable states of the associated nodes are made available.

Algorithms↗
PubMed2005

Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins.

In bacteria, whereas disruption methods have been improved recently, the use of plasmid complementation strategies are still subject to limitations, such as cloning difficulties, nonphysiological levels of gene expression, or a requirement for antibiotics as plasmid selection pressure. Moreover, because of the pleiotropic modifications of cell physiology often induced by plasmid-based complementation, these strategies may introduce biases when biological process such as adhesion or biofilm formation are studied. We developed a plasmid-free approach that combines the lambda-red linear DNA recombination method with site-directed insertion of a repression and expression (RExBAD) cassette which places a functional pBAD promoter upstream of a target gene. We showed that this method permits both inactivation and modulation of most Escherichia coli gene expression, including expression of toxin and essential genes. We used this strategy to study adhesion and bacterial biofilms by placing the RExBAD cassette in front of the tra operon, encoding the DNA transfer and pilus genes on the F conjugative plasmid, and in front of flu, the antigen 43 (Ag43) autotransporter adhesin-encoding gene. In silico analysis revealed the existence of 10 genes with homology to the Ag43 gene that were good candidates for genes that encode putative new adhesins in E. coli. We used the RExBAD strategy to study these genes and demonstrated that induction of expression of four of them is associated with adhesion of E. coli to abiotic surfaces. The potential use of the RExBAD approach to study the function of cryptic or uncharacterized genes in large-scale postgenomic functional analyses is discussed.

Adhesins, Escherichia coli↗
PubMed1987

Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene. We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA. Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, alpha-specific mating defects, and failure to homozygous diploids to sporulate. In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase. An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL10-lacZ fusion and maltase. The mating defects of MAT alpha ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of alpha-factor; no deficiency of MAT alpha 2 RNA was detected. We showed that ssn6 partially restored invertase expression in a cyr1-2 mutant, although ssn6 was clearly not epistatic to cyr1-2. We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). Possible functions of the SSN6 product are discussed.

Amino Acid Sequence↗
PubMed2003

The contribution of E2F-regulated transcription to Drosophila PCNA gene function.

E2F proteins control cell cycle progression by predominantly acting as either activators or repressors of transcription. How the antagonizing activities of different E2Fs are integrated by cis-acting control regions into a final transcriptional output in an intact animal is not well understood. E2F function is required for normal development in many species, but it is not completely clear for which genes E2F-regulated transcription provides an essential biological function. To address these questions, we have characterized the control region of the Drosophila PCNA gene. A single E2F binding site within a 100-bp enhancer is necessary and sufficient to direct the correct spatiotemporal program of G1-S-regulated PCNA expression during development. This dynamic program requires both E2F-mediated transcriptional activation and repression, which, in Drosophila, are thought to be carried out by two distinct E2F proteins. Our data suggest that functional antagonism between these different E2F proteins can occur in vivo by competition for the same binding site. An engineered PCNA gene with mutated E2F binding sites supports a low level of expression that can partially rescue the lethality of PCNA null mutants. Thus, E2F regulation of PCNA is dispensable for viability, but is nonetheless important for normal Drosophila development.

Animals↗
PubMed2003

Multi-functional gene ASY/Nogo/RTN-X/RTN4: apoptosis, tumor suppression, and inhibition of neuronal regeneration.

ASY, also designated Nogo, RTN-X, or RTN4, is a reticulon family protein containing two transmembrane domains and a C-terminal double lysine endoplasmic reticulum (ER) retrieval motif. Three protein variants are synthesized from the cognate mRNAs produced by alternative splicing, and are expressed in almost all tissues, localizing predominantly in the ER. The ASY protein induces apoptosis in various cancer cells when overexpressed, whereas normal cells are relatively resistant to ASY-dependent apoptosis. Furthermore, transcription of this gene is suppressed in certain types of cancers, suggesting that ASY may act to suppress tumor development. Although the physiological function of this protein has not been well defined, Nogo-A protein, the large variant of ASY, has been shown to inhibit neuronal regeneration in the central nervous system. Therefore, the products of this gene may be multi-functional, regulating apoptosis, tumor development, and neuronal regeneration.

Alternative Splicing↗
PubMed2001

A new family of yeast vectors and S288C-derived strains for the systematic analysis of gene function.

The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2 Delta 0 ade8 Delta 0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.

Base Sequence↗
PubMed1992

Ordering gene function: the interpretation of epistasis in regulatory hierarchies.

The order of action of genes in a regulatory hierarchy that is governed by a signal can often be determined by the method of epistasis analysis, in which the phenotype of a double mutant is compared with that of single mutants. The epistatic mutation may be in either the upstream or the downstream gene, depending on the nature of the two mutations and the type of regulation. Nevertheless, when the regulatory hierarchy satisfies certain conditions, simple rules allow the position of the epistatic locus in the pathway to be determined without detailed knowledge of the nature of the mutations, the pathway, or the molecular mechanism of regulation.

Animals↗
PubMed2005

Modulating Hox gene functions during animal body patterning.

With their power to shape animal morphology, few genes have captured the imagination of biologists as the evolutionarily conserved members of the Hox clusters have done. Recent research has provided new insight into how Hox proteins cause morphological diversity at the organismal and evolutionary levels. Furthermore, an expanding collection of sequences that are directly regulated by Hox proteins provides information on the specificity of target-gene activation, which might allow the successful prediction of novel Hox-response genes. Finally, the recent discovery of microRNA genes within the Hox gene clusters indicates yet another level of control by Hox genes in development and evolution.

Animals↗
PubMed1999

The 5' untranslated regions of the rat A2A adenosine receptor gene function as negative translational regulators.

The rat A2A adenosine receptor (A2A-R) gene contains two promoters, P1 and P2, which produce transcript 1 and transcript 2, respectively. These transcripts differ in the lengths of their 5' untranslated regions (5'UTR1: 514 bp, initiated from P1; 5'UTR2: 221 bp, initiated from P2) but encode the same protein. In the present study, we demonstrate that transcript 2 is present in various tissues at different levels, whereas transcript 1 is found only in the striatum. In the striatum, the level of transcript 2 is approximately 300-fold higher than that of transcript 1. The 5'UTR of both transcripts suppresses the expression of A2A-R and a firefly luciferase reporter gene at the translational level; this suppression is not observed after mutational inactivation of an "out-of-frame" upstream AUG codon. Translational suppression by the 5'UTR was also confirmed in cells using a bicistronic strategy. Collectively, these data suggest that P2 is the major promoter of the rat A2A-R gene. The 5'UTR of the rat A2A-R gene exerts an inhibitory effect on translation by an upstream open reading frame. Because the 5'UTR of the A2A-R gene possesses strong interspecies homology, translational suppression may be a general mechanism by which the expression of the A2A-R gene is regulated.

Animals↗
PubMed1979

Gene 24-controlled osmotic shock resistance in bacteriophage T4: probable multiple gene functions.

By use of mixed infections with conditional lethal mutations in the head genes and an osmotic shock-resistant mutant we have demonstrated that osmotic shock resistance is controlled by gene 24. Using acrylamide gel electrophoresis combined with the "immune replicate" technique, we confirmed the positions of gene products 24 and 24* (P24 and P24*). In this paper we have still used the notation "P24," etc., for designating the product of gene 23, etc., although we prefer and use in general the designation "gp23" as introduced by Casjens and King (Annu. Rev. Biochem. 44:585, 1975). The reason for using the old notation is because the illustrations were prepared several years ago.) P24 ts showed a significantly slower mobility. Both osmotic shock-resistant and -sensitive mature phages contain 24*. Giants constructed with the Osr phage showed the same surface lattice as normal phage. Through temperature-shift experiments with 24(tsL90) alone and in combinations, we studied the phages which are matured after the shift to permissive temperature in the absence of new protein synthesis. Our results strongly suggest that only a fraction of the total phage complement of gene 24-controlled proteins is involved in determining the phenotype of shock resistance, and the remainder is necessary to mature the head.

Coliphages↗
PubMed1999

Flik, a chick follistatin-related gene, functions in gastrular dorsalisation/neural induction and in subsequent maintenance of midline Sonic hedgehog signalling.

We have targetted the chick gene Flik with antisense oligodeoxynucleotide treatment at gastrular stages, when it is expressed in organiser-derived structures of the midline (K. Patel et al., 1996, Dev. Biol. 178, 327-342). A specific syndrome of deficient axial patterning and holoprosencephaly is produced. Most aspects of this syndrome can be understood as due to attenuation of dorsalising and neural-inducing signals during gastrulation, followed by failure to maintain the later signals from chordamesoderm/neural midline that pattern the mesodermal and neural cross sections during subsequent stages. Anatomical effects are first apparent at early neurula stages and correspond with what might be expected from a reduced counteraction of the ventralising Bone morphogenetic protein (BMP) pathway at the earlier stages, coupled with inadequate Sonic hedgehog (Shh) signalling subsequently. Delay in the clearing of BMP-4 RNA expression from the presumptive neural region at gastrulation is indeed seen, though chordin RNA expression within organiser derivatives remains normal. Subsequently, specific attenuation of chordamesoderm and neural midline Shh expression is observed. Brief preincubation of stage 4 chick blastoderms in supernatant from Xenopus oocytes that have been injected with Flik RNA prolongs and enhances the competence of their peripheral epiblast to respond to neural inductive signals from grafted Hensen's nodes. This effect specifically mimics that recently observed using microg/ml solutions of recombinant Follistatin (D. J. Connolly et al., 1999, Int. J. Dev. Biol., in press), further suggesting that Flik protein might act in vivo by somehow modulating activity of signalling pathways through BMP or other TGFbeta-related ligands. We discuss the significance of the observations in relation to recent ideas about neural induction, about possible redundancy in gene action, and about subsequent patterning of the axial cross section, suggesting that a Flik function in autocrine/paracrine maintenance of later midline Shh signalling represents a role of the gene separate from that in primary dorsalisation/neural induction.

Amino Acid Sequence↗
PubMed2006

Glucose activation of the glucagon receptor gene: functional dissimilarity with several other glucose response elements.

The glucagon receptor (GLR) expression is positively regulated by glucose. This regulation is allowed by the presence, in the promotor of the rat GLR gene, of a sequence feature similar to the two E-boxes motifs constituting the carbohydrate response elements (ChoRE) described for several glycolytic and lipogenic enzyme genes. Using reporter gene assays, we demonstrated here that, despite structural homologies with these ChoREs, the GLR gene glucose response element presents various functional dissimilarities. Testing glucose analogs, we demonstrated that, as for other genes, the glucose must be first phosphorylated. However, at variance with others homologue genes, our data showed the implication of the nonoxidative branch of the pentose phosphate pathway in the transmission of the glucose signal and lack of inhibition by adenosine monophosphate (AMP)-kinase. Furthermore, the activity of our reporter gene was strongly stimulated by butyrate, propionate, and acetate. This observation contrasts with fatty-acid-induced inhibition of the glucose activation, observed for all other genes containing homolog ChoREs. We also showed that glucose and butyrate influence the reporter gene expression via different features.

Animals↗
PubMed2001

DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer.

DAP-kinase is a pro-apoptotic Ca(2+) calmodulin-regulated serine/threonine kinase that participates in a wide array of apoptotic systems initiated by interferon-gamma, TNF-alpha, activated Fas, and detachment from extracellular matrix. It was isolated by an unbiased functional approach to gene cloning aimed at hitting central mediators of the apoptotic process. This 160 Kd protein kinase is localized to actin microfilaments and carries interesting modules such as ankyrin repeats and the death domain. The death promoting effects of DAP-kinase depend on its intact catalytic activity, the correct intracellular localization, and on the presence of the death domain. A few mechanisms restrain the killing effects of the protein in healthy cells. The enzyme's active site is negatively controlled by an adjacent CaM regulatory domain whose effect is relieved by binding to Ca(2+)-activated calmodulin. A second mode of autoinhibition engages the serine-rich C-terminal tail, spanning the last 17 amino acids of the protein. A link between DAP-kinase and cancer has been established. It was found that the mRNA and protein expression is frequently lost in various human cancer cell lines. Analysis of the methylation status of DAP-kinase's 5' UTR in DNA extracted from fresh tumor samples, showed high incidence of hypermethylation in several human carcinomas and B cell malignancies. The anti-tumorigenic effect of DAP-kinase was also studied experimentally in mouse model systems where the re-introduction of DAP-kinase into highly metastatic mouse lung carcinoma cells who had lost the protein, strongly reduced their metastatic capacity. Thus, it appears that loss of DAP-kinase confers a selective advantage to cancer cells and may play a causative role in tumor progression. A few novel kinases sharing high homology in their catalytic domains with DAP-kinase have been recently identified constituting altogether a novel family of death promoting serine/threonine kinases.

Animals↗
PubMed1987

Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers.

An elastase-human growth hormone (hGH) fusion gene containing 205 base pairs of elastase 5' flanking region is expressed exclusively in pancreatic acinar cells of transgenic mice. This paper shows that the promoter region (-72 to +8) and the enhancer (-205 to -73) function independently of each other. The elastase enhancer can activate the heterologous mouse metallothionein gene and the hGH gene promoters; conversely, enhancers from the thymocyte-specific murine leukemia virus MCF13 and the metal regulatory elements from the metallothionein gene can activate the elastase promoter in a variety of cell types. Combinations of immunoglobulin and elastase enhancers with a heterologous promoter and the hGH gene result in expression in all of the tissues predicted by the sum of each enhancer acting alone. Thus these enhancer elements act independently of each other, suggesting that they do not have silencing activity in cells in which they are normally inactive.

Animals↗
PubMed1998

Functional gene transfer from intracellular bacteria to mammalian cells.

We provide evidence of direct transfer of functional DNA from bacteria to mammalian cells. An Escherichia coli K12 diaminopimelate auxotroph made invasive by cloning the invasin gene from Yersinia pseudotuberculosis transfers DNA after simple co-incubation, into a variety of mammalian cell lines. Transfer efficiency was enhanced in some cells by coexpression of the gene for listeriolysin from Listeria monocytogenes. Expression of the acquired genes occurs in both dividing and quiescent cells. The only requirement for bacteria to transfer genetic material into nonprofessional phagocytic cells and macrophages is the ability to invade the host cell.

Adhesins, Bacterial↗
PubMed1999

Cell fate specification in the Drosophila salivary gland: the integration of homeotic gene function with the DPP signaling cascade.

Salivary gland formation in the Drosophila embryo is linked to the expression of the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when SCR is expressed everywhere, salivary glands form in new places. However, not every cell that expresses Scr is recruited to a salivary gland fate. Along the anterior-posterior axis, the posteriorly expressed proteins encoded by the teashirt (tsh) and Abdominal-B (Abd-B) genes block SCR activation of salivary gland genes, and along the dorsal-ventral axis, the secreted signaling molecule encoded by decapentaplegic (dpp) prevents activation of salivary gland genes by SCR in dorsal regions of parasegment 2. We have identified five downstream components in the DPP signaling cascade required to block salivary gland gene activation. These components include two known receptors, the type I receptor encoded by the thick veins (tkv) gene and the type II receptor encoded by the punt (put) gene; two of the four known Drosophila members of the Smad family of proteins which transduce signals from the receptors to the nucleus, Mothers against dpp (Mad) and Medea (Med); and, finally, a large zinc-finger transcription factor encoded by the schnurri (shn) gene. These results reveal how anterior-posterior and dorsal-ventral patterning information is integrated at the level of organ-specific gene expression.

Animals↗
PubMed2001

Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.

The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens-specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.

Animals↗