[Gene expression, gene regulation and gene transfer in the inner ear].
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The Erwinia chrysanthemi pecT gene regulates pectinase gene expression.
A new type of Erwinia chrysanthemi mutant displaying a derepressed synthesis of pectate lyase was isolated. The gene mutated in these strains, pecT, encodes a 316-amino-acid protein with a size of 34,761 Da that belongs to the LysR family of transcriptional activators and presents 61% identity with the E. coli protein LrhA. PecT represses the expression of pectate lyase genes pelC, pelD, pelE, pelL, and kdgC, activates pelB, and has no effect on the expression of pelA or the pectin methylesterase genes pemA and pemB. PecT activiates its own expression. The mechanism by which PecT regulates pectate lyase synthesis is independent of that of the two characterized regulators of pectate lyase genes, KdgR and PecS. In contrast to most of the members of the LysR family, pecT is not transcribed in a direction opposite that of a gene that it regulates. pecT mutants are mucoid when grown on minimal medium plates and flocculate when grown in liquid minimal medium, unless leucine or alanine is added to the medium. Thus, pecT may regulate other functions in the bacterium.
Amino Acid Sequence↗
Efficient transfer of regulated genes in adipocytes and hepatoma cells by the combination of liposomes and replication-deficient adenovirus.
Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes, are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3-F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2-CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times more efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3-F442A adipocytes and FAO hepatoma cells with the lipofectAMINE/pSV2-CAT complex in the presence of replication-deficient recombinant type-5 adenovirus at 200 pfu/cell. CAT activity of transiently transfected cells was increased about 50-fold when compared to the calcium phosphate procedure. To determine whether this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a construct containing -2100 to +69 bp of the phosphoenolpyruvate carboxykinase gene fused to the CAT gene (pPL1-CAT). This construct was shown previously to be cAMP-responsive after calcium-phosphate-mediated transfection of adipocytes and hepatoma cells. 3T3-F442A or FAO cells in which pPL1-CAT was either transiently or stably transferred by lipofectAMINE and adenovirus responded to isoproterenol or cAMP, respectively, with a 2-3-fold increase in CAT activity. Therefore the association of liposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.
3T3 Cells↗
Expression profile of genes regulated by activity of the Na-H exchanger NHE1.
BACKGROUND: In mammalian cells changes in intracellular pH (pHi), which are predominantly controlled by activity of plasma membrane ion exchangers, regulate a diverse range of normal and pathological cellular processes. How changes in pHi affect distinct cellular processes has primarily been determined by evaluating protein activities and we know little about how pHi regulates gene expression. RESULTS: A global profile of genes regulated in mammalian fibroblasts by decreased pHi induced by impaired activity of the plasma membrane Na-H exchanger NHE1 was characterized by using cDNA microarrays. Analysis of selected genes by quantitative RT-PCR, TaqMan, and immunoblot analyses confirmed results obtained from cDNA arrays. Consistent with established roles of pHi and NHE1 activity in cell proliferation and oncogenic transformation, grouping regulated genes into functional categories and biological pathways indicated a predominant number of genes with altered expression were associated with growth factor signaling, oncogenesis, and cell cycle progression. CONCLUSION: A comprehensive analysis of genes selectively regulated by pHi provides insight on candidate targets that might mediate established effects of pHi on a number of normal and pathological cell functions.
Amino Acid Substitution↗
A cis element in the recombination activating gene locus regulates gene expression by counteracting a distant silencer.
We have identified a silencer and an antisilencing element that interact at a distance of 85 kilobases to regulate expression of the recombination activating genes Rag1 and Rag2 in thymocytes. Transgenic experiments showed that Rag promoter-proximal cis elements directed tissue-specific expression and that a Runx-dependent intergenic silencer suppressed expression in developing T cells. Deletion of the antisilencing element from the genomic Rag locus unmasked the intergenic silencer and abrogated Rag expression in developing CD4(+)CD8(+) T cells. We speculate that the Rag antisilencing element belongs to a class of cis elements that might be useful for genome diversification by activating genes encoded by otherwise silent transposable elements.
Animals↗
Identification of a novel lithium regulated gene in rat brain.
Differential display PCR was used to identify genes regulated by mood stabilizer lithium in rat cerebral cortex. A differentially displayed lithium regulated gene fragment was isolated in rat cerebral cortex after chronic treatment with lithium (1.69 g/kg, p.o. ) for three weeks. A 1216-nucleotide cDNA for a novel lithium regulated gene (NLRG) was isolated from a rat brain cDNA library with RACE (rapid amplification of 5' cDNA end) PCR using a prime from the differentially displayed NLRG gene fragment. The deduced protein sequence was 321 amino acids long, and shows a significant homology with yeast nitrogen permease regulator 2 (NPR2). NLRG expression induced by lithium was confirmed by Northern and slot blot analysis in rat cerebral cortex and neuroblastomaxglioma NG108-15 cells, respectively. In situ hybridization revealed that chronic treatment with lithium increased NLRG gene expression in frontal cortex and hippocampus, but not in striatum, hypothalamus and thalamus regions of rat brain. These results suggest a novel target for lithium which may be relevant to its mechanism of action.
Amino Acid Sequence↗
Regulating gene expression in transgenic animals.
Regardless of the field of application, the raison d'etre of transgenic animals is to study gene regulation and function. With increasing frequency, mammalian genes are being isolated with no concomitant knowledge of their function. The human genome mapping initiative will undoubtedly produce a cornucopia of such genes. While the merit of taking a transgenic route to study genes of unknown function is axiomatic, the choices of strategies for gene regulation in vivo may not be fully appreciated. This review will address two main points: first, the targeted and regulated expression of genes, and second, the structural and functional ablation of genes.
Animals↗
Gene regulation by O2 deprivation: an anoxia-regulated novel gene in Drosophila melanogaster.
Organisms, across the animal kingdom, vary in their tolerance or susceptibility to cell injury from O2 deprivation. In this study we have taken advantage of the genetically well studied fruit fly to dissect basic mechanisms underlying their ability to tolerate lack of O2. Using differential display and molecular techniques, we cloned and characterized a novel gene, named fau, which is up-regulated considerably following anoxia in Drosophila melanogaster. Northern blot analysis revealed that the transcript of this gene is approximately 0.9 kb in length with an open reading frame encoding a small hydrophilic protein ( approximately 14.4 kDa). This protein has no homology to previously described gene products but has many potential phosphorylation sites. In situ hybridization showed that this gene is located in region 7C-D on the Drosophila X-chromosome and its transcript concentrated in the lamina and cortical neurons of the Drosophila central nervous system (CNS). Transgenic flies showed that over-expression of fau significantly reduced the recovery time of the flies from anoxia. We conclude that (1) this study provided a framework on which the mechanisms underlying anoxia tolerance can be dissected in the fruit fly and (2) fau gene plays an important role in the regulation of tissue responsiveness to O2 deprivation.
Amino Acid Sequence↗
[Aspects of the problem of regulator genes in the genetics of development].
The concept of gene-regulator in the genetics of prokaryotes and eukaryotes is considered. On the basis of numerous data, a conclusion is drawn on the presence of gene-controlled substances in eukaryotes which activate and suppress cell differentiation at many levels from transcription to posttranslation. It is proposed to define the gene-regulator in eukaryotes as a gene controlling the phenotypic expression (rather than activity only) of other genes at different levels of regulation.
Animals↗
Crystallin genes: specialization by changes in gene regulation may precede gene duplication.
The crystallins account for 80-90% of the water-soluble proteins of the transparent lens. These diverse proteins are responsible for the optical properties of the lens and have been recruited from metabolic enzymes and stress proteins. They often differ among species (i.e. are taxon-specific) and may be expressed outside of the lens where they have non-refractive roles (a situation we call gene sharing). Crystallin recruitment has occurred by changes in gene regulation resulting in high lens expression. Duck lactate dehydrogenase/epsilon-crystallin and alpha-enolase/tau-crystallin are each encoded in single-copy genes, consistent with these enzymes acquiring a crystallin role, without loss of their nonlens metabolic function, by a change in gene regulation in the absence of gene duplication. The small heat shock protein/alpha-crystallins and avian argininosuccinate lyase/delta-crystallins were also recruited by a change in gene regulation leading to high lens expression, except this was followed by a gene duplication with further lens specialization of the alphaA and the delta1 (in chickens) crystallin genes. Cephalopod (squid and octopus) S-crystallins were recruited from glutathione S-transferase apparently after duplication of the original gene encoding the enzyme, although this remains uncertain. We speculate that one of the new genes (glutathione S-transferase/S11-crystallin) specialized for lens expression by a change in gene regulation and subsequently duplicated many times to form the lens-specialized, multiple S-crystallins that lack enzymatic activity. That similar transcription factors (e.g. Pax-6, retinoic acid receptors, maf, Sox, AP-1, CREB) regulate different crystallin genes suggest that common features of lens-specific expression have played a pivotal role for recruiting the diverse, multifunctional proteins as crystallins.
Animals↗
Identification of ligands and coligands for the ecdysone-regulated gene switch.
The ecdysone-inducible gene switch is a useful tool for modulating gene expression in mammalian cells and transgenic animals. We have identified inducers derived from plants as well as certain classes of insecticides that increase the versatility of this gene regulation system. Phytoecdysteroids share the favorable kinetics of steroids, but are inert in mammals. The gene regulation properties of one of these ecdysteroids have been examined in cell culture and in newly developed strains of ecdysone-system transgenic mice. Ponasterone A is a potent regulator of gene expression in cells and transgenic animals, enabling reporter genes to be turned on and off rapidly. A number of nonsteroidal insecticides have been identified that also activate the ecdysone system. Because the gene-controlling properties of the ecdysone switch are based on a heterodimer composed of a modified ecdysone receptor (VgEcR) and the retinoid X receptor (RXR), we have tested the effect of RXR ligands on the VgEcR/RXR complex. Used alone, RXR ligands display no activity on the ecdysone switch. However, when used in combination with a VgEcR ligand, RXR ligands dramatically enhance the absolute levels of induction. This property of the heterodimer has allowed the development of superinducer combinations that increase the dynamic range of the system.
Animals↗
Sequence, transcriptional mapping, and overexpression of p47, a baculovirus gene regulating late gene expression.
A 2.8-kb region of the Autographa californica nuclear polyhedrosis virus genome was sequenced and found to contain an open reading frame (p47) which was capable of rescuing a previously characterized temperature-sensitive mutant, ts317 (S. Partington, H. Yu, A. Lu, and E. B. Carstens, Virology 157:91-102, 1990). Transcriptional mapping demonstrated that an early 4.2-kb RNA encoded the p47 open reading frame and probably overlapped the 39K delayed-early gene. The p47 open reading frame was cloned behind the polyhedrin promoter in a baculovirus transfer plasmid, which was then used to prepare a recombinant baculovirus overexpressing the p47 polypeptide. The overexpressed polypeptide was used to prepare p47-specific monoclonal antibodies. These antibodies detected a polypeptide of 47 kDa in A. californica nuclear polyhedrosis virus-infected cells, demonstrating that p47 is expressed as an authentic viral product. The p47 gene product was localized to the nucleus of infected cells, supporting the hypothesis that it is involved in regulating viral transcription at late times postinfection.
Amino Acid Sequence↗
A highly sensitive and specific assay using a novel human growth hormone cDNA reporter gene regulated by the human interleukin-4 inducible germline epsilon transcript promoter.
We have successfully developed a highly sensitive and specific assay system for human interleukin-4 (IL-4) regulated gene expression. It is based on a human Jijoye cell line with the germline epsilon transcript promoter joined to the human growth hormone (hGH) cDNA. The germline epsilon transcript promoter is responsive to IL-4 and involved in immunoglobulin heavy chain class switching. We cloned hGH complementary DNA (cDNA) as the reporter gene instead of using conventional hGH genomic DNA which failed to generate any IL-4 inducible clone in human Jijoye cells. The two IL-4 inducible cell lines with the hGH cDNA reporter show high signal/noise ratio for IL-4-mediated induction (60-90 fold). The response to IL-4 is dose-dependent with ED50 of 10 pM. As expected, there is no response to other human cytokines and growth factors, as well as mouse IL-4. The mutant hIL-4 antagonist hIL-4.Y124D inhibits the induction mediated by native hIL-4. These IL-4 inducible cell lines provide a sensitive, specific assay system to study IL-4-regulated gene expression, and in particular the regulation of the germline epsilon promoter.
Animals↗
Identification of androgen-regulated genes in mouse kidney by representational difference analysis and random arbitrarily primed polymerase chain reaction.
The molecular nature of tissue-specific gene regulation by androgens has not been well defined, partly as a result of the variable expression and incomplete regulation of currently available gene models. We have therefore aimed to establish more informative models by identifying alternative genes whose expression is tightly and coordinately regulated by androgens. Female C57BL/6 mice were dosed with dihydrotestosterone- or sham-treated for 8 days, after which kidneys were removed and complementary DNA (cDNA) prepared. We then applied the subtractive hybridization techniques of random arbitrarily primed-PCR and PCR-coupled subtractive hybridization method of cDNA representational difference analysis to the isolated cDNA. In addition to well characterized androgen-regulated genes [e.g. KAP (kidney androgen-regulated protein)], we demonstrate the differential expression of six genes previously not known to be under androgen control. RNA levels of SA, Cytochrome P450 4B1, IL-6ST (interleukin-6 signal transducer), OATP (organic anion transporter), and a newly identified gene, MJAM, were up-regulated by androgen, while 16-alpha-hydroxylase was decreased. Expression of these transcripts was inhibited in dihydrotestosterone-treated females by flutamide and in males by castration, confirming their dependence on androgens. Although all the genes demonstrate tissue-specific regulation by androgen, SA showed both kidney specificity and absolute requirement for androgen for its expression. These newly identified androgen-regulated genes will constitute very useful models for studying the nature of tissue-specific gene regulation by androgens.
Androgen Antagonists↗
How advances in chromosome conformation capture (3C) methods are reshaping our understanding of gene regulation in hematopoiesis.
The three-dimensional organization of the DNA within the nucleus plays a key role in regulating gene expression. Over the past two decades, advances in chromosome conformation capture (3C) technologies, in tandem with other methods, have shown that the genome forms a complex structure at multiple scales. Early studies identified large-scale structures such as chromosome territories, compartments and topologically associating domains (TADs). As the resolution of 3C techniques has improved, it has become possible to identify contacts between regulatory elements in detail and more recently, it has become possible to define intricate structures within cis-regulatory elements. In this chapter, we review the development of 3C-based methodologies and discuss the strengths and limitations of the different approaches. We examine how these technologies have refined our understanding of genome organization and gene regulation. Recent high-resolution studies reveal that chromatin architecture extends beyond classical domain structures to include nanoscale organization. Integration of 3C data with super-resolution imaging and molecular dynamics simulations supports a model in which genome folding is governed by the biophysical properties of chromatin.
Animals↗
Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis.
Plants respond to day/night cycling in a number of physiological ways. At the mRNA level, the expression of some genes changes during the 24-hr period. To identify novel genes regulated in this way, we used microarrays containing 11,521 Arabidopsis expressed sequence tags, representing an estimated 7800 unique genes, to determine gene expression levels at 6-hr intervals throughout the day. Eleven percent of the genes, encompassing genes expressed at both high and low levels, showed a diurnal expression pattern. Approximately 2% cycled with a circadian rhythm. By clustering microarray data from 47 additional nonrelated experiments, we identified groups of genes regulated only by the circadian clock. These groups contained the already characterized clock-associated genes LHY, CCA1, and GI, suggesting that other key circadian clock genes might be found within these clusters.
Arabidopsis↗
Immunogenetic markers as probes for polymorphism, gene regulation and gene transfer in man--the Gm system in perspective.
The genetic markers of immunoglobulins (Ig) demonstrable by immunological methods have shown their usefulness as genetic probes. The study of these allotypes originally proved that Ig production is under conventional genetic control and also established that allelic exclusion is valid for the key molecules of the immune response. The codons responsible for the G1m(a) marker and their position in the human genome are precisely known. This knowledge implies that Gm typing may be used as a convenient and reliable means of following the fate of IgG constant gene segments. Anti-Gm's are common in rheumatoid arthritis. They appear early in the disease and may persist throughout life. The stimulus for their appearance has not yet been established. The anti-Gm's in the allegedly autoimmune disease rheumatoid arthritis are commonly and apparently paradoxically specific for Gm gene products of other persons. Another apparent paradox brought to light by Ig allotype research is the occasional appearance of non-nominal allotypes in contradiction to Mendelian laws. It is proposed that a plausible explanation for these two paradoxes is Ig gene transfer between individuals with viral vectors. Reasons for this proposal and some possible consequences of gene transfer in a polymorphic species are delineated. Immunogenetics and DNA technology in combination provide powerful tools to elucidate the fate of genes. A method allowing the assignment of G1m(a+) and G1m(a-) at the gene level by polymerase chain reaction analysis has recently been established and is briefly described.
Codon↗
Pine genes regulated by the necrotrophic pathogen Fusarium circinatum.
A targeted genomics approach was used to construct a cDNA array of potential pathogen-regulated genes for investigating host-pathogen interactions in pine trees (Pinus species). This array, containing a nonredundant set of 311 cDNAs, was assembled by combining smaller sets of cDNAs generated by differential display or suppression subtraction hybridization using a variety of pathogen treatments and elicitors. The array was probed to identify host genes regulated by Fusarium circinatum, a necrotrophic fungus that incited pitch canker disease on pine stems. A set of 29 cDNAs were induced during the disease state. Notably, cDNAs on the array that were derived from experiments with fusiform rust, incited by Cronartium quercuum f. sp. fusiforme (a biotrophic fungus) were unregulated by Fusarium. the results imply distinct genetic responses in pine to diseases incited by necrotrophs and biotrophs. This cDNA collection expands the genomics toolkit for understanding interactions between conifers and their microbial associates in forest ecosystems.
Base Sequence↗