Regulation Of Multiple Genes Research Articles

Activation of multiple growth regulatory genes following inducible expression of IRF-1 or IRF/RelA fusion proteins.

Interferon Regulatory Factor (IRF)-1 has been characterized as an important growth regulatory and immunomodulatory transcription factor. To further characterize the potential targets of IRF-1 antiproliferative activity, IRF-1 was expressed under the control of the tetracycline-inducible system in murine NIH3T3 cells. Due to their ability to mimic IRF-1 transactivator function, the regulatory potential of IRF-1/RelA and IRF-2/RelA fusion proteins consisting of the DNA binding domains of IRF-1 or IRF-2 fused to the transactivation domain of NF-kappaB RelA(p65) was also examined. Cells inducibly expressing IRF-1 or IRF/RelA in response to doxycycline treatment displayed significantly reduced growth rates compared to control cells, and inhibition of cell growth correlated directly with the level of transgene expression. Interestingly, IRF-1 and IRF/RelA expression also induced a low level of apoptosis, as detected by microscopic analyses. Furthermore, expression of the interferon inducible, double stranded RNA dependent kinase PKR was increased following IRF-1 or IRF/RelA induction. Most strikingly, induction of IRF-1 and IRF/RelA expression resulted in a significant increase in STAT1 (p91) protein and increased ISGF3 DNA binding activity, suggesting that IRF-1 tumor suppressor activity may involve a novel mechanism which activates the JAK-STAT pathway through STAT1. WAF1 levels were also constitutively elevated in IRF-1 and IRF-1/RelA cells. These studies demonstrate that inducible expression of the transactivation function of IRF-1 or IRF/RelA mediates tumor suppressor activity by inducing cell growth arrest, apoptosis and the differential activation of growth regulatory genes such as PKR, STAT1 and WAF1.

Molecular aspects of the epithelial phenotype.

Epithelia can be defined morphologically as tissues that line surfaces, and ultrastructurally with reference to their cells' apico-basal polarity and possession of specific cell-cell junctions. Defining the epithelial phenotype at a molecular level is more problematic--while it is easy to name proteins (e.g. keratins) expressed by a 'typical' epithelium, no known molecules are expressed by every epithelium but by no other tissues. Cells can differentiate to and from the epithelial state as part of normal development, as a response to disease or when manipulated in culture. Many factors (matrix components, adhesion molecules, growth factors, transcription factors) have been identified that can trigger these transitions of phenotype in specific cases, but to date no general master regulators of the epithelial state have been found. The epithelial state may therefore be controlled by multiple regulatory genes so that there is no single molecule responsible for all of the diverse types of epithelium that exist in higher animals.

Role of mga in growth phase regulation of virulence genes of the group A streptococcus.

To determine whether growth phase affects the expression of mga and other virulence-associated genes in the group A streptococcus (GAS), total RNA was isolated from the serotype M6 GAS strain JRS4 at different phases of growth and transcript levels were quantitated by hybridization with radiolabeled DNA probes. Expression of mga (which encodes a multiple gene regulator) and the Mga-regulated genes emm (which encodes M protein) and scpA (which encodes a complement C5a peptidase) was found to be maximal in exponential phase and shut off as the bacteria entered stationary phase, while the housekeeping genes recA and rpsL showed constant transcript levels over the same period of growth. Expression of mga from a foreign phage promoter in a mga-deleted GAS strain (JRS519) altered the wild-type growth phase-dependent transcription profile seen for emm and scpA, as well as for mga. Therefore, the temporal control of mga expression requires its upstream promoter region, and the subsequent growth phase regulation of emm and scpA is Mga dependent. A number of putative virulence genes in JRS4 were shown not to require Mga for their expression, although several exhibited growth phase-dependent regulation that was similar to mga, i.e., slo (which encodes streptolysin O) and plr (encoding the plasmin receptor/glyceraldehyde-3-phosphate dehydrogenase). Still others showed a markedly different pattern of expression (the genes for the superantigen toxins MF and SpeC). These results suggest the existence of complex levels of global regulation sensitive to growth phase that directly control the expression of virulence genes and mga in GAS.

Identification of amdX, a new Cys-2-His-2 (C2H2) zinc-finger gene involved in the regulation of the amdS gene of Aspergillus nidulans.

The acetamidase-encoding amdS gene of Aspergillus nidulans has been shown to be controlled by multiple regulatory genes. A new gene, amdX, involved in amdS regulation was identified during the characterization of a translocation affecting amdS control. The amdX gene is predicted to encode a 1150-amino-acid polypeptide which contains two Cys-2-His-2 (C2H2) zinc finger DNA-binding motifs. Insertional inactivation of amdX and the phenotypes of transformants containing multiple copies of the amdX gene show that it is an activator of amdS expression. A fusion protein containing the AmdX zinc fingers was found to bind to sequences in the 5' region of amdS which overlap binding sites for the CreA and AmdA regulatory proteins. Evidence is presented for AmdX acting at these sites in vivo.

Asbestos induces nuclear factor kappa B (NF-kappa B) DNA-binding activity and NF-kappa B-dependent gene expression in tracheal epithelial cells.

Nuclear factor kappa B (NF-kappa B) is a transcription factor regulating expression of genes intrinsic to inflammation and cell proliferation--features of asbestos-associated diseases. In studies here, crocidolite asbestos caused protracted and dose-responsive increases in proteins binding to nuclear NF-kappa B-binding DNA elements in hamster tracheal epithelial (HTE) cells. This binding was modulated by cellular glutathione levels. Antibodies recognizing p65 and p50 protein members of the NF-kappa B family revealed these proteins in two of the DNA complexes. Transient transfection assays with a construct containing six NF-kappa B-binding DNA consensus sites linked to a luciferase reporter gene indicated that asbestos induced transcriptional activation of NF-kappa B-dependent genes, an observation that was confirmed by northern blot analyses for c-myc mRNA levels in HTE cells. Studies suggest that NF-kappa B induction by asbestos is a key event in regulation of multiple genes involved in the pathogenesis of asbestos-related lung cancers.

Transcriptional regulation of gene expression: mechanisms and pathophysiology.

Transcription factors are key mediators of the genetic programs that underlie human development and physiology. Mutations in genes that encode transcription factors or in DNA sequences to which these factors bind may adversely affect gene expression and result in disease. Mutations in genes encoding transcription factors often have pleiotropic effects because each transcription factor is involved in the regulation of multiple genes. For several transcription factors, germline mutations have been shown to result in malformation syndromes whereas somatic mutations in the same genes contribute to the multistep process of tumorigenesis. The study of transcription factors and their involvement in human disease thus provides insight into the molecular mechanisms underlying human development, physiology, dysmorphology, and oncology.

The daughterless gene product in Drosophila is a nuclear protein that is broadly expressed throughout the organism during development

The daughterless (da) gene in Drosophila functions in the regulation of at least three significant developmental pathways: sex determination, neurogenesis and oogenesis. As a member of the helix-loop-helix (HLH) family of DNA binding proteins, the da gene product appears to act as a transcription factor. Based on the genetic and molecular characterization of da, it has been proposed that the da protein (Da) functions as a generic member of this family, serving throughout development as a necessary binding partner for an assortment of other HLH proteins. As a result of temporally and/or spatially restricted expression, these binding partners would provide some regulatory specificity to the functional transcription complex. In order to participate in this way in the regulation of multiple genes, Da must be expressed in numerous times and places during development. Using anti-Da antibodies, we validate two predictions of this scenario of Da function: (1) Da protein is not only nuclear localized, but also associated with chromosomes in vivo; and (2) Da protein is widely distributed, both spatially and temporally, throughout development. With regard to the essential role of maternal da + in progeny sex determination little, if any, Da protein is synthesized in the maternal germline. This suggests that the female-specific germline function of da + is provided to the zygote as maternally synthesized RNA that becomes translated early in embryogenesis.

Autoregulatory factors and communication in actinomycetes.

The ability to produce a wide variety of secondary metabolites and a mycelial form of growth that develops into spores are two biological aspects characteristic of the gram-positive bacterial genus Streptomyces. Secondary metabolism and cell differentiation are controlled by diffusible low-molecular-weight chemical substances called autoregulators. A-factor, the representative of the autoregulators, triggers streptomycin production and aerial-mycelium formation in Streptomyces griseus. A-factor exerts its regulatory function with the aid of a receptor protein that itself acts as a repressor-type regulator. The A-factor signal via the A-factor-receptor protein is transferred to downstream genes, such as streptomycin-production genes and sporulation genes, through multiple regulatory genes in a complex regulatory cascade. Thus, A-factor can be termed a "microbial hormone." This review deals with the A-factor-regulatory cascade as a model system for other autoregulators. The biosynthesis of A-factor, the structures and characteristics of other autoregulators, and the importance of these autoregulators in the ecosystem are also included.

Affinity-purified CCAAT-box-binding protein (YEBP) functionally regulates expression of a human class II major histocompatibility complex gene and the herpes simplex virus thymidine kinase gene.

Efficient major histocompatibility complex class II gene expression requires conserved protein-binding promoter elements, including X and Y elements. We affinity purified an HLA-DRA Y-element (CCAAT)-binding protein (YEBP) and used it to reconstitute Y-depleted HLA-DRA in vitro transcription. This directly demonstrates a positive functional role for YEBP in HLA-DRA transcription. The ability of YEBP to regulate divergent CCAAT elements was also assessed; YEBP was found to partially activate the thymidine kinase promoter. This functional analysis of YEBP shows that this protein plays an important role in the regulation of multiple genes.

Genetic regulation and photocontrol of anthocyanin accumulation in maize seedlings.

The flavonoid pathway leading to anthocyanin biosynthesis in maize is controlled by multiple regulatory genes and induced by various developmental and environmental factors. We have investigated the effect of the regulatory loci R, B, and Pl on anthocyanin accumulation and on the expression of four genes (C2, A1, Bz1, and Bz2) in the biosynthetic pathway during an inductive light treatment. The results show that light-mediated anthocyanin biosynthesis is regulated solely by R; the contributions of B and Pl are negligible in young seedlings. Induction of the A1 and Bz2 genes by high fluence-rate white light requires the expression of a dominant R allele, whereas accumulation of C2 and Bz1 mRNA occurs with either a dominant or recessive allele at R. A1 and Bz2 mRNA accumulate only in response to high fluence-rate white light, but Bz1 is fully expressed in dim red light. Some C2 mRNA is induced by dim red light, but accumulation is far greater in high fluence-rate white light. Furthermore, expression from both dominant and recessive alleles of the regulatory gene R is enhanced by high fluence-rate white light. Seedlings with a recessive allele at R produce functional chalcone synthase protein (the C2 gene product) but accumulate no anthocyanins, suggesting that, in contrast to the R-mediated coordinate regulation of C2 and Bz1 observed in the aleurone, C2 expression in seedlings is independent of R and appears to be regulated by a different light-sensitive pathway.

Virology, immunology, and clinical course of HIV infection.

Human immunodeficiency virus (HIV), the cause of AIDS, was the second retrovirus to be associated with human diseases. The unique life cycle of retroviruses involves reverse transcription of their RNA to DNA, which is incorporated into their host's genes. HIV, despite its small genome, contains multiple structural and regulatory genes, the latter controlling the rate of viral replication. Experimental vaccines are aimed at inhibiting binding of HIV to host cells. Azidothymidine (AZT) and other nucleoside analogues inhibit the reverse transcriptase enzyme. HIV induces profound and specific immunosuppression by depleting helper lymphocytes that bear the cluster determinant 4 (CD4, formerly T4). The clinical course of HIV infection is mainly determined by the number of CD4 lymphocytes in the blood. A comprehensive, multidisciplinary approach to patient management can reduce the adverse medical, psychosocial, and economic impact of AIDS.

Signal transduction in the phosphate regulon of Escherichia coli.

Anumber of genes in E. coli, including phoA, pstS, phoE, and ugpB, are related to transport and assimilation of phosphate and are under the same physiological and genetic controL. They constitute a single pho regulon and are regulated by multiple regulatory genes that constitute a cascade regulatory network. In this system, the modulator protein PhoR undergoes autophosphorylation in the presence of ATP and acts as a protein kinase for the activator protein PhoB. Phosphorylation of PhoB enhances its binding activity to the pho box and activates transcription from the pho promoters.

Regulation of the expression of some genes for enzymes of glutathione metabolism in hepatotoxicity and hepatocarcinogenesis

The reversible stage of tumor promotion, which follows the stage of initiation and precedes that of progression in multistage carcinogenesis, is a unique example of reversible toxicity in biological systems. In order to study the molecular mechanisms involved in the action of promoting agents during this stage, the regulation of the expression of genes for two enzymes of glutathione metabolism, γ-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P), was studied under several different conditions of promotion during multistage hepatocarcinogenesis in the rat. Promotion by phenobarbital caused an increased expression of both of these genes in altered hepatic focal lesions, although this was somewhat more variable in the case of the GGT gene. C.I. Solvent Yellow 14, an industrial dye, served as an effective promoting agent. Feeding this dye resulted in a dramatic increase in the expression of GST-P, but not that of GGT in altered hepatic foci. Factors in crude, cereal-based diets inhibited the stage of promotion by diethylnitrosamine, but enhanced promotion by phenobarbital in a synergistic manner. In contrast, at least one purified diet had the converse effect during this stage. The mRNA levels of GST-P were uniformly elevated dramatically in reversible nodules and neoplasms of rat liver that had been induced by diethylnitrosamine and phenobarbital promotion. In contrast, the level of GGT mRNA was somewhat variable, with an occasional neoplasm exhibiting almost a background level of expression of this gene. Therefore, the altered regulation of multiple genes in hepatocytes during the stage of promotion can vary with the promoting agent itself; this process may be related to the heterogeneous gene expression seen in hepatic neoplasms. A possible role for specific DNA sequences in the 5′ flanking regions of such genes is considered. In addition, a cDNA clone to the mRNA of human liver GGT was isolated and sequenced. The homology of the coding sequence of the human liver GGT mRNA to that of rat kidney GGT mRNA was striking.

Multiple regulatory genes control expression of a gene family during development of Dictyostelium discoideum.

Mutant strains of Dictyostelium discoideum carrying dis mutations fail to transcribe specifically the family of developmentally regulated discoidin lectin genes during morphogenesis. The phenotypes of these mutants strongly suggested that the mutations reside in regulatory genes. Using these mutant strains, we showed that multiple regulatory genes are required for the expression of the lectin structural genes and that these regulatory genes (the dis+ alleles) act in trans to regulate this gene family. These regulatory genes fall into two complementation groups (disA and disB) and map to linkage groups II and III, respectively. A further regulatory locus was defined by the identification of an unlinked supressor gene, drsA (discoidin restoring), which is epistatic to disB, but not disA, and results in the restoration of lectin expression in cells carrying the disB mutation. Mutant cells carrying the drsA allele express the discoidin lectin gene family during growth and development, in contrast to wild-type cells which express it only during development. Therefore, the suppressor activity of the drsA allele appears to function by making the expression of the discoidin lectins constitutive and no longer strictly developmentally regulated. The data indicate that normal expression of the discoidin lectins is dependent on the sequential action of the disB+, drsA+, and disA+ gene products. Thus, we described an interacting network of regulatory genes which in turn controls the developmental expression of a family of genes during the morphogenesis of D. discoideum.

Multiple regulatory genes control expression of a gene family during development of Dictyostelium discoideum.

Mutant strains of Dictyostelium discoideum carrying dis mutations fail to transcribe specifically the family of developmentally regulated discoidin lectin genes during morphogenesis. The phenotypes of these mutants strongly suggested that the mutations reside in regulatory genes. Using these mutant strains, we showed that multiple regulatory genes are required for the expression of the lectin structural genes and that these regulatory genes (the dis+ alleles) act in trans to regulate this gene family. These regulatory genes fall into two complementation groups (disA and disB) and map to linkage groups II and III, respectively. A further regulatory locus was defined by the identification of an unlinked supressor gene, drsA (discoidin restoring), which is epistatic to disB, but not disA, and results in the restoration of lectin expression in cells carrying the disB mutation. Mutant cells carrying the drsA allele express the discoidin lectin gene family during growth and development, in contrast to wild-type cells which express it only during development. Therefore, the suppressor activity of the drsA allele appears to function by making the expression of the discoidin lectins constitutive and no longer strictly developmentally regulated. The data indicate that normal expression of the discoidin lectins is dependent on the sequential action of the disB+, drsA+, and disA+ gene products. Thus, we described an interacting network of regulatory genes which in turn controls the developmental expression of a family of genes during the morphogenesis of D. discoideum.

Regulation of a Neurospora crassa extracellular RNase by phosphorus, nitrogen, and carbon derepressions.

A new extracellular RNase, designated N4, was detected in culture filtrates from Neurospora crassa and its regulation was studied. Limitation of a nutrient obtainable from RNA alone was not sufficient to cause enzyme derepression. The addition of RNA to the medium had no inductive effect, but the addition of exogenous protein caused enzyme production. With protein in the medium, N4 was derepressible for all three elemental nutrients obtainable from RNA: carbon, nitrogen, and phosphorus. Successful carbon derepression required the addition of a small amount of proteolytic activity to the cultures, as has been reported for the carbon-derepressible proteases of N. crassa. Exogenous protein affected RNase production before translation. Effects of the exogenous protein appeared similar to those previously reported for N. crassa protease induction. N4 was under the control of the nit-2 and nuc-1 gene products. nit-2 and nuc-1 mutants were unable to derepress enzyme synthesis for nitrogen and phosphorus limitation, respectively; however, these mutants responded like wild types to the other two states of derepression. Enzyme synthesis was constitutive in the preg mutant. Results indicate that the transcription of the N4 structural gene responds to multiple regulatory gene products from different regulatory circuits and that external protein affects the synthesis of classes of hydrolases other than proteases.

The Ah Locus, A Multigene Family Necessary for Survival in A Chemically Adverse Environment: Comparison With the Immune System

Publisher Summary This chapter discusses the drug-metabolizing enzyme systems, presents the Ah system, and provides data for multiple Ah-structural gene products. A cytosolic receptor is regarded as the major product of the Ah-regulatory genes. Sucrose-density gradient analysis following dextran–charcoal treatment is among the most reliable methods for characterizing an Ah receptor. The aryl hydrocarbon hydroxylase (AHH) fluorescent assay is simple and extremely sensitive. This assay, using benzo[α]pyrene as the substrate in vitro, is most commonly used as the biochemical marker for the Ah locus in laboratory animal and human studies. When males and females of four inbred strains were housed together and allowed to breed randomly for 46 to 48 months, the AHH inducibility by 3-methylcholanthrene of weanlings between 18 and 22 generations approximated the distribution found normally for out-bred or random-bred mouse strains. These data indicate the involvement of multiple Ah regulatory genes and a “natural selection” tendency of the induction process to “drift” toward lower intensity in populations having heterogeneous genetic input.