Showing papers on "Regulation of gene expression published in 1986"

Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.

Abstract: We have examined the regulation of somatostatin gene expression by cAMP in PC12 rat pheochromocytoma cells transfected with the rat somatostatin gene. Forskolin at 10 microM caused a 4-fold increase in somatostatin mRNA levels within 4 hr of treatment in stably transfected cells. Chimeric genes containing the somatostatin gene promoter fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase were also induced by cAMP in PC12 cells. To delineate the sequences required for response to cAMP, we constructed a series of promoter deletion mutants. Our studies defined a region between 60 and 29 base pairs upstream from the transcriptional initiation site that conferred cAMP responsiveness when placed adjacent to the simian virus 40 promoter. Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP. cAMP responsiveness was greatly reduced when the somatostatin fusion genes were transfected into the mutant PC12 line A126-1B2, which is deficient in cAMP-dependent protein kinase 2. Our studies indicate that transcriptional regulation of the somatostatin gene by cAMP requires protein kinase 2 activity and may depend upon a highly conserved promoter element.

Role of ion flux in the control of c-fos expression.

Abstract: There has been much interest in the biochemical and biophysical processes that couple extracellular signals to alterations in gene expression. While many early events associated with the treatment of cells with growth factors have been described (for example, ion flux and protein phosphorylation), it has proved difficult to establish biochemical links to gene expression. Recently, the study of such genomic control signals has been facilitated by the demonstration that the c-fos proto-oncogene is rapidly and transiently induced by treatment of several cell types with polypeptide growth factors and other growth modulating substances. In one particular system it has been shown that nerve growth factor (NGF) causes a transient induction of c-fos in the phaeochromocytoma cell line PC12, within 15 min. Furthermore, the magnitude of this induction can be modulated with pharmacological agents such as peripheral-type benzodiazepines (BZDs). Thus, the study of c-fos expression in PC12 cells could yield valuable clues to the coupling mechanisms linking cell surface activation to genomic events. Here we demonstrate that c-fos is induced in PC12 cells either by receptor-ligand interaction or by agents or conditions that effect voltage-dependent calcium channels.

The purification of eukaryotic polypeptides synthesized in Escherichia coli.

Abstract: Over the last 13 years, manipulation of DNA in vitro has developed from the transfer of genetic information between prokaryotic organisms (Cohen et al., 1973) to a technology which facilitates efficient and controlled production of proteins in foreign hosts. A significant feature of these developments is the ability to express eukaryotic genes in prokaryotes such as Escherichia coli (Harris, 1983; Wetzel & Goeddel, 1983). The supply of many eukaryotic polypeptides which have potential clinical or industrial use is often limited by their low natural availability. Gene cloning and expression in E. coli can provide a more abundant source of these polypeptides. The mode of gene expression affects the location of the proteins produced. The proteins may either be located in the cytoplasm of E. coli or secreted through the cell membrane. Eukaryotic genes cloned in frame with synthetic or bacterial nucleic acid sequences can be expressed as hybrid products in the cell cytoplasm. Transcription, from bacterial promoters, and translation, yield fusion proteins which include bacterial or synthetic polypeptide sequences in addition to the eukaryotic polypeptide. An alternative approach which locates proteins in the cytoplasm is direct expression, where bacterial promoters and terminators are used in the transcription of the foreign gene alone. In E. coli an ATG, or occasionally a GTG, sequence must precede the gene coding sequence, for translation initiation. Thus the primary products oftranslation possess an N-terminal methionine residue. E. coli possesses enzymes which catalyse the efficient removal of the methionine residues from natural proteins when required, but these enzymes do not work with the same efficiency on recombinant polypeptides and therefore directly expressed proteins may possess an unnatural N-terminal methionine residue. Finally, gene sequences which include a leader or signal sequence cloned in frame with the eukaryotic genes, when transcribed and translated can direct secretion of the eukaryotic polypeptides through the bacterial cell membrane. From the increasing number of reports of eukaryotic polypeptide synthesis in E. coli it is clear that the mode ofexpression affects not only the efficiency ofproduction, but the nature of the polypeptide product itself. In general, recombinant polypeptides accumulate to higher levels of total cell protein when expressed intracellularly than when secreted, but many of the polypeptide products located in the cytoplasm are insoluble and aggregated. The consequent isolation and purification techniques required are the subject of this Review.

Gene regulation by proteins acting nearby and at a distance

Abstract: Transcription of genes can be controlled by regulatory proteins that bind to sites on the DNA either nearby or at a considerable distance. Recent experiments suggest a unified view of these apparently disparate types of gene regulation.

A cyclic AMP- and phorbol ester-inducible DNA element.

Abstract: Many cellular processes are regulated by hormones and neurotransmitters which interact with cell-surface receptors to produce intracellular second messengers that activate protein kinases. Cyclic (c) AMP is a second messenger whose intracellular level is determined by receptor-mediated activation or inhibition of adenylate cyclase. Phorbol esters directly activate protein kinase C, a Ca2+ and phospholipid-dependent protein kinase and a component of a different second messenger system, the phosphatidylinositol pathway. Proenkephalin messenger RNA levels are regulated in response to cAMP analogues, activators of adenylate cyclase, nicotinic agonists and depolarization, suggesting that expression of the gene encoding proenkephalin is regulated by trans-synaptic events involving cell-surface-receptor activation. Here we report that cAMP analogues and activators of adenylate cyclase regulate a proenkephalin-chloramphenicol acetyl transferase fusion gene when transiently expressed in tissue culture cells. Phorbol ester regulates the fusion gene in a similar fashion, but requires the presence of phosphodiesterase inhibitors for large effects. The DNA sequences required for regulation by both cAMP and phorbol ester map to the same 37-base pair (bp) region located 107-71 bp 5' to the mRNA cap site of the proenkephalin gene. This highly conserved region is composed of three closely related 12-bp sequences and has properties similar to those of previously characterized transcriptional enhancers.

Human growth hormone as a reporter gene in regulation studies employing transient gene expression.

Abstract: The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium.

Regulation of exoprotein gene expression in Staphylococcus aureus by agr

Abstract: Insertion of the erythromycin-resistance transposon Tn551 into the Staphylococcus aureus chromosome at a site which maps between the purB and ilv loci has a pleiotrophic effect on the production of a number of extracellular proteins. Production of alpha, beta and delta hemolysin, toxic shock syndrome toxin (TSST-1) and staphylokinase was depressed about fifty-fold while protein A production was elevated twenty-fold. Hybridization analysis showed that the defect in expression of TSST-1 and alpha hemolysin was at the transcriptional level. Inability of the mutant strain to express either a cloned TSST-1 gene or the chromosomal gene indicates that the transposon has inactivated a trans-active positive control element. This element has been designated agr for accessory gene regulator.

Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells

Abstract: A retrovirus-derived vector called self-inactivating (SIN) vector was designed for the transduction of whole genes into mammalian cells. SIN vectors contain a deletion of 299 base pairs in the 3' long terminal repeat (LTR), which includes sequences encoding the enhancer and promoter functions. When viruses derived from such vectors were used to infect NIH 3T3 cells, the deletion was transferred to the 5' LTR, resulting in the transcriptional inactivation of the provirus in the infected cell. Introduction of a hybrid gene (human metallothionein-promoted c-fos) into cells via a SIN vector was not associated with rearrangements and led to the formation of an authentic mRNA transcript, which in some cases was induced by cadmium. SIN vectors should be particularly useful in gene transfer experiments designed to study the regulated expression of genes in mammalian cells. Absence of enhancer and promoter sequences in both LTRs of the integrated provirus should also minimize the possibility of activating cellular oncogenes and may provide a safer alternative to be used in human gene therapy.

Regulation of insulin-like growth factor I gene expression by growth hormone.

Abstract: Recombinant clones containing exon 3 of the insulin-like growth factor I (IGF-I) gene were isolated from a mouse genomic library. These sequences were used to generate an RNA probe, which was used in a solution hybridization assay to quantitate IGF-I mRNA in various murine tissues as a function of growth hormone status. The liver is the major site of IGF-I synthesis and the level of IGF-I mRNA is regulated about 10-fold by growth hormone in the growth hormone-deficient lit/lit mouse. Nuclear run-on assays were used to show that growth hormone regulation is manifested in part at the transcriptional level. Growth hormone also affects the size distribution of hepatic IGF-I mRNAs. Pancreas showed the highest non-hepatic expression, but every tissue analyzed contained some IGF-I mRNA. Expression was not growth hormone-dependent in most non-hepatic tissues.

Differential expression of myc family genes during murine development

Abstract: The myc family of cellular oncogenes contains three known members. The N-myc and c-myc genes have 5'-noncoding exons, strikingly homologous coding regions, and display similar oncogenic potential in an in vitro transformation assay. The L-myc gene is less well characterized, but shows homology to N-myc and c-myc (ref. 6; also see below). c-myc is expressed in most dividing cells, and deregulated expression of this gene has been implicated in the development of many classes of tumours. In contrast, expression of N-myc has been found only in a restricted set of tumours, most of which show neural characteristics; these include human neuroblastoma, retinoblastoma and small cell lung carcinoma (SCLC). L-myc expression has so far been found only in SCLC. Activated N-myc and L-myc expression has been implicated in oncogenesis; for example, although N-myc expression has been found in all neuroblastomas tested, activated (greatly increased) N-myc expression, resulting from gene amplification, is correlated with progression of the tumour. We now report that high-level expression of N- and L-myc is very restricted with respect to tissue and stage in the developing mouse, while that of c-myc is more generalized. Furthermore, we demonstrate that N-myc is not simply a neuroectoderm-specific gene; both N- and L-myc seem to be involved in the early stages of multiple differentiation pathways. Our findings suggest that differential myc gene expression has a role in mammalian development and that the normal expression patterns of these genes generally predict the types of tumours in which they are expressed or activated.

Recombinant human tumor necrosis factor increases mRNA levels and surface expression of HLA-A,B antigens in vascular endothelial cells and dermal fibroblasts in vitro

Abstract: Recombinant human tumor necrosis factor (TNF), purified to greater than 99% homogeneity, increases surface expression of class I major histocompatibility complex (MHC) antigens to a maximum of 9-fold on cultured human endothelial cells (HEC) and human dermal fibroblasts (HDF). The increase is concentration dependent (peak 20-100 units/ml) and time dependent (nearly maximal by 4 days); expression remains elevated in the continued presence of TNF and requires greater than 7 days to return to basal levels upon TNF withdrawal. The increase in surface expression appears to result from increases in steady-state mRNA levels for the class I antigens, although the increase in mRNA is proportionately greater than for surface expression. No surface expression of or mRNA for class II MHC antigens is detectable in either control or TNF-treated HEC or HDF. These effects are similar to those produced by leukocyte or fibroblast (type I) interferons (IFNs). The protein synthesis inhibitor cycloheximide (CHX), when added coincidentally with type I IFNs, leads to superinduction of mRNA for class I MHC antigens and, unexpectedly, leads to the appearance of mRNA for class II MHC antigens. CHX has no effect by itself upon mRNA levels for class I or class II MHC antigens, nor does it modulate the increases in mRNA produced by immune (type II) IFN. Most interesting, CHX blocks the increase in mRNA for class I MHC antigens induced by TNF. Thus TNF appears to act on MHC gene expression through a newly synthesized protein intermediate. Our results provide direct evidence that TNF can modulate gene expression in normal (untransformed) cell types and contribute to understanding the complex nature of MHC gene regulation. Finally, they suggest that TNF may act in vivo as an immunoregulatory molecule.

Upstream regions of the human cardiac actin gene that modulate its transcription in muscle cells: presence of an evolutionarily conserved repeated motif

Abstract: Transfection into cultured cell lines was used to investigate the transcriptional regulation of the human cardiac actin gene. We first demonstrated that in both human heart and human skeletal muscle, cardiac actin mRNAs initiate at the identical site and contain the same first exon, which is separated from the first coding exon by an intron of 700 base pairs. A region of 485 base pairs upstream from the transcription initiation site of the human cardiac actin gene directs high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated myotubes of the mouse C2C12 muscle cell line, but not in mouse L fibroblast or rat PC-G2 pheochromocytoma cells. Deletion analysis of this region showed that at least two physically separated sequence elements are involved, a distal one starting between -443 and -395 and a proximal one starting between -177 and -118, and suggested that these sequences interact with positively acting transcriptional factors in muscle cells. When these two sequence elements are inserted separately upstream of a heterologous (simian virus 40) promoter, they do not affect transcription but do give a small (four- to fivefold) stimulation when tested together. Overall, these regulatory regions upstream of the cap site of the human cardiac actin gene show remarkably high sequence conservation with the equivalent regions of the mouse and chick genes. Furthermore, there is an evolutionarily conserved repeated motif that may be important in the transcriptional regulation of actin and other contractile protein genes.

Regulation of the assembly and expression of variable-region genes.

Abstract: During the past several years, it has become increasingly evident that the regulation of immunoglobulin (Ig) gene assembly and expression is intrinsically related to the progression of B-cell precursors to the B-cell differentiation stage. The general focus of this review is to describe in detail the mechanism of Ig variable-region gene assembly and to explore the possible regulatory mechanisms that the cell exploits to control these genomic rearrangement events. Because assembly of the variable region of the T-cell antigen-receptor genes appears to be mediated by the same molecular elements, the general principles we describe should apply to both Ig and T cell-receptor variable-region genes. The immune system is capable of responding to an almost infinite number of antigenic challenges by producing a tremendous diversity of antibody specificities. Each antibody molecule consists of heavy (H) and light (L) immunoglobulin polypeptide chains. The carboxy terminus of H and L chains is a region of constant amino acid sequence. The constant region of the H chain is involved in a variety of effector functions, such as Fe-receptor binding and complement fixation. The amino terminus of both these chains contains a region of variable amino acid sequences (designated the variable region) that are usually unique to the chains in a given antibody molecule; the variable regions of a single H and L chain interact to form an antigen-binding pocket. It is now clear that the DNA sequences encoding

Regulation of genome rearrangement events during lymphocyte differentiation.

Abstract: Analyses of A-MuLV transformed cell lines have provided fundamental insights into the molecular mechanisms which control the rearrangement events leading to the expression of specific antigen receptor genes. These studies have clearly indicated that tissue-specific, developmental stage-specific, and allelically excluded assembly of Ig H and L chain and TCR variable region genes are very strictly regulated processes and, furthermore, that this regulation probably is effected at the level of the accessibility of the individual sets of V gene segments to a common recombinase. More preliminary studies have also suggested that accessibility targeting may be involved in the regulation of directed Ig H chain class-switch recombination events. Currently, we do not understand the nature of "accessible" DNA sequences and we have little understanding of the molecular mechanisms by which Ig (and potentially TCR) chains mediate the regulation of specific recombination events by signaling changes in the accessibility of the various loci. However, an ideal model system for the analysis of these questions is currently available in the form of A-MuLV transformed pre-B cell lines which, in a properly regulated fashion, undergo all of the various recombination events associated with the pre-B stage of B cell differentiation.

Diversity of murine gamma genes and expression in fetal and adult T lymphocytes

Abstract: The search for the genes encoding the T-cell receptor alpha and chains revealed a third gene, T gamma (ref. 1), which shares with t T alpha (refs 2-7) and T beta (refs 8-15) genes a number of structure features, including somatic rearrangement during T-cell development. T gamma gene expression appears to be unnecessary in son mature T cells and is at its greatest in fetal thymocytes encouraging speculation that T gamma has a role in T-cell development and may be involved in the recognition of polymorphic major histocompatibility complex (MHC) products during thymic education. One argument against the participation of T gamma in such a process has been its apparently limited diversity, due to the small number of gene segments available for rearrangement. We here describe the identification of additional T gamma V-gene segments and demonstrate that they can be rearranged to previously identified J- and C-gene segments and are expressed in fetal thymocytes. In addition we describe a variety of patterns of T gamma mRNA processing which may be significant for T gamma gene regulation.

High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines.

Abstract: Southern blot-hybridization analysis of DNAs from human tumors demonstrated amplification of the epidermal growth factor (EGF) receptor gene in 10 of 12 squamous cell carcinoma cell lines tested and in none of 18 tumor cell lines of nonsquamous cell carcinomas. The degree of amplification in the squamous cells varied from 2- to 50-fold relative to the epidermal keratinocyte. Hybridization analysis of the RNA showed that the amplification of the EGF receptor gene is accompanied with an increase of the 5.6 kilobases of EGF receptor mRNA. Scatchard plot analysis and sodium dodecyl sulfate-polyacrylamide gel analysis of the EGF receptor revealed that the synthesis of the EGF receptor is also greater in the cells with amplified EGF receptor gene. In contrast, Southern blot analysis of DNAs of primary tumors showed that incidence of amplification of the EGF receptor gene in squamous cells (1 of 6) was almost as frequent as in nonsquamous cells (1 of 4). These results show that amplification of the EGF receptor gene is commonly found in various tumors. In addition, our data suggest that primary squamous cell carcinomas with amplified EGF receptor gene may readily adapt to growth in tissue culture.

The hypogonadal mouse: reproductive functions restored by gene therapy.

Abstract: The hypogonadal (hpg) mouse lacks a complete gonadotropin-releasing hormone (GnRH) gene and consequently cannot reproduce. Introduction of an intact GnRH gene into the genome of these mutant mice resulted in complete reversal of the hypogonadal phenotype. Transgenic hpg/hpg homozygotes of both sexes were capable of mating and producing offspring. Pituitary and serum concentrations of luteinizing hormone, follicle-stimulating hormone, and prolactin were restored to those of normal animals. Immunocytochemistry and in situ hybridization showed that GnRH expression was restored in the appropriate hypothalamic neurons of the transgenic hpg animals, an indication of neural-specific expression of the introduced gene.

A developmentally stable chromatin structure in the human β-globin gene cluster

Abstract: The DNase I-hypersensitive sites in the human embryonic beta-globin gene region have been mapped in erythroid-enriched fractions of disaggregated fetal livers, in adult nucleated red blood cells, and in fetal brain tissue. Our analysis of a region extending 11 kilobases (kb) 5' of the epsilon-globin gene reveals many minor nuclease-hypersensitive sites and one major site located 6.1 kb upstream of the epsilon-globin gene. All of these hypersensitive sites are erythroid-specific, and the major site is stable throughout erythroid development. As assayed by nuclear runoff transcription, little or no epsilon-globin gene expression is detectable in fetal or adult erythroid cells. Thus, the presence of the major hypersensitive site 5' of the epsilon-globin gene in both fetal and adult erythroid cells demonstrates that this site is not specifically correlated with transcription of the gene or with a particular stage of development. Rather, this site may reflect an early event in erythroid differentiation. In addition, DNase I has been used to probe the overall sensitivity of epsilon-globin chromatin in fetal erythroid cells. Our findings indicate that the epsilon-globin gene as well as the other genes in the beta-globin cluster reside within the chromatin domain that is more DNase I-sensitive than "bulk" chromatin.

Regulation of nitrogen fixation genes.

Abstract: INTRODUCTION AND O VERVIEW . . . . . . . . . .. . . . . . . .. . . . .. .. . . . . . 567 MECHAN ISM OF nif REGUL ATION 569 NifA and NtrC Require NtrA for Promoter Activation 569 NifA and NtrC are Structurally and Functionally Related 570 nif and ntr Promoters Have a Characteristic Primary Structure ....... 570 Role of NtrA in nif and ntr Gene Activation ........ ........ 571 Regulation of the glnA-ntrBC Operon 572 Regulation of NifA Synthesis and Activity in K. pneumoniae 574 Regulation of NifA Synthesis in R. meliloti 575 Mechanism of NifA Activation of nif Promoters 576 COMPARI SON OF N ifAlNtrC ACTI VATION WITH OTHER PO SITI VE REGUL ATORY SySTEM S ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580 New Sigma Factors 580 DNA-Binding Activator Proteins 581 Far Upstream Binding Sites 582 Significance of ntrinif Consensus Sequences 584 Homology of NtrC, NifA , and NtrB with Other Regulatory Proteins 584 CON CLUSION S........ . . . . .. ... ........ 585

Discontinuous Transcription and Antigenic Variation in Trypanosomes

Abstract: The main theme of this review is the discontinuous synthesis of mRNAs in trypanosomes. This novel process was discovered in the unicellular eukaryote Trypanosoma brucei, but it is probably a general feature of the order of Kinetoplastida, to which several other major human pathogens belong. Discontinuous RNA synthesis involves a sequence of 35 nucleotides (nt) found at the 5' end of all trypanosome mRNAs analyzed. The 35-nt sequence is encoded in arrays of 1.35-kb repeats that are clustered in the genome. The primary transcript of the 1.35-kb repeat is an RNA of 140 nt that carries the 35-nt sequence at its 5' end. The 35-nt sequence is transferred from the 140-nt precursor to pre-mRNAs made elsewhere in the genome. The process has not yet been reconstructed in vitro, and whether transfer involves priming of pre-mRNA synthesis, RNA-RNA ligation followed by splicing, trans-splicing, or more than one of these mechanisms, is still unknown. Circumstantial evidence makes priming the least likely of these alternatives. Why it is advantageous to trypanosomes to make their mRNAs in such an unusual fashion is unclear. As yet, there is no evidence for discontinuous synthesis of mRNAs in organisms other than kinetoplastid flagellates. The mini-exon sequence was first found in mRNAs for Variant-specific Surface Glycoproteins (VSGs), and the control of the synthesis of these proteins is a second theme of this review. Silent VSG genes may be activated by their duplicative transposition to a telomeric expression site. The transposition process looks like a gene conversion, mediated by short blocks of sequence homology. Activation of the transposed gene is due to its insertion into an active transcription unit, i.e. to promoter addition. Telomeric VSG genes can also be activated without duplication. This can occur by a reciprocal translocation in which a silent telomeric gene exchanges position with a gene residing in an active expression site. A VSG gene may also be activated without detectable translocation, however, by the transcriptional activation of the silent expression site in which it is located. How this occurs is still unknown, because the transcription units are so long that the promoter for pre-mRNA synthesis has not yet been reached by chromosome walking. A simple mechanism in which a mobile promoter moves between telomeres has been rendered unlikely by the demonstration that two telomeric transcription units can be simultaneously active when one of them is interrupted by a large DNA insertion.(ABSTRACT TRUNCATED AT 400 WORDS)