Showing papers on "Gene expression published in 1988"

SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat.

Abstract: We developed a novel promoter system, designated SR alpha, which is composed of the simian virus 40 (SV40) early promoter and the R segment and part of the U5 sequence (R-U5') of the long terminal repeat of human T-cell leukemia virus type 1. The R-U5' sequence stimulated chloramphenicol acetyltransferase (CAT) gene expression only when placed immediately downstream of the SV40 early promoter in the sense orientation. The SR alpha expression system was 1 or 2 orders of magnitude more active than the SV40 early promoter in a wide variety of cell types, including fibroblasts and lymphoid cells, and was capable of promoting a high level of expression of various lymphokine cDNAs. These features of the SR alpha promoter were incorporated into the pcD-cDNA expression cloning vector originally developed by Okayama and Berg.

Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB.

Abstract: A nuclear protein, CREB, has been isolated from rat brain and shown to stimulate transcription of the cyclic AMP-responsive gene somatostatin as a dimer. Biochemical analysis suggests that dimerization and transcriptional efficacy of CREB protein in vitro are regulated by phosphorylation. These findings demonstrate that cellular signals can modulate gene expression by regulating the covalent modification of pre-existing nuclear factors.

Wound Macrophages Express TGF-α and Other Growth Factors in Vivo: Analysis by mRNA Phenotyping

Abstract: The presence of macrophages is required for the regeneration of many cell types during wound healing. Macrophages have been reported to express a wide range of mitogenic factors and cytokines, but none of these factors has been shown in vivo to sustain all the wound-healing processes. It has been suggested that transforming growth factor-alpha (TGF-alpha) may mediate angiogenesis, epidermal regrowth, and formation of granulation tissue in vivo. Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, TGF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1. The expression of these transcripts was determined by a novel method for RNA analysis in which low numbers of mouse macrophages were isolated from wound cylinders, their RNA was purified and reverse-transcribed, and the complementary DNA was amplified in a polymerase chain reaction primed with growth factor sequence-specific primers. This single-cell RNA phenotyping procedure is rapid and has the potential for quantification, and mRNA transcripts from a single cell or a few cells can be unambiguously demonstrated, with the simultaneous analysis of several mRNA species. Macrophages from wounds expressed TGF-alpha antigen, and wound fluids contained TGF-alpha. Elicited macrophages in culture also expressed TGF-alpha transcripts and polypeptide in a time-dependent manner after stimulation with modified low-density lipoproteins and lipopolysaccharide endotoxin, which are characteristic of the activators found in injured tissues.

A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors.

Abstract: The amyloid proteins isolated from neuritic plaques and the cerebrovasculature of Alzheimer's disease are self-aggregating moieties termed A4 protein1 and βprotein2,3, respectively. A putative A4 amyloid precursor (herein termed A4695) has been characterized by analysis of a human brain complementary DNA4. We report here the sequence of a closely related amyloid cDNA, A4751,distinguished from A4695 by the presence of a 168 base-pair (bp) sequence which adds 57 amino acids to, and removes one residue from, the predicted A4 695protein. The peptide predicted from this insert is very similar to the Kunitz family of serine proteinase inhibitors. The two A4-specific messenger RNAs are differentially expressed: in a limited survey, A4751 mRNA appears to be ubiquitous, whereas A4695 mRNA has a restricted pattern of expression which includes cells from neuronal tissue. These data may have significant implications for understanding amyloid deposition in Alzheimer's disease.

Abnormalities in Structure and Expression of the Human Retinoblastoma Gene in SCLC

Abstract: Small cell lung cancer (SCLC) has been associated with loss of heterozygosity at several distinct genetic loci including chromosomes 3p, 13q, and 17p. To determine whether the retinoblastoma gene (Rb) localized at 13q14, might be the target of recessive mutations in lung cancer, eight primary SCLC tumors and 50 cell lines representing all major histologic types of lung cancer were examined with the Rb complementary DNA probe. Structural abnormalities within the Rb gene were observed in 1/8 (13%) primary SCLC tumors, 4/22 (18%) SCLC lines, and 1/4 (25%) pulmonary carcinoid lines (comparable to the 20 to 40% observed in retinoblastoma), but were not detected in other major types of lung cancer. Rb messenger RNA expression was absent in 60% of the SCLC lines and 75% of pulmonary carcinoid lines, including all samples with DNA abnormalities. In contrast, Rb transcripts were found in 90% of non-SCLC lung cancer lines and in normal human lung. The finding of abnormalities of the Rb gene in SCLC and pulmonary carcinoids (both neuroendocrine tumors) suggests that this gene may be involved in the pathogenesis of a common adult malignancy.

Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells

Abstract: Mutational inactivation of the retinoblastoma susceptibility (RB) gene has been proposed as a crucial step in the formation of retinoblastoma and other types of human cancer. This hypothesis was tested by introducing, via retroviral-mediated gene transfer, a cloned RB gene into retinoblastoma or osteosarcoma cells that had inactivated endogenous RB genes. Expression of the exogenous RB gene affected cell morphology, growth rate, soft agar colony formation, and tumorigenicity in nude mice. This demonstration of suppression of the neoplastic phenotype by a single gene provides direct evidence for an essential role of the RB gene in tumorigenesis.

Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression.

Abstract: Mature virions of herpes simplex virus type 1 contain an activating factor that primes transcription from the five virally encoded immediate early (IE) genes. This activator is specified by a 65-kD polypeptide termed VP16. The action of VP16 is mediated through cis-regulatory elements located in regions adjacent to each IE gene. Although VP16 is normally introduced into cells by infecting virions, its trans-activating function can also be observed by cotransfecting cells with a plasmid that encodes VP16 along with a reporter gene driven by IE cis-regulatory sequences. We have used such an assay to examine the function of mutant forms of VP16. Our results provide tentative identification of two domains of VP16 that are crucial to its role in the induction of IE gene expression. One domain is located within the carboxy-terminal 78 amino acids of VP16 and is characterized by its acidity. Another domain, located in a more amino-terminal region of the protein, appears to tailor the specificity of VP16 for IE genes. According to the results presented in this and the accompanying paper, we predict that VP16 achieves IE gene specificity via protein: protein, rather than protein: DNA, interaction.

Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes

Abstract: Regulation of expression of specific genes by antisense RNA is a naturally occurring mechanism in bacteria1,2, although gene regulation by this mechanism has not yet been observed in higher eukaryotes. However, antisense RNA has been shown to reduce expression of specific genes when injected into frog oocytes3 and Drosophila embryos4. Inhibition of expression of artificially introduced genes has been demonstrated by transient expression of antisense RNA constructs in mammalian cells5,6, and plant protoplasts7, and by stable expression in transgenic plants8. Here, we report a striking inhibition of expression of the endogenous, developmentally regulated gene for polygalacturonase in stably transformed tomato expressing antisense RNA.

Introns increase transcriptional efficiency in transgenic mice.

Abstract: Experiments were designed to test the effect of introns on gene expression in transgenic mice. Four different pairs of gene constructs, which were identical except that one member of each pair lacked all introns, were compared for expression of mRNA after introduction into the murine germ line by microinjection of fertilized eggs. The expression of two chimeric genes, made by fusing either the mouse metallothionein I or the rat elastase 1 promoter/enhancer to the rat growth hormone gene, was assayed in fetal liver or pancreas, respectively, while two natural genes, an oligonucleotide-marked mouse metallothionein I gene and the human beta-globin gene, were assayed in fetal liver. In each case there was, on average, 10- to 100-fold more mRNA produced from the intron-containing construct. Moreover, mRNA levels were proportional to the relative rates of transcription that were measured in isolated nuclei. However, when the expression of the two mouse metallothionein I gene-based constructs was tested after transfection into cultured cells, little difference was observed. These observations suggest that introns play a role in facilitating transcription of microinjected genes and that this effect may be manifest only on genes exposed to developmental influences.

Abscisic acid and water-stress induce the expression of a novel rice gene.

Abstract: We have identified a novel rice gene, called RAB 21, which is induced when plants are subject to water-stress. This gene encodes a basic, glycine-rich protein (mol. wt 16,529) which has a duplicated domain structure. Immunoblots probed with antibodies raised against beta-galactosidase/RAB 21 fusion protein detect RAB 21 protein only in cytosolic cell fractions. RAB 21 mRNA and protein accumulate in rice embryos, leaves, roots and callus-derived suspension cells upon treatment with NaCl (200 mM) and/or the plant hormone abscisic acid (10 microM ABA). The effects of NaCl and ABA are not cumulative, suggesting that these two inducers share a common response pathway. Induction of RAB 21 mRNA accumulation by ABA is rapid (less than 15 min in suspension cells) and does not require protein synthesis, indicating that preformed nuclear and/or cytosolic factors mediate the response to this hormone. We have characterized the RAB 21 gene by determining the complete nucleotide sequence of a nearly full-length cDNA and corresponding genomic copy, and by mapping the start site of its major transcript. The proximal promoter region contains various GC-rich repeats.

Developmental Expression of PDGF, TGF-α , and TGF-β Genes in Preimplantation Mouse Embryos

Abstract: Control of growth and differentiation during mammalian embryogenesis may be regulated by growth factors from embryonic or maternal sources. With the use of single-cell messenger RNA phenotyping, the simultaneous expression of growth factor transcripts in single or small numbers of preimplantation mouse embryos was examined. Transcripts for platelet-derived growth factor A chain (PDGF-A), transforming growth factor (TGF)-alpha, and TGF-beta 1, but not for four other growth factors, were found in whole blastocysts. TGF-alpha, TGF-beta 1, and PDGF antigens were detected in blastocysts by immunocytochemistry. Both PDGF-A and TGF-alpha were detected as maternal transcripts in the unfertilized ovulated oocyte, and again in blastocysts. TGF-beta 1 transcripts appeared only after fertilization. The expression of a subset of growth factors in mouse blastocysts suggests a role for these factors in the growth and differentiation of early mammalian embryos.

Inactivation of the retinoblastoma susceptibility gene in human breast cancers.

Abstract: Mutational inactivation of the retinoblastoma susceptibility (RB) gene, a recessive cancer gene, has been implicated in the genesis of retinoblastoma and certain other human neoplasms. This gene is now shown to be inactivated in two of nine human breast cancer cell lines examined. The RB gene of one cell line had a homozygous internal duplication of a 5-kilobase region containing exons 5 and 6. The RB messenger RNA transcript was correspondingly lengthened, and its translation was probably terminated prematurely due to a shifted reading frame. The other cell line had a homozygous deletion of the RB gene that removed the entire gene beyond exon 2. The RB gene product, pp110RB, was not detectable in either cell line by immuno-precipitation with specific antibodies. These findings are significant in relation to proposed genetic mechanisms of breast cancer formation.

Complexity of the early genetic response to growth factors in mouse fibroblasts.

Abstract: Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.

Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes.

Abstract: Freshly isolated adult rat hepatocytes exhibit a flat, extended morphology when cultured on dried rat tail collagen in the presence of growth factors; they actively synthesize DNA and express high levels of cytoskeletal mRNAs and proteins (actin, tubulin, cytokeratins, vinculin, alpha-actinin, and desmoplakin), while exhibiting low levels of liver-specific mRNAs (albumin, alpha 1-inhibitor III, and alpha 1-antitrypsin) and limited synthesis and secretion of albumin. Hepatocytes cultured on hydrated gel matrix from the Engelbreth-Holm-Swarm (EHS) mouse tumor form small spherical aggregates and exhibit low DNA, cytoskeletal mRNA, and protein synthesis, while at the same time exhibiting elevated liver-specific mRNAs and albumin production; these cells, therefore, more nearly conform to the program of gene expression seen within the normal animal. Hepatocytes on hydrated rat tail collagen resemble those on dry collagen when cultured at low density, but at high density they form compact trabecular aggregates, synthesize negligible amounts of DNA, and maintain a pattern of gene expression resembling that of hepatocytes seeded on the EHS matrix. If cell morphology is compact, as on EHS or on hydrated rat tail collagen when densely populated, DNA synthesis and expression of cytoskeletal genes are low, while liver-specific mRNAs are abundant. When cells are extended the opposite is the case. Without the growth supplement DNA synthesis is low throughout but gene expression is little affected. These studies point to the importance of cell-cell and cell-matrix interactions in determining the differentiated phenotype of hepatocytes, and they reveal an inverse relationship between cytoskeletal and liver-specific protein expression.

Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements.

Abstract: Human AP-2 is a sequence-specific DNA-binding protein that interacts with inducible viral and cellular enhancer elements to stimulate transcription of selected genes. Here, we report the isolation and characterization of a human cDNA clone containing the entire protein-coding region of AP-2. The deduced primary amino acid sequence of AP-2 does not contain a domain resembling any previously identified DNA binding motif. However, an interesting feature of the AP-2 protein is a clustered arrangement of proline and glutamine residues that have been found recently within the activation domains of other transcription factors. Expression of the AP-2 clone in bacteria yields a protein that binds to DNA and activates transcription in vitro in a comparable manner to native human AP-2. Transfection of cDNA clones into Drosophila cells indicates that the AP-2 gene product can also activate gene expression in vivo in a DNA template-dependent manner. Expression of endogenous AP-2 is repressed in a hepatoma cell line and stimulated following retinoic-acid-induced differentiation of a human teratocarcinoma cell line. This indicates that AP-2 may be a transcription factor involved in the control of developmentally regulated gene expression.

A human T cell-specific molecule is a member of a new gene family.

Abstract: We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.

Glucocorticoids selectively inhibit the transcription of the interleukin 1 beta gene and decrease the stability of interleukin 1 beta mRNA

Abstract: Transcription of the interleukin 1 beta (IL-1 beta) gene was studied by mRNA hybridization with a cDNA probe in the human promonocytic cell line U-937. Phorbol ester and lipopolysaccharide increased the steady-state level of IL-1 beta mRNA. Glucocorticoids markedly decreased IL-1 beta mRNA levels by two mechanisms. Transcription of the IL-1 gene was inhibited, as shown by in vitro transcription assays with nuclei isolated from glucocorticoid-treated cells. Moreover, kinetic analyses and pulse-labeling of mRNAs showed that glucocorticoids selectively decrease the stability of IL-1 beta mRNA, without affecting the stability of beta-actin and FOS mRNAs. Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects.