Beta Gene Transcription Research Articles

Cloning and chromosome mapping of human retinoid X receptor ?: selective amino acid sequence conservation of a nuclear hormone receptor in mammals

The murine retinoid X receptor beta (mRXR beta) is a nuclear hormone receptor that activates transcription of murine major histocompatibility complex (MHC) class I genes in response to retinoic acid. In this study, the human RXR beta gene was mapped onto the short arm or centromeric region of chromosome 6 (6pter-q13), which also harbors the MHC. Chromosomal localization was performed by Southern hybridization of genomic DNA from human rodent cell hybrids with the mRXR beta gene as a probe. In addition, a full-length cDNA clone encoding a human RXR beta was isolated by nucleic acid screening of a human cDNA library with a fragment of the mRXR beta gene as a probe. Comparison of the nucleotide-coding sequences of the human and the murine RXR beta revealed a predominance of third base substitutions, resulting in selective conservation of the predicted amino acid sequence of the proteins. The overall sequence homology was 97.6% on the amino acid level as opposed to 91.6% on the nucleotide level. In Northern hybridization experiments with the human cDNA as a probe, RXR beta gene transcripts were detected in a variety of human tumor cell lines, regardless of whether these cell lines expressed MHC class I genes.

Rapid inverse changes in alpha 1B- and beta 2-adrenergic receptors and gene transcripts in acutely isolated rat liver cells.

In vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an alpha 1-receptor (alpha 1AR) to a beta 2-receptor (beta 2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time-dependent changes in alpha 1- and beta 2-adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of alpha 1AR and beta 2AR, and the steady state levels of alpha 1BAR and beta 2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in alpha 1AR density, and a 70% decrease in alpha 1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol-induced but not in glucagon- or forskolin-induced cAMP accumulation, no significant change in beta 2AR density, and a twofold increase in beta 2AR mRNA levels. Exposure of cells to cycloheximide, 2 microM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced beta 2AR densities, while the decrease in alpha 1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in alpha 1BAR mRNA and increase in beta 2AR mRNA levels and corresponding inverse changes in the synthesis of alpha 1BAR and beta 2AR which account, at least in part, for the rapid conversion from alpha 1- to beta 2-adrenergic glycogenolysis.

Inhibition of expression of major histocompatibility complex class II molecules in macrophages infected with Leishmania donovani occurs at the level of gene transcription via a cyclic AMP-independent mechanism.

Among the important pleiotropic responses to gamma interferon (IFN-gamma) during the activation of macrophages (M phi) is the increased expression of major histocompatibility complex class II genes. In the present study, infection with Leishmania donovani was shown to inhibit in parallel the induction by IFN-gamma of H-2 A beta gene transcription, class II mRNA accumulation, and H-2 Ad protein expression in cells of the murine macrophage cell line P388D1. Treatment of P388D1 cells with either the adenylate cyclase activator cholera toxin or the protein kinase A activator N6-2'-O-dibutyryl cyclic AMP (dibutyryl cAMP) similarly inhibited the induction by IFN-gamma of class II protein expression, and in parallel with Leishmania infection, cholera toxin inhibited the induction of mRNA for the H-2 A alpha and H-2 A beta proteins. Concentrations of intracellular cAMP were significantly increased in cholera toxin-treated cells but not in leishmania-infected cells. These findings indicate that at least one mechanism by which Leishmania infection attenuates the activation of M phi by IFN-gamma involves selective, transcriptional inhibition of major histocompatibility complex class II genes via a cAMP-independent mechanism.

T-Cell Receptor β-Chain Gene Rearrangement and Expression During Human Thymic Ontogenesis

T-cell receptor (TCR) beta-chain proteins appear early (approximately 15th week of gestation) during human thymic ontogenesis. These beta-chain proteins, which appear before terminal deoxynucleotidyl transferase (TdT), could be an expression of a fully rearranged (V-D-J), incompletely rearranged (D-J), or germline TCR beta-chain gene. The aims of this study, performed from the 15th week onward, were the following: (1) to investigate whether or not TCR beta gene rearranges at an early stage during human thymic ontogenesis; (2) to investigate whether complete presumptive functional (1.3 kb) TCR beta gene transcript is present at these early stages of development, or if incomplete (1 kb) or germ-line (1.1 kb) transcripts are expressed; (3) to examine the phenotype of TCR beta-chain+ cells with two-color fluorescence using monoclonal antibody (MoAb) beta F1 and MoAbs that recognize CD1, CD2, CD3, CD4, CD8, CD5, and CD7 antigens (rabbit anti-calf TdT antiserum was used to detect TdT); and (4) to demonstrate whether or not beta gene N-diversity regions are detectable as early as the 15th week and whether or not N-nucleotide insertions correlate to TdT expression. Fifteen- to 22-week fetal thymuses and pediatric thymuses were investigated. We demonstrated that TCR beta-chain gene rearranged as early as the 15th week in human thymus and that a complete functional TCR beta gene transcript was expressed at these early stages of human development. No other analyses to date have investigated TCR beta gene expression in early human thymus using molecular biology techniques. No significant differences were detectable between phenotypic analysis of fetal and pediatric samples, except for TdT expression, which appeared after the 20th week. Essentially all mCD3+ (OKT3+) cells were beta-chain+ at the different weeks investigated. A significant percentage of CD1+ cells were beta-chain+, and the percentage increased along with the age of development. After the 20th week, we identified three main populations: TdT+, cCD3+, beta F-(early thymic precursors); TdT+, CD1+, beta F1+ (intermediate maturity cortical thymocytes); and TdT-, mCD3+, beta F1++ (mature medullary thymocytes). Given these values, we may consider beta-chain expression an ordered process. beta gene N-nucleotide insertions were correlated to TdT expression, since N-regions increased considerably after the 20th week. A further increase of N-nucleotide insertions was detected from the 22nd week to the 32nd week.

T-cell receptor beta-chain gene rearrangement and expression during human thymic ontogenesis

T-cell receptor (TCR) beta-chain proteins appear early (approximately 15th week of gestation) during human thymic ontogenesis. These beta- chain proteins, which appear before terminal deoxynucleotidyl transferase (TdT), could be an expression of a fully rearranged (V-D- J), incompletely rearranged (D-J), or germline TCR beta-chain gene. The aims of this study, performed from the 15th week onward, were the following: (1) to investigate whether or not TCR beta gene rearranges at an early stage during human thymic ontogenesis; (2) to investigate whether complete presumptive functional (1.3 kb) TCR beta gene transcript is present at these early stages of development, or if incomplete (1 kb) or germ-line (1.1 kb) transcripts are expressed; (3) to examine the phenotype of TCR beta-chain+ cells with two-color fluorescence using monoclonal antibody (MoAb) beta F1 and MoAbs that recognize CD1, CD2, CD3, CD4, CD8, CD5, and CD7 antigens (rabbit anti- calf TdT antiserum was used to detect TdT); and (4) to demonstrate whether or not beta gene N-diversity regions are detectable as early as the 15th week and whether or not N-nucleotide insertions correlate to TdT expression. Fifteen- to 22-week fetal thymuses and pediatric thymuses were investigated. We demonstrated that TCR beta-chain gene rearranged as early as the 15th week in human thymus and that a complete functional TCR beta gene transcript was expressed at these early stages of human development. No other analyses to date have investigated TCR beta gene expression in early human thymus using molecular biology techniques. No significant differences were detectable between phenotypic analysis of fetal and pediatric samples, except for TdT expression, which appeared after the 20th week. Essentially all mCD3+ (OKT3+) cells were beta-chain+ at the different weeks investigated. A significant percentage of CD1+ cells were beta- chain+, and the percentage increased along with the age of development. After the 20th week, we identified three main populations: TdT+, cCD3+, beta F-(early thymic precursors); TdT+, CD1+, beta F1+ (intermediate maturity cortical thymocytes); and TdT-, mCD3+, beta F1++ (mature medullary thymocytes). Given these values, we may consider beta-chain expression an ordered process. beta gene N-nucleotide insertions were correlated to TdT expression, since N-regions increased considerably after the 20th week. A further increase of N-nucleotide insertions was detected from the 22nd week to the 32nd week.

Modulation of Cell Proliferation and Cytokine Production in Acute Myeloblastic Leukemia by Interleukin-1 Receptor Antagonist and Lack of its Expression by Leukemic Cells

Interleukin-1 (IL-1) is spontaneously produced by acute myeloblastic leukemia (AML) cells. IL-1 also induces synthesis of colony-stimulating factors (CSFs) and sustains leukemic growth. An IL-1-specific inhibitor has been recently purified and cloned; this molecule binds to IL-1 receptors but has no IL-1 activity, fulfilling the characteristics of an IL-1 receptor antagonist (IL-1ra). Because high-affinity binding sites for IL-1ra were shown on AML cells by radioligand binding studies, we studied the effect of IL-1ra on the proliferative activity of blast cells isolated from 16 cases of AML. In each case, spontaneous proliferation was inhibited by addition of the IL-1ra in a dose-dependent manner (1 to 100 ng/mL). Culture supernatants of unstimulated leukemic cells contained IL-1 beta and granulocyte-macrophage CSF (GM-CSF), but when incubated with the IL-1ra, a reduction or disappearance of GM-CSF was observed in 8 of 10 cases, whereas spontaneous IL-1 production was reduced in four of seven cases. By Northern hybridization, IL-1 beta gene transcripts were shown in 20 of 23 AML cases, whereas IL-1ra-specific messenger RNA was present in only two of the patients studied. These data show a role for IL-1 in the spontaneous proliferation and cytokine production of AML cells and suggest that an imbalanced synthesis of IL-1 and of its natural receptor antagonist may contribute to the unrestricted growth of AML cells.

Modulation of cell proliferation and cytokine production in acute myeloblastic leukemia by interleukin-1 receptor antagonist and lack of its expression by leukemic cells

Interleukin-1 (IL-1) is spontaneously produced by acute myeloblastic leukemia (AML) cells. IL-1 also induces synthesis of colony-stimulating factors (CSFs) and sustains leukemic growth. An IL-1-specific inhibitor has been recently purified and cloned; this molecule binds to IL-1 receptors but has no IL-1 activity, fulfilling the characteristics of an IL-1 receptor antagonist (IL-1ra). Because high-affinity binding sites for IL-1ra were shown on AML cells by radioligand binding studies, we studied the effect of IL-1ra on the proliferative activity of blast cells isolated from 16 cases of AML. In each case, spontaneous proliferation was inhibited by addition of the IL-1ra in a dose- dependent manner (1 to 100 ng/mL). Culture supernatants of unstimulated leukemic cells contained IL-1 beta and granulocyte-macrophage CSF (GM- CSF), but when incubated with the IL-1ra, a reduction or disappearance of GM-CSF was observed in 8 of 10 cases, whereas spontaneous IL-1 production was reduced in four of seven cases. By Northern hybridization, IL-1 beta gene transcripts were shown in 20 of 23 AML cases, whereas IL-1ra-specific messenger RNA was present in only two of the patients studied. These data show a role for IL-1 in the spontaneous proliferation and cytokine production of AML cells and suggest that an imbalanced synthesis of IL-1 and of its natural receptor antagonist may contribute to the unrestricted growth of AML cells.

Human herpesvirus 6 induces interleukin-1 beta and tumor necrosis factor alpha, but not interleukin-6, in peripheral blood mononuclear cell cultures.

The human herpesvirus 6 (HHV-6) is known to interact intimately with cells of the immune system. Here we report that HHV-6 is a potent inducer of interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) in cultures of peripheral blood mononuclear cells. In contradistinction, HHV-6 has no effect on IL-6 synthesis. Maximal IL-1 beta and TNF-alpha gene transcription, as detected by polymerase chain reaction amplification analysis, is observed at 12 and 6 h postinfection, respectively. Release of IL-1 beta and TNF-alpha into the culture supernatants peaked at 24 h and gradually decreased with time. Heat-inactivated virus was unable to stimulate IL-1 beta and TNF-alpha syntheses, whereas UV-irradiated virus retained the full monokine-inducing potential of the native particle. Preincubation of viral preparation with neutralizing anti-HHV-6 antibody resulted in the abrogation of this cytokine-inducing effect, whereas treatment of cells with phosphonoacetic acid (an inhibitor of viral DNA polymerase activity) had no effect on the ability of the virus to stimulate monokine release. These results indicate that HHV-6 can exert a strong immunomodulatory effect by stimulating the cells of myeloid lineage to produce these cytokines.

Thyrotropin-releasing hormone regulation of thyrotropin .beta.-subunit gene expression involves intracellular calcium and protein kinase C

Our previous studies demonstrated TRH stimulation of TSH beta gene expression in rat pituitary cell cultures and GH3 tumor cells in a transient expression assay. To begin to characterize the gene-proximal elements of the pathways involved in TRH stimulation of TSH beta gene transcription, we examined the effects of factors that increase intracellular calcium concentration, [Ca2+]i, or activate protein kinase C on TSH beta promoter activity in transfected GH3 cells. TPA, a tumor-promoting phorbol ester, stimulated a dose-dependent increase in TSH beta promoter activity at 8 h similar to TRH (2-3-fold). TPA did stimulate protein kinase C activation without [Ca2+] mobilization. The calcium ionophore ionomycin increased cytoplasmic free [Ca2+] by stimulating both calcium influx and release from internal stores without affecting protein kinase C. Ionomycin also stimulated a dose-dependent increase (2-fold) in TSH beta promoter activity at 8 h. However, the voltage-dependent Ca2+ channel agonist Bay K 8644, which increased influx of extracellular calcium, had little or no effect on TSH beta gene expression until 48 h (5-fold). Similar effects on prolactin/mRNA levels were observed in these cells. Effects of these factors were not additive, suggesting a common pathway(s) to stimulate gene expression. Inhibition of intracellular calcium mobilization by treatment with 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) inhibited ionomycin effects on gene expression without affecting phorbol ester activity, and, conversely, inhibition of protein kinase C activity by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) or TPA desensitization blocked TPA effects without affecting ionomycin activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of myosin gene expression in mouse cardiac muscle.

Expression of the two isoforms of cardiac myosin heavy chain (MHC), MHC alpha and MHC beta, in mammals is regulated postnatally by a variety of stimuli, including serum hormone levels. Less is known about the factors that regulate myosin gene expression in rapidly growing cardiac muscle in embryos. Using isoform-specific 35S-labeled cRNA probes corresponding to the two MHC genes and the two myosin alkali light chain (MLC) genes expressed in cardiac muscle, we have investigated the temporal and spatial pattern of expression of these different genes in the developing mouse heart by in situ hybridization. Between 7.5 and 8 d post coitum (p.c.), the newly formed cardiac tube begins to express MHC alpha, MHC beta, MLC1 atrial (MLC1A), and MLC1 ventricular (MLC1V) gene transcripts at high levels throughout the myocardium. As a distinct ventricular chamber forms between 8 and 9 d p.c., MHC beta mRNAs begin to be restricted to ventricular myocytes. This process is complete by 10.5 d p.c. During this time, MHC alpha mRNA levels decrease in ventricular muscle cells but continue to be expressed at high levels in atrial muscle cells. MHC alpha transcripts continue to decrease in ventricular myocytes until 16 d p.c., when they are detectable at low levels, but then increase, and finally replace MHC beta mRNAs in ventricular muscle by 7 d after birth. Like MHC beta, MLC1V transcripts become restricted to ventricular myocytes, but at a slower rate. MLC1V mRNAs continue to be detected at low levels in atrial cells until 15.5 d p.c. MLC1A mRNA levels gradually decrease but are still detectable in ventricular cells until a few days after birth. This dynamic pattern of changes in the myosin phenotype in the prenatal mouse heart suggests that there are different regulatory mechanisms for cell-specific expression of myosin isoforms during cardiac development.

Induction of interleukin-1 ? and ? gene transcription in mouse peritoneal exudate cells after intraperitoneal infection with herpes simplex virus-1

Macrophages have been shown to play a determining role in the immune defense against herpes simplex virus-1 (HIV-1) intraperitoneal infection in the mouse. In the present study, the effect of HSV-1 infection on interleukin-1 alpha and beta gene transcription in peritoneal exudate cells was investigated. HSV-1 infection was found to induce interleukin-1 alpha and beta gene transcription in these cells. Induction of the interleukin-1 beta gene initiated 6 h postinfection (p.i.) and terminated at 48 h p.i. In contrast, interleukin-1 alpha RNA was detectable at high levels at 6h p.i. but not at 24 h p.i. Inactivation of virus prior to infection prevented HSV-1-induced IL-1 gene induction, indicating that only infectious virus is able to stimulate IL-1 gene transcription. These findings are discussed in relation to the role of macrophages in the immunological mechanisms of defense against HSV-1 infection.

Molecular cloning and characterization of human pyruvate dehydrogenase beta subunit gene.

A genomic clone encompassing the entire gene for the human pyruvate dehydrogenase beta subunit (PDH beta) has been isolated by screening a leukocyte genomic library with a nick-translated human foreskin fibroblast PDH beta cDNA probe. The 18-kilobase clone was characterized by restriction enzyme analysis, extensive DNA sequencing, and primer-extension analysis. The PDH beta structural gene is composed of 10 exons and 9 introns. All intron-exon splice junctions follow the GT/AG rule. The Alu family was found in introns 2 and 8. The 5' flanking region of the PDH beta gene contains a "CAAT" consensus promoter sequence but no "TATA" sequence. Primer-extension analysis indicated that the PDH beta gene transcription start site is an adenine residue located 132 bases upstream from the initiation codon in exon 1.

Interactions of Thyrotropin-Releasing Hormone, Phorbol Ester, and Forskolin-Sensitive Regions of the Rat Thyrotropin-β Gene

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Discordant Effects of Aging on Prolactin and Luteinizing Hormone-β Messenger Ribonucleic Acid Levels in the Female Rat*

To examine the molecular genetic basis for the age-related increase in PRL secretion and decrease in LH production in the rat, we measured steady state levels of PRL and LH beta mRNA in pituitary homogenates and cell lysates from monolayer adenohypophyseal cultures. These mRNA levels were compared with the corresponding levels of immunoreactive PRL and LH in sera and culture media. Paired groups (n = 4-10/group) of intact and 4-week ovariectomized mature (6-7 months old) and old (23-25 months old) female Wistar rats were studied. Serum PRL levels were 550% higher in intact old vs. mature rats (P less than 0.001), whereas the corresponding pituitary homogenate levels of PRL mRNA were similar (P greater than 0.4). Medium PRL concentrations were 230% greater (P less than 0.006) whereas cell lysate concentrations of PRL mRNA were unaltered (P greater than 0.2) in monolayer cultures from intact old vs. mature rats. Serum PRL levels were 650% higher (P less than 0.003) and pituitary homogenate PRL mRNA levels were slightly increased (P less than 0.04) in ovariectomized old vs. mature rats. Neither serum LH values (P greater than 0.07) nor pituitary homogenate LH beta mRNA levels (P greater than 0.1) differed in intact old and mature rats, whereas the corresponding medium concentrations of LH were reduced (P less than 0.001). Ovariectomized old vs. mature rats exhibited reductions in serum (P less than 0.02) and medium (P less than 0.001) LH concentrations, as well as in pituitary homogenate (P less than 0.002) and cell lysate (P less than 0.006) LH beta mRNA levels. Thus, these data revealed coordinate decreases with age in LH beta mRNA and LH secretion, particularly in ovariectomized rats, suggesting an age-related alteration at or before LH beta gene transcription. These findings parallel observations on other genes whose products change with age. In contrast, the observation that the increased secretion of PRL in old rats is accompanied by little or no increase in PRL mRNA is novel and suggests that age-related alterations in PRL gene expression proceed through a posttranscriptional mechanism.

Coordinate regulation of transforming growth factor beta gene expression and cell proliferation in hamster lungs undergoing bleomycin-induced pulmonary fibrosis.

The number of mesenchymal cells, as well as their ability to synthesize extracellular matrix (ECM) components, greatly increase in the interstitium of fibrotic lungs. We have previously shown that the transcription of type I procollagen and fibronectin genes in the lungs is preferentially elevated during the early stages of bleomycin-induced pulmonary fibrosis (Raghow, R., S. Lurie, J. M. Seyer, and A. H. Kang. 1985, J. Clin. Invest. 76:1734-1739. Since a cytokine-like transforming growth factor beta (TGF beta) that is capable of enhancing mesenchymal cell proliferation and ECM synthesis could be potentially involved in this process, we investigated the temporal relationship between the regulation of TGF beta gene transcription and cellular proliferation in the bleomycin-treated hamster lungs. We observed a transient 5-7-fold increase in the accumulation of TGF beta transcripts, a concomitant 3-4-fold elevation in the cellular proliferation, and 8-10-fold stimulation of DNA synthesis in these lungs; all three parameters peaked around day 10 after bleomycin administration. Based on these results, we conclude that regulation of TGF beta gene expression may contribute significantly to the early events that lead to bleomycin-induced pulmonary fibrosis.

Binding of thyroid hormone receptors to the rat thyrotropin-beta gene.

Negative regulation of rat TSH beta gene expression by thyroid hormone is mediated largely by decreased transcription of the gene. This is apparently mediated by a cis-acting element which has been localized to a 57-basepair fragment spanning the second transcriptional start site of the rat TSH beta gene. We have investigated whether thyroid hormone receptors bind specifically to DNA sequences in this region of the gene. We compared binding of native T3 receptor to the TSH beta gene sequences and to the rat GH (rGH) gene T3 response element (TRE), and examined the ability of two different forms of in vitro synthesized T3 receptor to bind to the TSH beta gene. The avidin-biotin complex DNA binding assay was used to examine sequence-specific binding of the receptor. [125I]T3-labeled receptor in GH3 cell nuclear extracts bound to a site within the first exon of TSH beta and also to a region immediately upstream of the second transcriptional start site of the gene. In addition, the Hc-erbA beta and r-erbA alpha-1 forms of the T3 receptor each bound to TSH beta and rGH sequences, demonstrating that both alpha- and beta-forms of T3 receptor can bind to TREs exerting either positive or negative transcriptional regulation. Competition experiments showed that both native and in vitro synthesized T3 receptor bound to the first exon of TSH beta with an affinity slightly less than that for the rGH TRE. The two receptor-binding sites of the rTSH beta gene show sequence similarity to adjacent regions of the rGH TRE. These data indicate that negative regulation of rat TSH beta gene transcription may be effected by direct binding of the T3-receptor complex to one or both of the binding sites flanking the second transcriptional start site.

T-cell receptor genes expression in B-cell leukaemias.

Using Northern-blot analysis we have studied the expression of T-cell receptor (TCR) alpha, beta and gamma chain genes in primary cells obtained from 36 leukaemic patients. Fifteen patients had myeloid leukaemias, two had T-cell leukaemias, and 19 leukaemias corresponding to various stages of B-lymphocyte differentiation. We observed that truncated TCR beta mRNAs were produced in B-cells at relatively high levels even in the absence of detectable gene rearrangements. Ti alpha mRNAs of abnormal size were also frequently found. Such transcripts were not detectable in total RNA extracted from leukaemic myeloid cells. Factors allowing Ti alpha and beta gene transcription must be active in leukaemic pre-B and B cells but not in myeloid cells. Neither B-lineage nor myeloid leukaemias expressed TCR gamma gene at detectable levels.

The Transcriptional Response of the Human Chorionic Gonadotropin β-Subunit Gene to cAMP is Cycloheximide Sensitive and is Mediated bycis-Acting Sequences Different from that Found in the α-Subunit Gene

Cyclic AMP stimulates transcription of the genes encoding the alpha- and beta-subunits of human CG (hCG). Although the cis-acting cAMP response element (CRE) of the alpha-subunit gene has been extensively characterized, relatively little is known about the putative response element of the hCG beta gene. Here, we demonstrate that cAMP stimulation of hCG beta gene transcription requires ongoing protein synthesis, whereas cAMP stimulation of alpha-subunit gene transcription does not. These results suggest that cAMP-stimulated transcription of the alpha and hCG beta genes may involve different cis-acting elements and trans-acting factors. Accordingly, we have constructed a series of deletion mutants consisting of fragments of the hCG beta promoter-regulatory region linked to the bacterial chloramphenicol acetyl transferase gene. To potentiate detection of a CRE, we constructed vectors containing the Rous sarcoma virus enhancer to augment transcriptional activity of the hCG beta-promoter. We also used vectors designed to determine whether regions containing a putative CG beta CRE could confer cAMP responsiveness to a heterologous promoter. These constructs were evaluated for activity by transfection into choriocarcinoma (BeWo) cells. Transient expression of these vectors identifies a broad region which renders transcription of the chloramphenicol acetyl transferase gene responsive to cAMP. Surprisingly, division of the hCG beta 5'-flanking and untranslated regions into smaller fragments led to loss of cAMP responsiveness, suggesting that the hCG beta CRE may be comprised of multiple domains dispersed across the proximal 650 base pairs of CG beta 5'-flanking and untranslated sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion of Mls‐reactive T cells in H‐2‐compatible but Mls‐incompatible bone marrow chimeras

The cellular basis of tolerance induction to Mls-encoded antigens in radiation bone marrow (BM) chimeras has been investigated in two H-2-compatible strain combinations of AKR/J (H-2k, Thy-1.1, Mls-Ia) and C3H/He (H-2k, Thy-1.2, Mls-IIa). Sequential appearance of host- and donor-derived T cell subsets and T cell receptor gene messages occurred in the peripheral lymphoid organs of both irradiated AKR/J mice reconstituted with C3H/He BM cells (C3H/He-AKR/J chimera) and irradiated C3H/He mice reconstituted with AKR/J BM cells (AKR/J-C3H/He chimera). A large number of cells expressing T cell receptor gamma genes were detected in spleen on day 21 after reconstitution, while the normal level of alloreactivity was first detected in the spleen on day 56 after reconstitution in correlation with the appearance of appreciable levels of Thy-1 high cells and T cell receptor alpha and beta gene transcripts. T cells bearing V beta 6, that is strongly correlated with reactivity to antigens encoded by the Mls-Ia genetic locus, were virtually abolished in spleen on day 56 in both C3H/He-AKR/J chimera and AKR/J-C3H/He chimera. Furthermore, expression of V beta 3 gene transcripts, that are important for recognizing Mls-IIa, was undetected either in the peripheral lymphoid cells of AKR/J-C3H/He chimera or in those of C3H/He-AKR/J chimera. These results suggested that clonal elimination of self-reactive T cells bearing V beta 3 or V beta 6 was induced by both host-derived radioresistant cells and donor-derived repopulating cells in the thymus of radiation BM chimeras.

Thyroid Hormones Regulate Rat Thyrotropinβ Gene Promoter Activity Expressed in GH3Cells

Thyroid hormones suppress the synthesis of TSH in part by decreasing the rate of alpha and TSH beta gene transcription. Cis-acting DNA sequences present in the rat TSH beta subunit gene that are induced in transcriptional regulation by thyroid hormone have been identified by deletion-mutation and transient expression studies. Plasmid expression vectors were constructed including 2900, 900, 204, 77, 17 base pairs (bp) of 5'-flanking sequence and exon (5'-untranslated sequence, transcriptional start sites) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. The transfected chimaeric plasmids demonstrated expression (with TSH beta DNA sequences in the 5'- to -3'-but not 3'- to -5'-orientation) in both a clonal pituitary cell line, GH3, and primary pituitary cell cultures, both of which are responsive to thyroid hormones. T3 (10(-11) M to 10(-7) M) treatment of transfected cells produced a dose-dependent decrease in CAT expression with a maximal 70% decrease at 10(-8) M. While a decrease in the basal level of expression was noted with progressive removal of both 5'-flanking and intronic sequences adjacent to exon 1, the fold-decrease in response to T3 was equivalent even in the 57 bp construct. In contrast, T3 had no effect on CAT expression directed by the promoter of the herpes simplex virus thymidine kinase gene. Thus, the rat TSH beta gene 5'-flanking region can direct heterologous gene expression in GH3 cells and contains sequences which have properties of a putative cis-active T3 responsive regulatory element(s).2+he