Enhancer-like Element Research Articles

The Human Leukocyte Antigen A2 Interferon-stimulated Response Element Consensus Sequence Binds a Nuclear Factor Required for Constitutive Expression

Both constitutive and interferon-inducible enhancer-like elements have been identified previously in the promoter of human leukocyte antigen (HLA) class I genes. One of these sites is termed the interferon-stimulated response element (ISRE). We have tested the function of an ISRE consensus sequence in the human HLA class I gene HLA-A2 and confirmed previous studies that showed that the HLA-A2 ISRE consensus sequence does not mediate a response to interferons. However, deletion of the ISRE consensus sequence caused a several-fold reduction in the constitutive expression of the HLA-A2 gene in K562 and Jurkat cells. Mobility shift assays performed with the HLA-A2 ISRE revealed the presence of a constitutive binding protein (ISRE/CBP). This protein binds specifically to the HLA-A2 ISRE sequence, and binding is not efficiently competed by the ISRE sequences of the HLA-B7 or ISG54 genes. Substitution of the HLA-B7 or ISG54 ISRE sequences for the HLA-A2 ISRE sequence caused a severalfold reduction in the constitutive expression of the HLA-A2 gene. Mass determinations showed the ISRE/CBP to be 105 kDa, different than any previously characterized ISRE binding proteins. We propose that ISRE/CBP is a novel positive transcriptional regulatory factor for the HLA-A2 gene that may contribute to the differential expression of HLA-A versus HLA-B genes.

Purification of a heteromeric CCAAT binding protein from Neurospora crassa.

Expression of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene is controlled by two upstream enhancer-like elements designated URSam alpha and URSam beta. URSam alpha is localized between - 1.3 and - 1.4 kb with respect to the major transcriptional start site. Deletion of a 90 bp sequence containing this element resulted in the loss of approximately 50% of normal glutamate dehydrogenase expression. Gel mobility shift analysis indicated that a nuclear protein from Neurospora binds in a specific manner to sequences within the 90 bp fragment. We have now used a combination of ion-exchange and affinity chromatography to purify this nuclear protein, which we call Am Alpha Binding protein (AAB). The activity was monitored by gel shift analysis. The protein was purified more than 14,000-fold with a yield of approximately 7%. The purified protein appears as a heteromer on denaturing polyacrylamide gel electrophoresis, with only two strong bands visible in silver-stained preparations. One band has an apparent molecular mass of 40 kDa, the other appears as a doublet with an apparent molecular mass of 30 kDa. DNAse I protection analysis indicated a protected region consisting of 30 bp, which contains a CCAAT pentanucleotide motif. Mutagenesis of the CCAAT motif abolished the binding of AAB to the DNA fragment.

Sequence comparison of three mammalian type-X collagen promoters and preliminary functional analysis of the human promoter

The mechanism(s) controlling the specific expression of the type-X collagen (COL10A1)-encoding gene in the growth plate of developing long bones is not known. In preparation for identifying and characterizing the 5'-regulatory sequences and transcription factors which control mammalian Col10al gene expression, we have isolated and sequenced the first exon and 5′ flanking promoter regions of bovine Col10al and human COL10al. Sequence comparisons, including those previously published for mouse Coll0al, highlighted a number of conserved domains within the promoter and upstream elements. Reporter cat gene (encoding chloramphenicol acetyltransferase, CAT) constructs containing 5′-regulatory sequences of human COL10al (hCOL10al) were transfected into primary cultures of foetal bovine growth plate chondrocytes producing COL10A1 and non-producing epiphyseal cartilage chondrocytes. Constructs containing up to 900 bp of promoter sequence exhibited low levels of CAT production in expressing cells and non-expressing cells. Addition of a further 1.5 kb of upstream sequence resulted in a dramatic increase in CAT production in expressing cells only. The results demonstrate the presence of enhancer-like elements between 900 bp and 2.4 kb upsteam of the transcription start point(s) of hCOL10al, which is distinctly different from that reported for the chick.

Transgenic Systems in Studying Myelin Gene Expression

The myelin sheath is produced by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). It is necessary to introduce DNA into these cells to study gene regulation and the functions of myelin proteins. Since these cells are labile to commonly used techniques to introduce DNA, a transgenic system must be used for these studies. Myelin basic protein (MBP) and myelin proteolipid protein (PLP) are both highly abundant in CNS myelin, but are also produced in Schwann cells. Various lengths (6.5 kb to 256 bp) of the promoter region of the (classic) MBP gene directed oligodendrocyte-specific expression of the reporter gene in transgenic mice, but no expression was seen in Schwann cells. Promoter regions of the PLP gene (4.2-2.4 kb) seem to contain all the information required for correct spatiotemporal expression, but the level of expression was low. The first intron of the PLP gene is a candidate for the location of the enhancer-like element. Studies on MBP mutant mice carrying wild-type MBP gene or cDNA clearly demonstrated that one function of MBP is to make myelin lamellae compact by fusing the cytoplasmic surfaces of oligodendrocytes into the form of major dense lines. However, functional analysis of PLP gene products using a similar strategy produced confusing results. The wild-type PLP gene introduced into jimpy mutant mice (one of the PLP mutants) did not complement the mutant phenotype. Moreover, overexpression of the PLP gene itself (in wild-type background) was shown to produce a phenotype similar to that of the mutants, including arrest in oligodendrocyte maturation and hypomyelination. Thus, PLP gene products play a fundamental role in oligodendrocyte maturation as well as in the stabilization of myelin structure, and its expression must be tightly regulated.

Complex Cell Type-Specific Transcriptional Regulation by the Promoter and an Intron of the Mouse P-Cadherin Gene: (P-cadherin/cell adhesion molecule/transcriptional regulation).

Expression of cell-cell adhesion molecule cadherins is dynamically regulated during development. We describe the structure of the promoter of the mouse P-cadherin gene and its transcriptional activity. The P-cadherin promoter had a tandem of B2 repetitive elements showing structural similarities to the E-cadherin gene promoter. Transcriptional properties of the P-cadherin promoter were surveyed along about 10 kbp of the 5° flanking region by CAT assay in several cell lines expressing or not expressing P-cadherin mRNA. The P-cadherin promoter exhibited complex profiles of transcriptional activity. When the promoter was introduced into P-cadherin positive cell lines, its activity greatly varied with the cell lines. Moreover, this promoter was active even in some of P-cadherin negative cell lines. Analyses of 5° deletions of the promoter suggest that multiple elements are differentially used by different cell lines. We also detected enhancer-like elements in the 2nd intron, which can activate both the P-cadherin and thymidine kinase promoters. The activity of each enhancer varied with cell lines, being independent of endogenous P-cadherin expression. These results suggest that P-cadherin gene transcription is regulated by highly complex combinations of elements in the promoter and the 2nd intron studied here and other regions for the cell-type specific expression.

Putative "MDR enhancer" is located on human chromosome 20 and not linked to the MDR1 gene on chromosome 7.

The physiologic expression of the human multidrug resistance MDR1 gene product P-glycoprotein is controlled in a tissue- and cell-specific manner, but the regulatory mechanisms have not been characterized in great detail. Studies by Kohno et al. [(1990) J Biol Chem 265:19690-19696] suggested that a tissue-specific enhancer element located approximately 10 kb upstream from the major MDR1 transcription start site may act to increase the levels of transcription in cultured adrenal and kidney cells. Using this putative "MDR enhancer" as a probe, we isolated a 14 kb DNA fragment from a genomic DNA library prepared from human fetal liver. The restriction map and partial nucleotide sequence of this DNA fragment were consistent with the previously described data obtained for a similar piece of genomic DNA derived from human placenta by Kohno et al. (ibid.). Pulsed-field gel electrophoresis of large genomic DNA fragments, however, showed that the DNA sequences, including the putative "MDR enhancer," were not linked to the MDR1 gene. Fluorescence in situ hybridization analysis revealed that this enhancer-like element is located on chromosome 20 at band q13.1 and is, therefore, distinct from the MDR locus on chromosome 7, band q21.1. Thus, this putative regulatory element does not modulate the tissue specificity of expression of the MDR1 gene in vivo, but may play a role in the regulation of expression of another, so far unknown gene.

Characterization of the internal promoter of simian foamy viruses

Simian and human foamy viruses (HFV and SFV), genetically related members of the spumavirus genus of retroviruses, have complex genome structures which encode the gag, pol, and env genes for virion proteins as well as additional open reading frames. One of these open reading frames is a viral transactivator, encoded by genes designated taf for SFV and bel-1 for HFV, which augments transcription directed by the long terminal repeat (LTR) through cis-acting targets in the U3 domain of the LTR. Recently, an internal transcriptional promoter has been identified in sequences within the 3' end of the HFV env gene (M. Lochelt, W. Muranyi, and R. M. Flugel, Proc. Natl. Acad. Sci. 90:7317-7321, 1993). We have demonstrated by using transient expression assays in several tissue culture cell lines and by analyzing viral transcripts in infected cells that SFV-1 from a rhesus macaque and SFV-3 from an African green monkey also encode an internal promoter in the env gene. Transcription directed by the internal promoters of SFV-1 and SFV-3 is activated by the taf-1 and taf-3 gene products, respectively, in several cell types. The importance of a TATA box for the SFV-1 internal promoter was established by site-specific mutagenesis, and the 5' ends of transcripts initiating in the internal promoter have been determined. cis-acting sequences in the SFV-1 env gene required for the response to taf-1 are contained within a 121-bp element located 5' to the TATA box in the internal promoter. This taf-1-responsive element in the internal promoter functions in a position- and orientation-independent fashion in a heterologous promoter and thus has the properties of an enhancer which depends on taf-1 activity. Alignments reveal that the SFV-1 internal promoter and the SFV-1 LTR have little sequence relatedness. Cross-transactivation studies show that the transactivators of SFV-1 and HFV function on the internal promoter and LTR of the homologous virus but not on the heterologous virus. In summary, the genomes of simian and human foamy viruses direct viral transcription through both the promoter in the LTR and an internal promoter within the env gene, and each promoter contains unique enhancer-like elements regulated by the viral transactivator.

Functional role of the cis-acting elements in human monocyte chemotactic protein-1 gene in the regulation of its expression by phorbol ester in human glioblastoma cells.

The monocyte chemotactic protein-1 (MCP-1) is a 76 amino acid protein that specifically attracts monocytes. The expression of MCP-1 gene can be induced by lipopolysaccharides (LPS), phorbol esters (TPA) and several cytokines. However, how they regulate MCP-1 gene expression is not known. We tested whether the two putative TPA-responsive elements (TREs) and one kappa B enhancer-like region found in the MCP-1 promoter region, are involved in this regulation of MCP-1 gene expression. The 5' untranslated region of MCP-1 gene was linked to chloramphenicol acetyl transferase (CAT) reporter gene and transfected into human glioblastoma cells in which endogenous MCP gene expression was found to be stimulated by TPA and tumor necrosis factor-alpha (TNF-alpha). The 128 bp 5'-flanking region containing one TRE was adequate for basal promoter activity but the presence of both TREs in the MCP-1 promoter region were needed to give TPA responsive enhancement (2.5 fold) of expression of the marker gene. Mutations in either of the TRE's could abolish the TPA induction of CAT expression. Replacement of the kappa B enhancer-like element with a TRE-like sequence caused a 10-fold enhancement of CAT expression by TPA treatment. Random mutation of kappa B enhancer-like element did not affect CAT expression or its TPA induction. None of the MCP promoter constructs showed significant increase in CAT expression by treatment with tumor necrosis factor-alpha (TNF-alpha). This result suggested that the TNF regulation of MCP-1 gene involves other parts of the gene besides the proximal 5' flanking region.

Functional Analysis of the Promoters of Phenylalanine Ammonia-Lyase Genes in Pea

Pycnospore germination fluid of Mycosphaerella pinodes, a fungus pathogenic on pea, contains both an elicitor and a suppressor of the accumulation of pisatin, a major phytoalexin of pea. Transcription of the genes encoding key enzymes in the biosynthesis of pisatin, namely PAL (a gene encoding phenylalanine ammonia-lyase) and CHS (a gene encoding chalcone synthase), was shown to be activated upon the treatment of pea epicotyl tissues with the fungal elicitor and suppressed upon treatment with the fungal suppressor. To investigate the mechanisms underlying activation and suppression of plant defense genes by signal molecules secreted by a fungal pathogen and other stresses, such as ultraviolet (UV) light, we constructed chimeric genes composed of the 5'-flanking regions of two members of the PSPAL family (the genes encoding phenylalanine ammonia-lyase in Pisum sativum) fused to a bacterial gene for chloramphenicol acetyltransferase. Then, the cis-regulatory elements necessary for elicitor-mediated activation and suppressor-mediated suppression were examined in pea protoplasts. Functional analysis of 5' nested deletions of PSPAL1 and PSPAL2 suggested that an enhancer-like element is located in the TATA-distal region (-2,196 to -406) in PSPAL2. A cis-acting element(s) responsible for elicitor-mediated activation was found in the TATA-proximal region (-340 to -95 in PSPAL1; -406 to -158 in PSPAL2), in which the consensus sequence motifs known as box 1, box 2 and box 4 [Yamada et al. (1992) Plant Cell Physiol. 33: 715, Lois et al. (1989) EMBO J. 8: 1641] were present in close proximity. Furthermore, both promoters were activated by UV light but were partially suppressed in response to the fungal suppressor.

Identification of the promoter region of chicken anemia virus (CAV) containing a novel enhancer-like element.

The single promoter region in the cloned genome [Noteborn et al., J. Virol. 65 (1991) 3131-3139] of chicken anemia virus (CAV) in chicken T-cells was analysed via CAT assays. A unique region containing four or five near-perfect direct repeats (DR) of 21 bp with one 12-bp insert was proven to be the main transcription-activation element, with enhancer-like characteristics. PCR studies revealed that CAV isolates from across the world all contained this promoter sequence. Electrophoretic mobility-shift assays (EMSA) showed that individual DR units, as well as the 12-bp insert, can bind to nuclear factors of chicken T-cells. Competition assays revealed that the DR units bound to factors other than the 12-bp insert. A synthetic oligodeoxyribonucleotide containing an SP1-box (5'-GGGCGG) could compete with factors binding to the 12-bp insert. Purified human SP1 was shown to have very strong affinity for the 12-bp insert.

Multiple promoters of human choline acetyltransferase and aromatic L-amino acid decarboxylase genes

The promoter regions of human choline acetyltransferase (ChAT) and aromatic L-amino acid decarboxylase (AADC) genes have been analyzed by transient transfection assays. AADC gene is transcribed from two alternative non-coding first exons, 1N and 1NN, expressed in pheochomocytoma and hepatoma cells, respectively. 5′ flanking sequences of exon 1 N (from 9000 to 147 bp) display promoter activity in SK-N-BE neuroblastoma cells, but not in MC-I-XC cholinergic neuroepithelioma cells, and in AADC-rich non-neuronal cells. On the contrary, 5′ flanking sequences of exon 1 NN (from 1117 to 119 bp) display high promoter activity in human hepatoma cells HepG2, but not in SK-N-BE cells, suggesting high degrees of specificity of promoters N and NN for AADC-expressing neuronal and non-neuronal cells, respectively. Preliminary evidence suggests that leukemia inhibitory factor suppresses the activity of the neuronal promoter in cultured sympathetic neurons. Two alternative first exons, R and M, have been localized in human ChAT gene, and the corresponding promoters characterized in cholinergic PC12 and NG-108-15 cells, and in non-cholinergic neuro2A cells. Several positively or negatively acting cis elements have been localized in the two promoters, as well as a cAMP-inducible, enhancer-like element in the second intron. Among the various cell lines studied, there was no correlation between promoter activities and the expression of the endogenous ChAT gene, suggesting that the fine-tuning of ChAT gene expression is controlled by silencer elements which remain to be localized.

In vivo functional characterization of an ecdysone response enhancer in the proximal upstream region of the Fbp1 gene of D. melanogaster

Transcription of the D. melanogaster Fat-body-protein-1 (Fbp1) gene is induced by the steroid hormone 20-hydroxyecdysone and is restricted to the fat body tissue at the end of the third larval instar. The location and functional properties of the Fbp1 cis-acting regulatory sequences contained in the region from −1386 to +80 relative to the transcription start were examined by transformation using hybrid constructs with the Adh or lacZ genes as reporters. Regulatory element(s) required for the full level of transcription of the Fbp1 gene were located between positions −1386 and −138. Sequences between −138 and −68 were able to drive transcription from a heterologous minimal promoter in the fat body of late third instar larvae. Remarkably, these sequences also conferred 20-hydroxyecdysone inducibility and behaved as an enhancer-like element. These results provide the first functional characterization, at the level of the whole organism, using a direct in vivo ecdysone induction assay, of a discrete ecdysone response element.

The promoter of the Arabidopsis thaliana plastocyanin gene contains a far upstream enhancer-like element involved in chloroplast-dependent expression.

Plastocyanin is part of the photosynthetic electron transport chain in the chloroplast and is encoded in the nucleus. Expression of the Arabidopsis thaliana plastocyanin gene is organ specific: high mRNA levels are observed in young green parts of the plant. Furthermore, expression is dependent on the presence of light and functional chloroplasts. When grown in the presence of norflurazon under white light conditions, resulting in the photo-oxidative destruction of the chloroplast, plastocyanin mRNA levels are strongly reduced. A -1579 to -9 promoter fragment confers light-regulated and chloroplast-dependent expression to the beta-glucuronidase reporter gene in transgenic tobacco plants. This suggests that regulation takes place at the level of transcription. A plastocyanin promoter deletion series ranging from -1579 to -121 which was also tested in tobacco, revealed the presence of a strong positive regulating element (PRE) in the -1579 to -705 region. Deletion of this part of the promoter resulted in a approximately 100-fold reduction of GUS expression as measured in mature leaves. Surprisingly, this enhancer-like element was capable of stimulating transcription from a position downstream of its reporter. Moreover, it could also activate a truncated CaMV 35S promoter. Deletion of this element coincides with the loss of chloroplast-dependency of reporter gene expression, as judged by norflurazon treatment of transgenic seedlings. So, the activity of the PRE itself might depend on the presence of functional chloroplasts.

Regulation of the human cardiac/slow-twitch troponin C gene by multiple, cooperative, cell-type-specific, and MyoD-responsive elements.

The cardiac troponin C (cTnC) gene produces identical transcripts in slow-twitch skeletal muscle and in heart muscle (R. Gahlmann, R. Wade, P. Gunning, and L. Kedes, J. Mol. Biol. 201:379-391, 1988). A separate gene encodes the fast-twitch skeletal muscle troponin C and is not expressed in heart muscle. We have used transient transfection to characterize the regulatory elements responsible for skeletal and cardiac cell-type-specific expression of the human cTnC (HcTnC) gene. At least four separate elements cooperate to confer tissue-specific expression of this gene in differentiated myotubes; a basal promoter (between -61 and -13) augments transcription 9-fold, upstream major regulatory sequences (between -68 and -142 and between -1319 and -4500) augment transcription as much as 39-fold, and at least two enhancer-like elements in the first intron (between +58 and +1028 and between +1029 and +1523) independently augment transcription 4- to 5-fold. These enhancers in the first intron increase myotube-specific chloramphenicol acetyltransferase activity when linked to their own promoter elements or to the heterologous simian virus 40 promoter, and the effects are multiplicative rather than additive. Each of the major myotube regulatory regions is capable of responding directly or indirectly to the myogenic determination factor, MyoD.A MyoD expression vector in 10T1/2 cells induced constructs carrying either the upstream HcTnC promoter elements or the first intron of the gene 300- to 500-fold. Expression was inhibited by cotransfection with Id, a negative regulator of basic helix-loop-helix transcription factors. The basal promoter contains five tandem TGGGC repeats that interact with Sp1 or an Sp1-like factor in nuclear extracts. Mutational analysis of this element demonstrated that two of the five repeat sequences were sufficient to support basal level muscle cell-specific transcription. Whereas the basal promoter is also critical for expression in cardiac myocytes, the elements upstream of -67 appear to play little or no role. Major augmentation of expression in cardiomyocytes is also provided by sequences in the first intron, but these are upstream (between +58 and +1028). The downstream segment of the first intron has no enhancer activity in cardiomyocytes. A specific DNA-protein complex is formed by this C2 cell enhancer with extracts from C2 cells but not cardiomyocytes. These observations suggest that tissue-specific expression of the HcTnC gene is cooperatively regulated by the complex interactions of multiple regulatory elements and that different elements are used to regulate expression in myogenic and cardiac cells.

Organization of the Murine T-Cell Receptor γ Locus

Murine T cells carry either of two antigen receptors on their surface: αβ or γδ T-cell receptors (TCR). The function of γδ T cells is not well understood. In this population, subsets exist with tissue-specific localization, with invariant variable regions, and with specificity for self antigens. We have analyzed the murine TCH γ locus by field inversion gel electrophoresis and cosmid cloning. The structure of the complete TCR γ locus, the first complete physical map of any mouse TCR locus, is reported here. The locus spans 205 kb and consists of four clusters of genes and gene segments. Each cluster contains one or more variable elements, one joining element, and one constant element. We demonstrate that the Cγ4 gene is distinguished from the other Cγ genes in that it has an additional hinge-encoding exon. New enhancer-like elements are identified in two of the clusters, downstream from the Cγ genes. The most upstream cluster has four V elements and resembles the human TCR γ locus. The four clusters probably arose via duplications.

Cis-active elements controlling lung cell-specific expression of human pulmonary surfactant protein B gene

Human surfactant protein B (SPB) is a 79-amino-acid hydrophobic protein that enhances the surface active properties of pulmonary surfactant. SPB is expressed in nonciliated bronchiolar and alveolar type II cells of the respiratory epithelium, and its expression increases markedly late in gestation. In the present study, a human pulmonary adenocarcinoma cell line, H441, was used in both functional and biochemical assays to identify DNA sequences controlling lung cell-specific expression of the SPB gene. DNase I hypersensitive studies demonstrated two distinct regions of lung cell-specific hypersensitivity located proximal to the SPB promoter and within the eighth intron of the gene. To functionally define these DNA sequences, a series of plasmid vectors were constructed in which segments of the human SPB gene and 5'-flanking sequence were linked to a CAT reporter gene and assayed for expression in lung and nonlung cell lines. Whereas far upstream and intronic sequences did not contain enhancer-like elements, a 259-base pair DNA segment (base pair -218 to +41) was sufficient to support lung cell-specific expression. DNase I footprinting demonstrated that this pulmonary epithelial cell-specific promoter fragment contained five nuclear protein-binding sites, two of which bound lung cell-specific nuclear protein complexes. These results suggest that the pulmonary epithelial cell-specific expression of SPB is determined, in part, by both ubiquitous and cell type-specific protein-DNA interactions within the proximal promoter region.

Stable expression of antibiotic resistance genes using a promoter fragment of the U1 snRNA gene

As U1 snRNA is produced in all mammalian cell types, antibiotic resistance genes driven by this promoter would be ideally suited as genetic selection markers. However, although the U1 snRNA gene is transcribed by RNA polymerase II, its native product is not a messenger RNA, but a splicing cofactor. To test whether this promoter could nevertheless produce a functional mRNA, sensitive reporter genes expressing resistance to the antibiotics hygromycin-B and bleomycin were constructed with either the U1 snRNA promoter or the SV40 early promoter. Resistant cell lines could only be obtained with constructs equipped with a functional polyadenylation signal. With the U1 snRNA promoter about three times fewer colonies were obtained than with the SV40 early promoter. Another potential advantage of the U1 snRNA promoter is that, in contrast to the promoters commonly used to express genetic selection markers, the enhancer-like element contained in the U1 snRNA promoter had only a minimal stimulative effect, only detectable with the most sensitive methods, on an adjacent mRNA-producing gene. The U1 snRNA promoter was also capable of expressing bleomycin resistance in the context of a self-inactivating retrovirus vector, whereby it was discovered that the mouse 3T3 cells used in this experiment were 10 times more sensitive to bleomycin than human or hamster cell lines.

Expression and control of PDGF stimulatory loops in the developing placenta

Summary In order to explain the high proliferative potential of human placentalcytotrophoblast cells, we have examined the possible involvement of platelet-derived growth factor (PDGF) and its receptors; using in vivo and primary cell culture approaches. Extravillous, term cytotrophoblast cells were found to express the PDGF-B gene and furthermore, primary cultures of cytotrophoblast cells purified from the decidua basalis produced significant amounts of PDGF-B protein. A significant proportion of these cells also expressed the PDGF α- and β-receptors, although expression of these two receptors appeared to be mutually exclusive. One subpopulation expressed α-type receptors at the cell surface, whereas a quite distinct subpopulation expressed β-type receptors, which could only be detected intracellularly. The intracellular PDGF β-receptors immuno-precipitated from these cells were physically indistinguishable from PDGF β-receptors from ligand-stimulated fibroblast cells and furthermore, were found to be associated with tyrosine-phosphorylated substrates which are characteristic of PDGF-activated PDGF β-receptors. This data suggests that PDGF β-receptors, but not α-receptors are constitutively activated by endogenously produced PDGF-B protein in these cytotrophoblast cells. It is our proposal, therefore, that the proliferation of these cells involves a PDGF autostimulatory loop, which is limited at the receptor expression level. In order to characterize the molecular mechanisms which control the component genes of such an autostimulatory loop, we have identified an unusual, highly promoter-specific enhancer-like element in the 1 st intron of the PDGF-B gene.

Transcriptional regulation of the murine k-FGF gene in embryonic cell lines

Previous studies have shown that embryonal carcinoma (EC) cells express the fibroblast growth factor k-FGF; however, there is a large decrease in the expression of this gene when EC cells differentiate. In addition, it has been shown that differentiation of mouse F9 EC cells reduces the expression of a reporter gene under the control of both the putative human k-FGF promoter and an enhancer-like element that is located in the third exon of the k-FGF gene. Given the low degree of sequence similarity between the human k-FGF gene and the murine k-FGF gene upstream of the transcription start site, it was unclear whether human sequences mimic fully the regulation of the k-FGF gene in mouse cells. To address this question, we have examined the expression of gene constructs containing various regions of the murine k-FGF gene in two mouse EC cell lines and one mouse embryonic stem (ES) cell line. Our results demonstrate that the mouse 5′ flanking region, like the human 5′ flanking region, cannot support expression of the reporter gene. In both EC cell lines and the ES cell line, expression of the reporter gene is elevated 10- to 100-fold by the addition of a 316-bp region taken from the third exon of the murine k-FGF gene. In addition, we provide evidence that octamer binding proteins are involved in the regulation of the k-FGF gene. Last, this study has identified regions upstream of the transcription start site that appear to regulate the expression of the murine k-FGF gene in EC cells and in ES cells.

Multiple ocs-like elements required for efficient transcription of the mannopine synthase gene of T-DNA in maize protoplasts.

Regulatory elements controlling transcriptional activity of the mannopine synthase 2' promoter (mas 2') were defined by analysis of deletion mutants in transient expression assays in maize protoplasts. Deletion of the region between -305 and -290 containing sequence similarity to the octopine synthase (ocs) promoter element reduced activity by 67% compared to wild type activity. Less than 1% of the activity remained in 5' deletions downstream of -153. Inclusion of various heterologous enhancer-like sequences immediately upstream of position -325 increased activity by up to 7.5-fold. Insertion of the -325 to -275 sequence alone, or in combination with heterologous enhancer-like elements, restored activity of some of the 5'-deletion mutants. Restoration of activity was not obtained with mutants deleted past position -127. Our results suggest that a single class of nuclear proteins from maize interact with high affinity at elements designated mas b (-306 to -275; mas 1' element), d (-127 to -108), and e (-82 to -39; mas 2' element) as well as the 20 bp element from the ocs promoter. Although the binding site at mas d only appears to accommodate a single protein, this element has the potential to make a weak, but positive, contribution to the activity of the mas 2' promoter. The binding of nuclear proteins could not be demonstrated at mas a and c, both of which showed limited homology to the ocs element. Mutational evidence suggested that mas a and c may also contribute to mas 2' transcription.