Transcriptional Regulatory Sequences Research Articles

Characterization of the human and murine IL-7 genes.

IL-7 cDNA clones were used to isolate clones from the human IL-7 gene locus. Characterization of the clones revealed that the human IL-7 gene contains six exons, distributed over more than 33-kbp. An 18 amino acid insert found in human IL-7, for which no counterpart has yet been demonstrated in murine IL-7, is exactly encoded by exon 5 of the human gene. Clones were also isolated containing 5' flanking sequences and the first four exons of the murine IL-7 gene. RNase protection studies of murine IL-7 mRNA, as well as the sequences of 5'-terminal murine IL-7 cDNA clones obtained by anchored polymerase chain reaction cloning, indicate that the murine IL-7 gene initiates transcription at multiple sites within a 200-bp region. This region, and the sequence upstream of this region, appears to lack transcriptional regulatory sequences commonly found in eukaryotic promoters, including the TATA and CAAT sequences. However, the region lies within a CpG island, and contains potential recognition sequences for the "helix-loop-helix" class of DNA binding proteins.

Growth factors and oncogenes in human gastrointestinal carcinomas

Multi-autocrine loops of the epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), platelet-derived growth factor (PDGF) and TGF beta system are expressed in human gastrointestinal carcinomas. In esophageal and gastric carcinomas, they evidently play an important role in tumor progression. Gastrin, one of the major gut hormones, may also act as an autocrine growth factor for gastric and colonic carcinomas. The HST1 and INT-2 genes, belonging to the fibroblast growth factor gene family, are coamplified in approximately 50% of primary tumors and in all the metastatic tumors of esophageal carcinoma. TGF alpha and EGF are the ligands of the tumor cells that overexpress EGF receptor in esophageal carcinomas. The synchronous expression of EGF and its receptor, as well as TGF alpha and ras p21, is evidently correlated with the depth of tumor invasion, metastasis and prognosis of gastric carcinomas. Amplification of c-erbB-2 and EGF receptor genes has been observed in many metastatic sites of gastric carcinomas regardless of histological type. In addition to TGF alpha and EGF, TGF beta and PDGF A chain produced by tumor cells may stimulate collagen synthesis not only by fibroblasts but also by tumor cells themselves, resulting in extensive progression and diffuse fibrosis of scirrhous gastric carcinomas. Moreover, TGF alpha or EGF and estrogen may also play a cooperative role in the development of scirrhous gastric carcinoma. In colorectal carcinoma, it has been shown that the accumulation of several alterations in ras genes and p53 genes is most important for the conversion of adenoma to carcinoma. Critical genetic changes, including activation of oncogenes, mutation and deletion of tumor suppressor genes and disturbances in transcriptional regulatory sequences, may bring about aberrant expression of growth factors and their receptors in gastrointestinal carcinomas. The understanding of the significance of EGF-related growth factors in tumor progression provides a framework for a biological approach to the therapy of human gastrointestinal carcinomas. 8-Cl-cAMP, which inhibits expression of oncogenes and TGF alpha, may be useful not only for cancer therapy but also for the study of cell differentiation.

The transcription control region of the rat α-fetoprotein gene. DNA sequence and homology studies

The alpha-fetoprotein (AFP) gene, an important system for studying developmental and tissue-specific gene expression, is regulated mostly through the control of transcription. The promoter and cis-acting DNA elements which regulate the rat gene lie within a 7 kbp region upstream of the cap site. We have determined the sequence of this entire region. It contains several repetitive elements and a species-specific distribution of DNA methylation sites. We aligned our rat AFP sequence with fragmentary mouse and human AFP sequences to define blocks of highly conserved sequence, which we then analyzed for homology to known transcription regulatory sequences. Our analysis demonstrates that the regulatory region of the rat AFP gene is unusually complex.

Growth factors in progression of human esophageal and gastric carcinomas

Human esophageal and gastric carcinomas express multi-autocrine growth factors and hormones including epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and beta, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) and sex hormones. Overexpression of EGF, TGF-alpha and EGF receptor (EGFR) by tumor cells is closely correlated with the tumor invasion and patient prognosis. This is substantiated by the facts that EGF and TGF-alpha act as autocrine growth factors and then induce the expression of mRNAs for multi-growth factors and their receptors (EGF, TGF-alpha, EGFR, ERBB2, PDGF). Moreover, they stimulate the expression of metalloproteinase genes suggesting that EGF and TGF-alpha successively evoke cascade phenomena which are most convenient for tumor progression, invasion and metastasis. On the other hand, multiple oncogene alterations take place in the process of tumor progression. HST-1 and INT-2 genes which is a member of fibroblast growth factor gene family, are amplified in approximately 50% of primary tumors and all the metastatic tumors of esophageal carcinomas. The amplification of ERBB2 gene in metastatic gastric carcinomas is detected more frequently than in primary carcinomas. Overexpression of multi-growth factor-receptor systems might lead to genetical alterations. Scirrhous gastric carcinoma has vast fibrous stroma with rapid and extensive growth and exhibits high malignancy. Its fibrous stroma may account for synchronous overexpression of EGF, TGF-alpha, PDGF, IGF and TGF-beta by tumor cells. Most of well differentiated adenocarcinomas show overexpression of p 185ERBB2 and coexpression of p 185ERBB2, and EGFR evidently correlates with high malignancy. In conclusion, the accumulation and interaction of several growth factors produced by tumor cells are necessary for the progression of human esophageal and gastric carcinomas. They may be attributed to genetic changes including activation of oncogenes, inactivation and deletion of anti-oncogenes and transcriptional regulatory sequences.

A brain-specific transcription activator

We have identified a DNA binding protein, named BETA, that interacts with the same (B) transcriptional regulatory sequence as the known transcription factor NF-κB. BETA is found only in gray matter throughout the brain, and not in a variety of other rat tissues. Two binding sites for BETA are present adjacent to the promoter of the rat proenkephalin gene. Transfection of primary brain cultures that express BETA, with a reporter gene driven by the SV40 promoter linked to BETA DNA binding sites, results in transcriptional activation. We infer that that BETA is a brain-specific transcription activator.

Identification of a functional NF-kappa B binding site in the murine T cell receptor beta 2 locus.

We have identified a sequence in the TCR beta 2 locus that is homologous to the kappa B site in the Ig kappa light chain enhancer. This element, TCR beta-B, is located in the vicinity of previously identified T cell-specific DNase1 hypersensitive sites. Transfection analysis shows that a 60-bp fragment encompassing this site is preferentially active in T cells stimulated with phorbol esters or the HTLV-1 tax gene product compared with a B cell line that constitutively expresses NF-kappa B. Our results provide the first evidence for transcriptional regulatory sequences residing within the J beta 2-C beta 2 intron and suggest the possible involvement of these sequences in modulation of TCR beta gene expression upon cellular activation.

P-element-mediated enhancer detection applied to the study of oogenesis in Drosophila*

We have stained the ovaries of nearly 600 different Drosophila strains carrying single copies of a P-element enhancer detector. This transposon detects neighbouring genomic transcriptional regulatory sequences by means of a beta-galactosidase reporter gene. Numerous strains are stained in specific cells and at specific stages of oogenesis and provide useful ovarian markers for cell types that in some cases have not previously been recognized by morphological criteria. Since recent data have suggested that a substantial number of the regulatory elements detected by enhancer detection control neighbouring genes, we discuss the implications of our results concerning ovarian gene expression patterns in Drosophila. We have also identified a small number of insertion-linked recessive mutants that are sterile or lead to ovarian defects. We observe a strong correlation with specific germ line staining patterns in these strains, suggesting that certain patterns are more likely to be associated with female sterile genes than others. On the basis of our results, we suggest new strategies, which are not primarily based on the generation of mutants, to screen for and isolated female sterile genes.

P-element-mediated enhancer detection: a versatile method to study development in Drosophila.

We generated and characterized greater than 500 Drosophila strains that carry single copies of a novel P-element enhancer detector. In the majority of the strains, the beta-galactosidase reporter gene in the P-transposon responds to nearby transcriptional regulatory sequences in the genome. A remarkable diversity of spatially and temporally regulated staining patterns is observed in embryos carrying different insertions. We selected numerous strains as markers for different embryonic organs, tissues, and cells. Many of these strains should allow the study of complex developmental processes, such as nervous system development, which have not been convenient to analyze previously. Also, we present genetic evidence that some of the detected regulatory elements control nearby Drosophila genes. In light of our results, we discuss the diversity and complexity of cis-acting regulatory elements in the genome and the general applications of the enhancer detector method for the study of Drosophila development.

Structure and Expression of the Murine Slow/Cardiac Troponin C Gene

Cardiac troponin C (cTnC) is the calcium-binding subunit of the myofibrillar thin filament that regulates excitation-contraction coupling in cardiac muscle. We have utilized a novel polymerase chain reaction cloning procedure to isolate cDNA clones encoding murine cTnC. Murine cTnC is a 161-amino acid polypeptide that has been highly conserved during evolution. Southern blot analyses demonstrated that the cTnC gene is a member of a multigene family. Northern blot analyses revealed that the cTnC gene is expressed in murine cardiac tissue and slow skeletal muscle (soleus), but is not expressed in fast skeletal muscle (extensor digitorum longus and anterior tibialis) or in neonatal or adult brain, kidney, lung, liver, or testis. In addition, while the cTnC gene is not expressed in murine C2C12 myoblasts, differentiation of these cells into myotubes was shown to result in a dramatic induction of cTnC gene expression. A full length cTnC genomic clone was isolated from a murine genomic library by hybridization with a cTnC cDNA probe and structurally characterized by DNA sequence, primer extension, and S1 nuclease protection analyses. The cTnC gene is 3.4 kilobase pairs long and is composed of six exons. The introns do not appear to divide the gene into functional domains. Analysis of the 5'-flanking region of the gene revealed the presence of a consensus TATA box 24 base pairs 5' of the transcription start site. Despite the finding that the gene is expressed only in cardiac and slow skeletal muscle, it lacks the previously described CArG and M-CAT transcriptional regulatory sequence motifs that are involved in regulating the expression of a number of other myofibrillar genes.

Expression of mouse protamine 1 genes in transgenic mice.

Mouse protamine genes are expressed exclusively in spermatids. Mouse protamine 1 (mP1) transcriptional regulatory elements can target the expression of either marked mP1 transgenes or mP1 chimeric genes to spermatids in transgenic mice. Sequences between -40 and -465 bp relative to the transcription start site are required for expression in spermatids, whereas sequences 3' of the point of translation initiation are dispensable. mP1 transcriptional regulatory sequences were used to direct the expression of a toxic gene product to spermatids. The phenotypic consequences of toxin expression in spermatids are described.

Human papovavirus BK early gene regulation in nonpermissive cells

The human papovavirus BK latently infects a majority of the population worldwide, and its DNA has been found in human tumor tissue. BKV is known to be highly oncogenic in rodents, and is capable of transforming cells in vitro. Rearrangements in the transcriptional regulatory sequences controlling expression of the transforming early gene, T antigen, are known to affect both the tumorigenic and transforming properties of this virus. Little is known about the mechanism by which this occurs. We have examined several aspects of BKV early promoter/enhancer regulation in cell types which the virus transforms, baby hamster kidney (BHK) and newborn rat kidney (NRK) cells, and compare them to the same processes in monkey kidney CV1 cells. We find that BKV early transcriptional efficiency requires the same enhancer repeat elements in all three cell types, but that requirements for sequences to the early and late side of these repeats vary between these cells. While the BKV T antigen was found to repress early gene expression from the BKV early promoter in CV1 cells, this effect was lower in BHK cells and essentially absent in NRK cells. The impaired autoregulation observed in rodent cells may be the result of inefficient T antigen production in these cells. DNA replication from the BKV origin was not detected in either BHK or NRK cells. Finally, we find no correlation between the efficiency of the BKV early regulatory region in directing gene expression and the ability to transform NRK cells.

Transcriptional Regulatory Sequences of the Housekeeping Gene for Human Triosephosphate Isomerase

To examine the functional organization of the human triosephosphate isomerase (TPI) promoter, deletion, insertion, and linker scanning mutations were introduced into the TPI promoter of hybrid TPI/beta-globin genes. These genes were transiently expressed in mouse L and human HeLa cells, and the effect of each mutation on the frequency and position of transcription initiation was assayed by S1 nuclease transcript mapping. Multiple positive regulatory elements reside between positions -595 and +1 in L cells and -920 and -7 in HeLa cells and coordinately promote maximum hybrid gene transcription. These elements include an array of GC boxes (positions -126 to -48) that variably conform to the consensus Sp1-binding site, and a canonical TATA box (positions -27 to -21) that is essential for detectable levels of transcription. In an additive yet position-dependent fashion, the GC boxes function in cis to the TATA box to control both the frequency and position of transcription initiation. Additional positive elements reside upstream of position -131 and are required for full promoter function. Also, an inhibitory element(s) residing between position -7200 and either -2800 in L cells or -920 in HeLa cells reduces transcription approximately 7-fold relative to the level of transcription achieved with the maximally active promoter.

Molecular cloning and sequencing of the Mtv-1 LTR: Evidence for a LTR sequence alteration

The vertically transmitted Mtv-1 provirus is the primary causative factor of mammary neoplasia in certain C3Hf strains that lack the horizontally transmitted mouse mammary tumor virus (MMTV). The studies here report the molecular cloning of the germ line 4.5 kb Mtv-1 3' EcoRI fragment and sequencing of the 3' Mtv-1 LTR. The Mtv-1 LTR sequence is closely related to the 5' Mtv-11 LTR sequence also reported here, as well as to known Mtv-8 and MMTV LTR sequences in the portion of MMTV and Mtv-8 LTRs previously demonstrated to contain transcriptional regulatory sequences. A 91 bp unique sequence region, Mtv-1 bp 862 to 952, exists in the Mtv-1 LTR, which is upstream of the sequence homology with the MMTV transcriptional regulatory domain. The Mtv-1 unique sequence region is distinct from a 117 bp sequence, bp 862 to 978, in the Mtv-11 LTR sequence as well as reported Mtv-8 and MMTV LTR sequences, and is present in the germ line Mtv-1 5' and 3' LTR-containing restriction fragments. S1 nuclease mapping experiments of C3Hf/Se mammary tumor poly(A) RNA with the cloned Mtv-1 and Mtv-11 LTRs exhibited a specific set of S1 protected fragments demonstrating that Mtv transcripts which accumulate in C3Hf spontaneous mammary tumors are encoded by the Mtv-1 provirus. Mtv-1 LTR sequence transcription

Structural Organization of the Human Mitochondrial Cytochrome c1 Gene

The structural organization of the entire human nuclear encoded gene for mitochondrial cytochrome c1 was determined by analyzing a clone obtained from an EMBL3 genomic DNA library. The gene spans 2.4-kilobase pairs and contains seven exons interrupted by six introns of relatively small sizes. All intron/exon splice junctions follow the GT/AG rule. The 5'-flanking region of the gene lacks typical transcriptional regulatory sequence elements such as TATA and CAAT boxes but contains seven putative GC boxes (Sp1 binding sites) and several sequences that resemble another type of the Sp1 responsive element, the enhancer core consensus sequence, the AP-1 responsive element, and the cAMP- and phorbol ester-inducible element. The region also contains a 15-nucleotide sequence highly homologous to the AP-4 consensus sequence and to those in the 5'-flanking regions of the genes for two enzymes associated with respiratory function, the beta subunit of human ATP synthase and chicken 5-aminolevulinate synthase. The presequence, which is essential for the transport of the cytochrome c1 precursor into mitochondria, is encoded in both the first and second exons, and the nucleotide sequence corresponding to the presequence is separated by the first intron. This is the first example of a leader sequence coding for a presequence clearly separated into two parts by an intron.

Cloning of cDNAs coding for human HMG I and HMG Y proteins: both are capable of binding to the octamer sequence motif

In human B lymphocytes and placenta HMG I and its smaller isoform HMG Y are encoded by two distinct but structurally highly similar mRNAs which arise most likely by alternative splicing of a single primary transcript. Both have been cloned as cDNAs. On Northern blots an abundant mRNA species 2000 nucleotides in length was detected in all cell lines examined. Exclusively in erythroid cells an additional rare 3800 nucleotides long mRNA species was noted. In quiescent cells the mRNA levels of HMG I/Y were not significantly down-regulated. Southern blot analysis indicated that at least four genes are present per haploid human genome. Both proteins when expressed in bacteria bind specifically to A-T rich stretches of DNA suggesting that no posttranslational modifications are necessary for specific DNA binding. Interestingly, HMG I as well as HMG Y are capable of binding to the octamer transcriptional regulatory sequence motif.

Vectors for Drosophila P-element-mediated transformation and tissue culture transfection

We describe nine P-element vectors that can be used to study gene regulation and function in Drosophila. These vectors were designed for use in germline transformation and cell culture transfection assays. One set consists of five P elements that can be used to study transcriptional regulatory sequences. These vectors contain several unique restriction sites for insertion of a foreign promoter upstream from either a cat or lacZ reporter gene. Two of the β-galactosidase-coding vectors also require the insertion of a start codon for translation of the reporter enzyme and thus can be used to study translational regulatory sequences. The second set of P elements consists of four vectors that contain the Drosophila cytoplasmic actin 5C promoter and polyadenylation signals. Upon insertion of a foreign DNA segment, these vectors direct constitutive expression of the encoded RNA and protein.

Lineage-specific regulation of the vasoactive intestinal peptide gene in neuroblastoma cells is conferred by 5.2 kilobases of 5'-flanking sequence.

The expression of a transfected plasmid containing 5.2 kilobases (kb) of 5' regulatory DNA sequence of the human vasoactive intestinal peptide (VIP) gene attached to coding sequences of the reporter gene chloramphenicol acetyltransferase (CAT) was compared with endogenous VIP expression in subclones of the human neuroblastoma cell line SK-N-SH. These subclones vary widely in basal and inducible quantities of VIP and its precursor mRNA and can be interconverted under specified culture conditions. Endogenous VIP immunoreactivity, detectable in all subclones, was lowest in the neuronal subclone SH-SY-5Y, whereas 15- to 25-fold higher levels were observed in the epithelial-appearing SH-EP and intermediate SH-IN subclones. Treatment with 10 nM phorbol 12-myristate 13-acetate (PMA) stimulated VIP peptide levels approximately 5-fold in SH-SY-5Y cells but did not increase appreciably VIP levels in the other subclones. Treatment with 2.5 microM forskolin resulted in less than 50% stimulation of VIP expression in all subclones. Levels of mRNA encoding the VIP precursor generally paralleled these differences in VIP immunoreactivity. In cells transfected with the VIP/CAT fusion gene, CAT activity reflected closely these differences in basal VIP expression and the changes in response to PMA and forskolin. Deletion of 2.7 kb of the most upstream sequences resulted in an 80-90% reduction in basal CAT activity in SH-IN, but not SH-SY-5Y cells, and resulted in an 80% reduction in PMA stimulation in SH-SY-5Y cells. Deletion to within 74 nucleotides of the transcription start site resulted in CAT expression in SH-IN cells that was only 3% of that seen with the full 5.2-kb flanking sequences and further diminished the remaining PMA responsiveness in SH-SY-5Y cells. The data indicate that important cell-type-specific transcription regulatory sequences reside greater than 2.5 kb upstream from the VIP transcription start site.

Interaction of a Hepatoma-Specific Nuclear Factor with Transcription-Regulatory Sequences of the Human α-Fetoprotein and Albumin Genes

DNase I footprinting and gel mobility shift analysis showed that an HuH-7 hepatoma nuclear protein, termed AFP1, binds specifically to an AT-rich sequence, TGATTAATAATTACA, in domain B of the human alpha-fetoprotein enhancer. No such binding activity was found in HeLa cell nuclei. Transient transfection studies showed that a 54-base-pair region corresponding to the AFP1-binding site could stimulate the simian virus 40 early promoter to express a linked chloramphenicol acetyltransferase gene in an orientation-independent and cell-specific manner. The correlation between the binding of AFP1 and the stimulation of chloramphenicol acetyltransferase gene expression strongly suggests that specific interaction of AFP1 with the AT motif is important for cell-specific transcriptional enhancement. Competition gel mobility shift analysis revealed that similar AT-rich sequences with high affinities to AFP1 were also present in the promoters of the alpha-fetoprotein and albumin genes. These results suggest that AFP1 may function as a common regulatory factor in the transcription of the alpha-fetoprotein and albumin genes.

Interaction of a hepatoma-specific nuclear factor with transcription-regulatory sequences of the human alpha-fetoprotein and albumin genes.

DNase I footprinting and gel mobility shift analysis showed that an HuH-7 hepatoma nuclear protein, termed AFP1, binds specifically to an AT-rich sequence, TGATTAATAATTACA, in domain B of the human alpha-fetoprotein enhancer. No such binding activity was found in HeLa cell nuclei. Transient transfection studies showed that a 54-base-pair region corresponding to the AFP1-binding site could stimulate the simian virus 40 early promoter to express a linked chloramphenicol acetyltransferase gene in an orientation-independent and cell-specific manner. The correlation between the binding of AFP1 and the stimulation of chloramphenicol acetyltransferase gene expression strongly suggests that specific interaction of AFP1 with the AT motif is important for cell-specific transcriptional enhancement. Competition gel mobility shift analysis revealed that similar AT-rich sequences with high affinities to AFP1 were also present in the promoters of the alpha-fetoprotein and albumin genes. These results suggest that AFP1 may function as a common regulatory factor in the transcription of the alpha-fetoprotein and albumin genes.

An analysis of the macronuclear actin genes of Oxytricha.

We have cloned and sequenced a 1.6-kb macronuclear molecule encoding actin from the hypotrichous ciliate Oxytricha nova. High-stringency Southern hybridization to native and digested macronuclear DNA shows that there is only one 1.6-kb actin-encoding molecule in O. nova. The 227-nucleotide 5' leader sequence contains AT-rich stretches punctuated by short GC regions. The AT-rich regions contain TATA-like sequences. However, other known eukaryote transcription regulatory sequences were not found. The 249-nucleotide 3' trailer sequence is also AT-rich and does not contain any obvious known eukaryotic mRNA processing signals. Sequence comparison with a closely related species, O. fallax, shows an 87% sequence similarity in the coding regions and an almost total lack of similarity in the noncoding regions of the molecules. However, a few small sequence similarities and motifs appear in the noncoding regions of the actin-encoding molecules of these two species. The actin-encoding molecule of O. nova could encode a polypeptide 374 amino acids long, the same size as some vertebrate cytoplasmic actins. Contrary to a previous report, we show that the actin-encoding molecule of O. fallax also codes for a polypeptide 374 amino acids long.