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PubMed1998

Extreme position dependence of a canonical hormone response element.

Hormone response elements (HREs) are considered enhancers, activating transcription in a relatively position- and orientation-independent fashion. Upon binding to an HRE, steroid receptors presumably contact coactivators and/or proteins associated with the transcription initiation complex. As a receptor target site is moved further from a fixed position such as the TATA box, not only will the spatial separation of the receptor with respect to its interaction partners change, so will the orientation due to the rotation of the DNA helix. Additional constraints may be imposed by the assembly of DNA into chromatin. Therefore, we have endeavored to test rigorously the assertion that HRE action is position independent. We have constructed a series of 42 chloramphenicol acetyltransferase expression vectors that contain a single progesterone/glucocorticoid receptor-binding site separated from a TATA box by 4 to 286 bp. The enhancer activity of the HRE was assessed after transient transfection of progesterone receptor-expressing fibroblasts. We find that the position of the HRE has a dramatic influence on induction by progestins. When closely juxtaposed to the TATA box, the HRE was unable to support a hormone response, perhaps due to direct steric hindrance with the transcription initiation complex. Full activity was gained by moving the HRE 10 bp further from the TATA sequence. As the HRE was moved incrementally further, activity remained near maximal over the next 26 bp. HRE activity then declined over the subsequent 26 bp and remained low for another 2.5 helical turns. Surprisingly, a narrow window of HRE activity occurred at an HRE-TATA box separation of 90-100 bp. Little or no hormone-induced transcriptional activity was observed when the HRE was positioned further from the TATA box. The addition of a second HRE or a basal (nuclear factor-1) element failed to relieve this constraint. A similar series of experiments was carried out in a mammary carcinoma cell line that expressed high levels of both glucocorticoid and progestin receptors. Data in these cells indicate that glucocorticoids and progestins supported a similar HRE position-activity profile, but this pattern of HRE activity was quite distinct from that seen in fibroblasts. This may be indicative of cell type-specific interactions between steroid receptors and adapter/coactivator proteins or cell type-specific activities such as acetylases or deacetylases participating in the steroid response.

Base Sequence↗
PubMed1995

Promoter recognition in archaea is mediated by transcription factors: identification of transcription factor aTFB from Methanococcus thermolithotrophicus as archaeal TATA-binding protein.

At least two transcription factors, aTFB and aTFA, are required for accurate and faithful in vitro transcription of homologous templates in cell-free extracts from the methanogenic Archaeon Methanococcus thermolithotrophicus. We have recently shown that the function of aTFB can be replaced by eucaryal TATA-binding proteins. Here we demonstrate using template commitment experiments that promoter recognition in an Archaeon is mediated by transcription factors. The archaeal TATA box was identified as recognition site for binding of aTFB by gel shift analyses. aTFB binds also to the TATA box of adenovirus 2 major late promoter suggesting homology of eucaryal and archaeal TATA boxes. Our analyses provide evidence for a common molecular mechanism of transcription initiation by eucaryal RNA polymerases and archaeal RNA polymerase. They indicate also an evolutionary homology for aTFB and TBP.

Adenoviridae↗
PubMed1997

Characterization of the transcriptional regulator YY1. The bipartite transactivation domain is independent of interaction with the TATA box-binding protein, transcription factor IIB, TAFII55, or cAMP-responsive element-binding protein (CPB)-binding protein.

YY1 is a multifunctional transcription factor implicated in both positive and negative regulation of gene expression as well as in initiation of transcription. We show that YY1 is ubiquitously expressed in growing, differentiated, and growth-arrested cells. The protein is phosphorylated and has a half-life of 3.5 h. To define functional domains, we have generated a large panel of YY1 mutant proteins. These were used to define precisely the DNA-binding domain, the region responsible for nuclear localization, and the transactivation domain. The two acidic domains at the N terminus each provide about half of the transcriptional activating activity. Furthermore, the spacer region between the Gly/Ala-rich and zinc finger domains has accessory function in transactivation. YY1 has been shown previously to bind to TAFII55, TATA box-binding protein, transcription factor IIB, and p300. In addition, we identified cAMP-responsive element-binding protein (CBP)-binding protein as a YY1 binding partner. Surprisingly, these proteins did not bind to the domains involved in transactivation, but rather to the zinc finger and Gly/Ala-rich domains of YY1. Thus, these proteins do not explain the transcriptional activating activity of YY1, but rather may be involved in repression or in initiation.

3T3 Cells↗
PubMed1997

Identification of the human alpha6 integrin gene promoter.

The alpha6 integrin subunit couples with either the beta1 or the beta4 subunit to form a laminin receptor. alpha6 expression is cell-type-specific and generally is present at high levels in epithelial and endothelial cells. To study its gene regulation, we isolated a genomic clone containing the human alpha6 integrin gene promoter. It includes 3 kb of the upstream flanking region, the first exon (385 bp), and 9 kb of the first intron. The alpha6 promoter directs transcription initiation from a primary site 202 nucleotides from the translation initiation codon. Unlike most other integrin gene promoters, the alpha6 promoter has a TATA box (GATAAA), which is located 22 nucleotides upstream from the primary transcription initiation site. A 190-bp region upstream from the TATA box is highly rich (78%) in C and G nucleotides and contains several Sp1 and AP2 binding sequences. However, full promoter activity (in the presence of the SV40 enhancer) requires only 78 bp of this C/G-rich sequence upstream from the TATA box. Slightly upstream from the C/G-rich region are a steroid receptor binding homolog and an epithelial-cell-specific E-pal sequence. Another possible epithelial cell-specific binding sequence (Ker1) is found immediately downstream from the TATA box. Cell-type-specific activities of the promoter paralleled the alpha6 mRNA levels in four tested cell lines. In the presence of the SV40 enhancer, alpha6 promoter activity increased approximately four-fold in primary keratinocytes and in HT1080 fibrosarcoma cells and 30-fold in T47D breast carcinoma cells, but remained undetectable in K562 leukemia cells. Genomic analysis that compared alpha6-expressing with non-alpha6-expressing cells suggested that DNA methylation is not involved in the silencing of the alpha6 gene in alpha6-negative cells. DNase I footprint analysis confirmed the binding of Sp1 and AP2 to their cognate sequences. A nuclear extract of high-alpha6-expressing HBL-100 cells also produced significant binding to these sites, suggesting that the two transcription factors are probably involved in the positive regulation of the alpha6 promoter.

Amino Acid Sequence↗
PubMed1997

Transcription of the Acanthamoeba TATA-binding protein gene. A single transcription factor acts both as an activator and a repressor.

Transcription of the Acanthamoeba TATA-binding protein (TBP) gene is regulated by TBP promoter-binding factor (TPBF), a previously described transactivator that binds as a tetramer to the TBP Promoter Element (TPE) and stimulates transcription up to 10-fold in vitro. Here we report that TPBF also functions as a transcription repressor by binding to a negative cis-element, located between the TATA box and the transcription initiation site. The negative element, referred to as the nTPE, is structurally similar to the TPE, and its disruption increases the transcription potency of the TBP promoter. TPBF binds to the nTPE, as demonstrated by mobility shift assays. However, the binding affinity of TPBF for the nTPE is about 10-fold lower than for the TPE. When placed upstream of the TATA box, the nTPE has very little effect on transcription. However, it inhibits transcription when placed at several positions downstream of the TATA box. Mechanistic studies with the TBP promoter suggest that binding of TPBF to the nTPE not only prevents TBP from binding to the TATA box but also displaces bound TBP, thereby inhibiting further assembly of the preinitiation complex. These results suggest a mechanism in which the cellular TPBF concentration controls the level of TBP gene transcription and show that a single factor can be stimulatory, inhibitory, or neutral depending on the sequence and the context of its binding site.

Acanthamoeba↗
PubMed1993

Identification of multiple repeat sequences and transcription-related elements within introns 4, 8 and 9 of human Raf-1.

Oncogenic activation of the human c-raf-1 protein (Raf-1) occurs by truncation of its aminoterminus (regulatory domain) and retention of the carboxyterminus (kinase domain). To gain insight into the structure of human raf-1 genomic DNA corresponding to the regulatory domain of Raf-1, we analyzed the nucleotide sequences of introns 4, 8, and 9. Very high frequencies of the direct and indirect repeats, and A/T nucleotides were observed within introns 4, 8, and 9; and the DNA sequences, 5'-TTAGTCA-3' and 5'-TGATTCA-3', like the DNA recognition site of transcription factor AP-1, were identified within intron 4 of raf-1 in human normal and tumor DNAs. In addition, several transcriptional elements (CAAT box, TATA sequences, eukaryotic transcription initiation sites, and cellular transcriptional elements) were found within introns 4, 8, and 9 of human raf-1. These data suggest: 1) the high frequency of intron-specific repeats is a possible recombinational mechanism for derivation of truncated oncogenic forms of Raf-1; and 2) the multiple transcriptional elements within introns 4, 8, and 9 may have important implications in the regulation of raf-1 transcription and, therefore, its role in cell growth and proliferation.

Base Sequence↗
PubMed1988

Organization and structure of Volvox alpha-tubulin genes.

Southern analysis of Volvox genomic DNA revealed two genes homologous to Chlamydomonas reinhardtii alpha-tubulin cDNA. Restriction fragment length polymorphism analysis indicated that the two genes are not genetically linked. Clones representing one of the alpha-tubulin genes have been isolated from a genomic library of Volvox carteri f. nagariensis. A 3153 bp BamHI fragment containing the entire alpha-tubulin gene (1802 bp) plus 707 bp of the 5'- and 644 bp of the 3'-untranslated regions has been sequenced, revealing the following features: (1) the derived alpha-tubulin primary structure of 451 amino acids is highly conserved, differing in two residues from the alpha 1- and in two additional residues from the alpha 2-tubulin of C. reinhardtii; (2) in comparison to the C. reinhardtii genes, the Volvox alpha-tubulin gene contains a third intron; positions of the other two introns are precisely conserved; (3) codon usages are biased towards G or C, and against A, in the third position; 19 codons are absent from the alpha-tubulin coding sequence, and 5 of these are not used in any of 7 compiled Volvox genes; (4) transcription begins with an A, 30 bp downstream of the putative TATA box; upstream of the TATA box is a 14 bp sequence similar to consensus sequences found in all 4 C. reinhardtii tubulin genes and believed to regulate promoter function.

Amino Acid Sequence↗
PubMed1999

Isolation and Sequence Analysis of the Eel Cytochrome P450 1A1 Gene.

: The putative CYP1A1 gene was isolated from a genomic library of eel (Anguilla japonica) chromosomal DNA. Sequence analysis of 8184 bp showed that the clone obtained was the structural gene of CYP1A1, which consisted of seven exons and six introns in a region approximately 5800 bp in length, as well as a 5' upstream region of about 2300 bp. The initiation codon was found not in the first exon, but in the second exon, as was reported for the CYP1A genes of mammals and fish. The GC box, CAAT box, and TATA box were located proximately to the first exon, at 110, 62, and 38 bp upstream of the transcriptional start site, respectively. We found six core sequences of xenobiotic-responsive elements (XREs) in the 5' upstream region, and also one in the first intron. All the XRE core sequences in the 5' upstream region were unique in their localization in the sense strand. The XRE core sequence in the first intron was in the antisense strand.

Journal Article↗
PubMed1998

A new apparently functional IGVK gene (VkLa) present in some individuals only.

We describe a hitherto unknown functional IGKV gene, VkLa, belonging to the IGKV1 subgroup with exon 2 having only 94% similarity to the closest known IGKV gene, 1-13/1D-13 (L4/L18a). Genomic DNA sequences spanning from 5' of the decanucleotide box to 3' of the heptamer (649 bp) were cloned and sequenced from four individuals. The new gene encodes the conserved amino acids in the exons and contains no apparent defects in known regulatory intron sequences such as pd-box, dc-box, TATA-box, CCCT-elements, splice-sequences, initiation codon, and heptamer sequence. VkLa is therefore potentially functional and, correspondingly, we found transcripts of properly rearranged VkLa with somatical hypermutations. VkLa was found in 12 of 57 (21%) healthy Caucasians by a nested polymerase chain reaction and subsequent sequencing of exon 2. This finding shows that there is more inter-individual variation in the available IGKV gene repertoire than was hitherto assumed. Finally, we describe a minor correction in the IGKV1D-43 (L23) gene sequence.

Amino Acid Sequence↗
PubMed2006

Molecular cloning and characterization of a novel dehydrin gene from Ginkgo biloba.

A full-length cDNA encoding a dehydrin was cloned from the living fossil plant Ginkgo biloba by rapid amplification of cDNA ends (RACE). The cDNA, designated as GbDHN, was 813 bp long containing an open reading frame of 489 bp. The deduced GbDHN protein had 163 amino acid residues, which formed a 17 kDa polypeptide with a predicted isoelectric point (pI) of 5.75. GbDHN had an S-segment and a K-segment, indicative of dehydrins, but no Y-segments. Homology analysis indicated that the S-segment and K-segment of GbDHN shared identity with those of other reported dehydrins, indicating that GbDHN belonged to dehydrin superfamily. Genomic sequence of GbDHN was also cloned using genomic walker technology. By comparing genomic DNA with the cDNA, it was found that there was a 257-bp intron in this gene. Promoter analysis indicated that it contained six CAAT boxes, one TATA box, one ABRE box and one GC-motif in the 5'-flanking region. Southern blot analysis revealed that GbDHN belonged to a single copy gene family. RT-PCR analysis revealed that GbDHN constitutively expressed in stems and roots. The increased expression of GbDHN was detected when G. biloba seedlings were treated with exogenous abscisic acid (ABA), salt stress and drought stress. These results indicate that the GbDHN has the potential to play a role in response to ABA and environmental stresses that can cause plant dehydration.

Amino Acid Sequence↗
PubMed1990

Characterization of a putative promoter region of the human poly(ADP-ribose) polymerase gene: structural similarity to that of the DNA polymerase beta gene.

The 5'-flanking region of the human poly(ADP-ribose) polymerase gene was isolated and characterized. The nucleotide sequence of a part of the poly(ADP-ribose) polymerase gene completely matched that of the cDNA. The transcriptional initiation sites (cap sites) of this gene, located about 166-bp upstream from the translational initiation site, were identified by S1 mapping analysis. Neither CAAT box nor TATA box was found within 500-bp upstream from the cap sites of poly(ADP-ribose) polymerase gene. The 200-bp immediately upstream of the cap site had a high G+C content (76.5%) and contained double repeats of the sequence CCGCCC, putative Sp1 binding sites, and a palindromic structure. The 5'-flanking region of poly(ADP-ribose) polymerase gene also showed promoter activity in chloramphenicol acetyltransferase assay and structural similarity to that of DNA polymerase beta gene.

Base Sequence↗
PubMed1990

Characterization of the gene and an antigenic determinant of equine herpesvirus type-1 glycoprotein 14 with homology to gB-equivalent glycoproteins of other herpesviruses.

The gene encoding glycoprotein 14 (gp14) of equine herpesvirus type 1 was sequenced. Nucleotide sequence analysis revealed a complete transcription unit composed of a CAT box, a TATA box, a ribosome-binding sequence, a polyadenylation signal and an open reading frame (ORF) of 2940 bp transcribed from left to right. The amino acid (aa) sequence deduced from this ORF corresponded to that of a protein with 979 aa and had the characteristic features of membrane gp including a 20-aa signal sequence at the N terminus, a 743-aa surface domain, a 40-aa membrane anchoring region, a 108-aa hydrophilic cytoplasmic domain at the C terminus and eleven potential sites for N-linked glycosylation. An unusual feature of this protein was an exceptionally long (66aa) sequence, with a preponderance of hydrophilic residues, preceding the hydrophobic signal core. An antigenic determinant recognized by an anti-gp14 monoclonal antibody was present in the N terminus of the postulated surface domain. Comparison of gp 14 with the gp of other herpesviruses indicated that gp14 was highly homologous to corresponding gp of pseudorabies (gII), bovine herpesvirus (gI), varicella-zoster virus (gII), as well as of herpes simplex virus, Epstein-Barr virus and human cytomegalovirus (gB).

Amino Acid Sequence↗
PubMed1995

Genomic organization of the human alpha-adducin gene and its alternately spliced isoforms.

The cDNA for the human alpha-adducin gene has been cloned, and different alternately spliced forms have been identified. We report the complete genomic organization of the human alpha-adducin gene and these alternately spliced forms. The human alpha-adducin gene, spanning approximately 85 kb, consists of 16 exons ranging in size from 34 to 1892 bp. One of the spliced forms of the human alpha-adducin gene results from alternate use of the 5' splice donor site for exon 10, while another results in a truncated protein following insertion of 34 bp comprising exon 15, followed by a premature stop codon. This alternate spliced form of alpha-adducin is predicted to result in an altered carboxyl terminus that would eliminate a protein kinase and calmodulin binding site. Seven nucleotide substitutions and 4 insertion/deletions were also identified. The 5' region of the human alpha-adducin gene contains one Sp1 site, two AP2 sites, and two CAAT boxes. No TATA box was apparent, consistent with features of a housekeeping gene. We have mapped another cDNA within the first intron of the human alpha-adducin gene, suggesting overlapping genes in this 4p16.3 genomic region.

Alternative Splicing↗
PubMed2004

Reporter gene stimulation by MIDA1 through its DnaJ homology region.

MIDA1 was reported as a protein that can associate with Id1. Its N-terminus has homology to Z-DNA binding protein, Zuotin, that contains DnaJ motif, considered to interact with Hsp70s, and Id binding domain. In the present study, we found that MIDA1 stimulates the transcription of the co-transfected genes. This stimulation was independent of promoter specificity because it was observed in various transfected genes. MIDA1 enhanced formation of DNA-protein complexes with E-box or TATA box without its direct binding to DNA. Analysis with deletion mutants of MIDA1 showed that the short protein fragment containing DnaJ motif within Zuotin homology region is sufficient for the stimulation of transcription and we demonstrated that MIDA1 associates with Hsp70. These data suggest involvement of MIDA1 in the stimulation of transcription in concert with Hsp70/Hsc70 molecular chaperones, thus providing a link between Hsp70/Hsc70 molecular chaperones and components of the transcriptional machinery.

Amino Acid Motifs↗
PubMed1988

Organization of the gene encoding the insulin-like growth factor binding protein IBP-1.

Human genomic clones encompassing the tissue specific expressed gene IBP-1, an insulin-like growth factor binding protein were isolated and characterized. The gene is organized in four exons and spans 5.9 kb. S1 nuclease analysis determined a single transcription start site. The first exon and 5' flanking region are highly GC rich and located in a CpG island. The CpG island enclose the CAAT box, the TATA box, the transcription start site and a potential SP1 transcription factor binding site. The presumptive promoter region is characteristic for genes expressed in a tissue specific fashion. All signals required for cleavage/polyadenylation are located within exon IV, predicting a mRNA of 1.5 kb which is consistent with the size seen on RNA blots.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and analysis of the 5'-flanking region of the rat PP1 alpha gene.

We have cloned an 8 kbp genomic fragment of 5'-flanking region of the gene encoding the catalytic subunit of rat protein phosphatase 1 alpha. Neither CAAT box nor TATA box was detected but a 300 bp high GC region containing nine Sp1 transcription factor binding sites is present immediately upstream of the translation start site, demonstrating that PP1 alpha is a housekeeping gene. Luciferase reporter assay showed that transcription of PP1 alpha is controlled at the high GC region.

3T3 Cells↗
PubMed2001

Cloning and characterization of the 5'-flanking region of human cytokeratin 19 gene in human cholangiocarcinoma cell line.

BACKGROUND/AIMS: The regulatory mechanism of cell-specific gene expression in cholangiocytes has not been sufficiently evaluated. In this study, we characterized the 5'-flanking region of the cytokeratin 19 gene expressed specifically in cholangiocytes. METHODS: We cloned a 2952-bp fragment in the 5'-flanking region of cytokeratin 19 and evaluated the regulatory mechanism of gene expression in this region by assaying transient expression of reporter gene and DNA footprinting. RESULTS: This segment of the 5'-flanking region of the human cytokeratin 19 gene shows an intense transcriptional activity in the cholangioma cell line KMBC, which was about 10 times its activity in the osteosarcoma cell line Saos-2, which does not express cytokeratin 19. From the results of reporter assays, important transcription regulatory elements are considered to be located in the segments from -2249 to -2050 bp and from -732 bp to the first ATG, and six protein-binding sites were detected in the segment from -732 bp to the first ATG by the DNA footprinting technique. CONCLUSIONS: Sp1 site, CCAAT box, and TATA box were present in the segment from -374 to the first ATG, and they are considered to constitute a cholangiocyte-specific promoter.

Base Sequence↗
PubMed2001

Promoter analysis of the nuclear gene encoding the chloroplast glyceraldehyde-3-phosphate dehydrogenase B subunit of Arabidopsis thaliana.

The promoter of the nuclear gene, GAPB, which encodes the B subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GADPH) of Arabidopsis thaliana, was previously shown to contain four direct repeats (Gap boxes, located between -237 and -181) that were necessary but not sufficient for light-activated gene transcription. To identify additional elements located between the Gap boxes and TATA box, various GAPB promoter fragments driving the beta-glucuronidase (GUS) reporter gene were constructed in transgenic Arabidopsis. We found a 23 bp element (the XXIII element), centered at -119, that is essential for promoter activity. Mutations in the XXIII element abolished transcription of GAPB completely. Furthermore, we have identified three additional elements, PI, Tboxes, and PII that serve as positive modulators in the light-activated transcription of GAPB. Mutations in any of these three elements resulted in the reduction in light inducibility of the GAPB gene. The PI, XXIII, Tboxes and PII sequences are novel cis-acting elements that are not present in the closely related GAPA promoter or other promoters that are similarly regulated by light. In our current study, we found that transgenic Arabidopsis containing a GAPB promoter::GUS construct with all four Gap boxes deleted exhibited significant GUS expression albeit reduced to 42% of the optimal expression level. In contrast, in previous studies on transgenic tobacco, total abolishment of GUS expression was seen when the Gap boxes were deleted. This suggests that different trans-acting factors present in heterologous systems may result in variability of the expression of the transgene.

Agrobacterium tumefaciens↗