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IHF Institute for Hormone and Fertility Research, University of Hamburg (S.P., A.C.R., C.B.), 22529 Hamburg, Germany; Department of Medicine, University of Hamburg (S.P.), 20251 Hamburg, Germany; and Endokrinologikum Hamburg (H.M.S.), 22767 Hamburg, Germany
Address all correspondence and requests for reprints to: Dr. S. Petersenn, Division of Endocrinology, Medical Center, University of Essen, Hufelandstrasse 55, 45122 Essen, Germany. E-mail: . stephan.petersenn{at}uni-essen.de
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Introduction |
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Materials and Methods |
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FIXRII genomic DNA library (Stratagene, La Jolla, CA) was screened by adapting a PCR-based method (12). Aliquots containing approximately 10,000 plaque-forming units were distributed into each of the 96 wells of a microplate. Pools from each well in a column and from each well in a row and subsequently single wells were screened by PCR using SR5S1 and SR5A1. Positive wells were plated and screened with the digoxigenin-labeled probe. Hybridization and detection were performed using a DIG Luminescent Detection Kit (Roche, Mannheim, Germany) following the manufacturer’s protocol. The genomic inserts were subcloned into the NotI restriction site of the pBluescript SK+ plasmid (Stratagene) by a shotgun cloning approach. Sequences were compared with DNA databases using BLASTN (http://www.ncbi.nlm.nih.gov/BLAST). The nucleotide (nt) sequence data reported in this paper has been submitted to GenBank and assigned the accession number AY081193. Open reading frames were checked by the BESTORF program, and possible splice sites were identified with the RNASPL program (Solovyev, V. V., and A. A. Salamov, BCM Gene Finder, Sanger Center, UK; http://genomic.sanger.ac.uk). Putative transcription factor binding sites were identified using the TRANSFAC database (13), CpG islands were checked at the Webgene homepage (http://www.itba.mi.cnr.it/webgene) (14).
Determination of the transcription start site
An adapted inverse PCR method was used to clone 5'-cDNA regions (15). Total RNA from a human somatotropic pituitary tumor was reverse transcribed (51 C for 30 min, 95 C for 5 min) by use of 200 U SuperScript reverse transcriptase (Life Technologies, Inc., Karlsruhe, Germany) and 100 nM antisense primer SR5A2 (5'-CTG-CCA-GGT-TGA-GAA-TGT-AGA-TGT-3', position +250) in the presence of 1.5 M betaine. The second strand was synthesized with 50 U Escherichia coli DNA polymerase I and 1 U T4 DNA ligase; simultaneously RNA was degraded by 1 U ribonuclease H (RNase H; 16 C for 8 h, 75 C for 5 min; all enzymes from Life Technologies, Inc.). Blunt ends were generated (11 C for 15 min) with 10 U T4 DNA polymerase (Life Technologies, Inc.), 10 U RNase T1, and 0.25 U RNase A (Ambion, Inc., Austin, TX). After phenol/chloroform/isoamyl alcohol extraction, ligation of blunt ends was performed with 10 U T4 DNA ligase (Life Technologies, Inc.) at 16 C overnight. Inverse PCR reaction (95 C for 30 s, 62 C for 60 s, 72 C for 60 s; 40 cycles) included SR5S2 (5'-TCC-TCC-CCG-GGG-GCT-GCC-TCT-G-3', position +43) as sense primer and SR5A3 (5'-GTG-GAG-GCT-GGG-AAC-AGG-GGC-TCC-3', position +24) as antisense primer. PCR products were cloned into pCRII. Transcription start sites were determined by sequencing analysis and comparison of genomic sequence and SR5A2 sequence. The location of the start site was also analyzed by RT-PCR analysis using sense strand primers upstream of position +1, including SR5S9 (5'-GAC-CCG-GGA-GTC-GAG-CAG-GTG-AAG-3', position -6277 relative to the ATG start codon) and antisense primer SR5A6 (5'-GGG-GAG-CCC-CGA-TGT-CC -3', position -6142), as well as sense strand primer SR5S3 (5'-GAG-CCA-GTG-CCG-CGC-GGA-CAT-CG-3', position -6173) and antisense primer SR5A4 (5'-TGC-ACG-TCC-GCG-AAC-ACC-AGG-AGC-3', position +542). To overcome secondary structures in RNA due to the high G+C content of the analyzed sequence, we used C.therm. polymerase (Roche) for RT (60 C for 30 min in the presence of 5% dimethylsulfoxide).
RT-PCR
Human pituitary mRNA as well as total RNA from small intestine, lung, and spleen were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). Total RNA from human placenta was extracted. After RT-PCR was performed on 50 ng total or 5 ng polyadenylated cDNA using 50 nM of each sense and antisense primers, 0.75 U Pfu Turbo DNA polymerase (Stratagene), and 200 nM deoxy-NTP (94 C for 30 sec, 62 C for 30 sec, 72 C for 1 min; 35 cycles). The upstream primer used for RT-PCR of exon 1a was SR5S3, and that used for amplification of exon 1 was SR5S2. Downstream primer for both amplifications was SR5A4. Amplified fragments were detected by Southern blotting. A DNA fragment containing residues 43–542 of the human sst5 gene was cloned into pCRII using the TA-Cloning Kit (Invitrogen). Specific primers used were SR5S2 and SR5A4. The identity of the probe was confirmed by sequence analysis. A digoxigenin-labeled probe was synthesized by PCR using digoxigenin-11-deoxy-UTP (Roche). Hybridization and detection were performed using a digoxigenin Luminescent Detection Kit (Roche) following the manufacturer’s protocol. Digoxigenin-labeled DNA molecular weight marker II (Roche) was used for size comparison. RT-PCR for cyclin A2 or glyceraldehyde-3-phosphate dehydrogenase was used to assess the integrity of cDNA template in each sample.
Construction of luciferase expression vectors containing upstream sequence
Promoter constructs were obtained by PCR on human genomic DNA (Roche) using data for a clone from chromosome 16 (GenBank accession no. AL031706) for sense primer design. The antisense primer SR5A5 (5'-GAT-GTC-CGC-GCG-GCA-CTG-GCT-C-3', position +33 relative to the transcription start site) contained sequence derived from 5'-inverse PCR. Sense primers (position relative to the transcription start site; sequence) were SR5S4 (-1741; 5'-GGC-CAG-GGG-ACT-CCT-GCC-CAG-AAC-3'), SR5S5 (-1269; 5'-GTG-GGA-GGG-TGG-GAG-GGA-TAC-AGC-3'), SR5S6 (-991; 5'-AAG-GGA-GCT-GCT-GGC-GCC-TGT-3'), SR5S7 (-317; 5'-GCC-CCT-GAC-CCT-CTC-CCC-TTG-3'), and SR5S8 (-101; 5'-AGA-CGT-GGG-ACC-CGG-GAG-TCG-3'). Similar constructs containing sequences immediate upstream of the translation start codon were cloned. SR5A7 (5'-GTC-AGG-CTC-TGC-AAG-AGA-AGA-AGG-3', position -43 relative to the translation start codon) was used as the antisense primer, and SR5S10 (-1492; 5'-GGG-TCA-ACC-CAA-ACA-TCC-GGA-C-3') as the sense primer.
PCR products were cloned into pCRII-Blunt Vector (Invitrogen), released with a suitable restriction enzyme combination, and subcloned into pGL3-Basic.
Cell culture studies
Rat mammosomatotrope pituitary GH4 cells, monkey kidney COS-7 cells, and human chorion carcinoma cells JEG-3 were grown in DMEM (Life Technologies, Inc.) containing 10% FCS (Serva, Heidelberg, Germany), whereas human endometrium Skut-1B cells were grown in a 1:1 mixture of DMEM and Ham’s F-12 medium containing 10% FCS. All media were supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 37 C in 5% CO2. Cell lines were transfected in triplicate by means of calcium phosphate coprecipitation using the ProFection Mammalian Transfection System (Promega Corp., Madison, WI). Transfections included simian virus 40-ß-galactosidase promotor (Promega Corp.) as an internal control of transfection efficiency. The duration of treatment with various hormones was 48 h. Cells were harvested 64 h after transfection in reporter lysis buffer (Promega Corp.) for luciferase assay. Luciferase light units were normalized to the activity of ß-galactosidase. All experiments were performed in triplicate in at least three independent experiments, and data are expressed as the mean ± SEM.
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Genomic structure of the human sst5
To determine the transcription start site of the human sst5, total RNA obtained from a human somatotropic pituitary adenoma was used for 5'-inverse PCR. We analyzed 24 PCR clones, of which 16 demonstrated an extended 5'-untranslated region (5'-utr) of 94 nt, determining a cytosine residue as a major transcription start site (Fig. 1A
). The remaining clones contained various shorter fragments of the 5'-flanking region, possibly indicating further transcription start sites or artificial early termination of RT. To overcome secondary structures of the RNA due to the high G+C content, a thermostable reverse transcriptase was used in the presence of betaine.
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). Splice sites were identified at position -6125/-6124 and -28/-27. The newly identified upstream sequence was subsequently denoted exon 1a. The location of the transcription start site was confirmed by RT-PCR analysis using sense strand primers upstream of the translation start codon (Fig. 1C). A sense primer located 12 nt downstream of the transcription start site determined by us did allow amplification of a correct fragment by RT-PCR analysis (625 nt). In contrast, amplification of cDNA with a sense primer located 93 nt upstream of the transcription start site did not result in a specific fragment (136 nt). As the G+C-rich area upstream of the transcription start site may result in strong secondary structures of RNA, we performed RT at 60 C using thermostable reverse transcriptase. The correct amplification by PCR was confirmed using genomic DNA as a positive control. The identities of the amplified fragments were confirmed by sequence analysis. Examination of the transcribed sequence confirmed the previously determined open reading frame. A putative polyadenylation signal containing the homology sequence AATAAA was identified at +2568 in the 3'-utr. These data reveal that the human sst5 is encoded by 2 exons. Altogether, the human sst5 gene spans 8758 nt. In the somatotropic pituitary cell, 6090 nucleotides of intronic sequence are removed by splicing the pre-mRNA. The 5'-donor splice site of the intron showed the consensus sequence 5'-(C/A)AGgu(a/g)ag-3', the 3'-acceptor splice site showed the conserved consensus sequence 5'-(u/c)11n(u/c)ag(G/A)-3' (17). Exon 1a is composed of 67 nt of 5'-utr; exon 1 consists of 27 nt of 5'-utr, the complete open reading frame, and the 3'-utr. The sequence for the open reading frame of the human sst5 described in this report matches that reported by Yamada et al. (7), but differs slightly from that reported by Panetta et al. (6).
Characterization of the promoter region
A fragment containing 1741 nt 5' of the transcription start site was amplified by PCR from genomic lymphocyte DNA, subcloned, and sequenced (Fig. 2). No deviations from the sequence AL031706 were found. The promoter lacks consensus sites for TATA or CAAT boxes, YY1, or an initiator sequence, but the proximal 5'-untranscribed region was highly GC rich, with 90% content within the first 70 nt. Putative binding sites for basal transcription factors such as nuclear factor-1 were located at nt -1402 and -1250. SP1 sites were situated throughout the promoter at nt -1585, -1519, -1367, -1214, -921, -414, -288, -265, -244, -221, -139, -108, -42, and -24. Possible responsive elements for Oct-1 were found at nt -674, -484, and -431. Several binding sites for tissue-specific transcription factors were identified. A site for the pituitary-specific transcription factor Pit-1 was found at -457 nt and for Krox-20 at -822 nt. A site for the basic-helix-loop-helic protein pancreas specific transcription factor 1 implied in the development of pancreas was located at nt -795. A binding site for the muscle-specific transcription factor MyoD was identified at nt -384. Potential binding sites for hormone-regulated factors included half-sites for the thyroid hormone receptor at nt -888, coinciding with an RAR site, and at nt -1192, -958, and -573. A half-site for ER was identified at nt -77, and for GR at nt -1493, -1021, and -513. Activating protein-2 sites were found at nt -1553, -1386, -1172, -1141, -779, -640, -348, -173, and -60, and cAMP response element-binding protein sites were located at nt -331 and -156. CpG islands were identified in the promoter from nt -1119 to -568 and from nt -201 to -1 relative to the transcription start site.
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). RT-PCR using primers specific for exon 1 resulted in amplification of a 500-nt fragment from pituitary and placenta. Signals of low intensity were observed for RNA obtained from small intestine and lung, pointing to low expression in these tissues. No signal was obtained in spleen. The absence of expression in spleen was confirmed by Southern blotting using an sst5-specific probe. An additional PCR product for pituitary tissue does not represent sst5-specific sequences, as demonstrated by sequence analysis and Southern blotting.
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). The absence of expression of exon 1a in nonpituitary tissues was confirmed by Southern blotting. We therefore suggest differential promoter usage in the tissues analyzed.
Transient expression analysis of the 5'-flanking region
To determine whether the sst5 5'-flanking region can direct cell-specific expression, a fragment containing 1741 nt 5' of the transcription start site was inserted into a transient expression vector, pGL3-Basic, which contains luciferase as the reporter gene. The resulting plasmid (-1741hsst5/luc) was transiently transfected into various cultured cell lines. As we observed significant expression of sst5 in pituitary and placenta, we chose rat mammosomatotrope pituitary GH4 cells, human chorion carcinoma JEG-3 cells, and human endometrium Skut-1B cells for transfection studies. Expression of sst5 in these cells was demonstrated by RT-PCR (data not shown). Therefore, it can be inferred that the appropriate cell-specific transcriptional machinery is in place to drive the exogenous promoter if the appropriate sequence is provided. Monkey kidney COS-7 cells were chosen as a negative control, as we did not observe sst5 expression in these cells. As shown in Fig. 4A, 1741 nt of the human sst5 promoter directed significant levels of luciferase expression in GH4 cells, Skut-1B cells, and JEG-3 cells, but not in COS-7 cells. Therefore, 1741 nt of the human sst5 5'-flanking region direct cell-selective expression. The relative activities of pGL3-Control containing a simian virus 40 viral promoter in COS-7, JEG3, Skut-1B, and GH4 cells were 328-, 968-, 342-, and 37-fold, respectively.
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, left panel). The activity obtained with a minimal -101 promoter construct was 22-fold in GH4 cells (
), 29-fold in Skut-1B cells (
), and 21-fold in JEG-3 cells (
) compared with the promoterless control (Fig. 4B, right panel). Promoter activity was not enhanced after transfection of larger constructs. More specifically, the -317 construct demonstrated decreased activity in JEG-3 cells, which was similar for all larger constructs tested. In Skut-1B cells, activity of the -317 construct was similar to the -101 construct, but analysis of larger constructs again revealed decreased activity. In GH4 cells, decreased activity of both -317 and -991 constructs was found, whereas the larger constructs demonstrated activity comparable to that of the minimal -101 construct. These results suggest that the proximal 101 nt contain transcription factor-binding sites necessary for activation of the human sst5 promoter in all three cell lines tested. The region between -101 and -991 may contain negative elements, which are activated in variable ways in GH4, Skut-1B, and JEG-3 cells. To investigate the importance of the immediate proximal promoter region, we generated deletion constructs (
-51, -14) -101hsst5/luc, and (-51, -14) -1741hsst5/luc. The luciferase activity of these constructs was significantly decreased compared with that of the wild-type plasmids. Therefore, the region between nt -51 and nt -14 may contain elements that are required for activation of the human sst5 promoter. The negative control, pGL3-Basic without any 5'-flanking region, caused low luciferase activity in all cell lines.
To test whether the sequences immediate upstream of the translation start codon can be used as an alternative sst5 promoter in pituitary cells, a fragment containing 1492 nt 5' of the ATG codon was inserted into pGL3-Basic. As shown in Fig. 4A, -1492ATGhsst5 directed significant levels of luciferase expression in GH4 cells, but not in COS-7 cells. Therefore, the immediate 5'-flanking sequences of the sst5-coding region contain elements to drive the expression of sst5 in the GH4 rat pituitary cell line. As we have not yet determined the transcription start sites for sst5 in other cell types, we did not test the transcriptional activity of the immediate upstream sequences in these cell lines.
The pituitary-specific transcription factor Pit-1 is not involved in transcriptional activation of the human sst5 promoter in GH4 pituitary cells
Significant transcriptional activity of the sst5 5'-flanking region in GH4 rat pituitary cells suggests an interaction between pituitary-specific factors and the sst5 promoter. Other tissue-specific transcription factors may drive the -1741hsst5/luc construct in human chorion carcinoma JEG-3 cells and human endometrium Skut-1B cells. The expression of the POU domain transcription factor Pit-1 is strictly pituitary-specific and is required for transcription of the GH gene. COS-7 monkey kidney cells do not produce any significant amount of the specific transcription factor Pit-1. As shown in Fig. 5, Pit-1 did not enhance the activity of the -1741hsst5/luc construct in COS-7 cells. In contrast, the activity of 344 nt of the human GH promoter was significantly enhanced by Pit-1 cotransfection. These results suggest that pituitary-specific transcription factors other than Pit-1 may be responsible for the significant transcriptional activity of the sst5 promoter in GH4 pituitary cells.
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, treatment with 10-7 M phorbol 12-myristate-13-acetate, 13 nM IGF-1, 10-7 M hydrocortisone, or 10-9 M ß-E2 did not influence sst5 promoter activity. In contrast, 10-9 M thyroid hormone (T3) or 10-6 M forskolin significantly enhanced, and 10-8 M SRIF significantly inhibited, the activity of the -1741 sst5 promoter in GH4 rat pituitary cells.
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) and stimulation by forskolin (Fig. 7B) were maintained for all deletion constructs investigated, with no significant changes between the various constructs. Both SRIF and forskolin are thought to transduce their effects by control of the cAMP signal cascade. Therefore, cAMP-responsive elements may be located in the region between -101 and the transcription start site. Significant stimulation of the sst5 promoter by thyroid hormone was found for a minimal -317 construct (Fig. 7C). Activation by thyroid hormone is further enhanced for the -1741 promoter construct. Transcriptional regulation of the human sst5 promoter by thyroid hormone may require elements located between nt -1741 and -1269 and between nt -317 and -101.
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Discussion |
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Expression of sst5 has been demonstrated in various human tissues, including cerebellum, heart, small intestine, adrenal (5), pituitary (23), placenta (24), and testicular tissue (25), whereas no transcripts were found in thymus (26). We identified transcripts of exon 1 in pituitary and placenta and, to a lesser extent, in small intestine and lung, but not in spleen. Exon 1a transcripts present exclusively in the pituitary indicate specific transcriptional activity of the promoter identified. However, the absence of exon 1a transcripts in some tissues expressing exon 1 suggests the use of an alternative promoter. Previously, 2.2 kb of sequence located immediately upstream of the ATG start codon were suggested as a putative promoter for sst5 (16). The transcription start site was not determined in that study. The researchers describe significant transcriptional activity of 0.9-kb sequence immediately upstream of the ATG start codon in GH3 cells, but not in CHO ovary cells. In our study significant activity of 1492 nt of the immediate 5'-flanking sequences of the sst5-coding region was observed in GH4 rat pituitary cells. Therefore, the immediate upstream sequence of the human sst5 gene may also function as a promoter, depending on the tissue, species, and cell model studied.
For the mouse sst5, a single transcription start site was described by analysis of TtT-97 thyrotropic tumor RNA, which is located approximately 5.3 kb upstream of the ATG start codon (22). Other tissues were not tested. Tissue-specific alternative promoters have been described for the mouse sst2. Primer extension experiments using RNA from AtT-20 cells revealed three transcription initiation sites divided by introns larger than 25 kb (20). Whereas the first promoter was found to be active only in AtT-20 cells, usage of the second promoter was demonstrated in brain, pituitary, adrenals, and pancreas. The third promoter was additionally used in lung, kidney, and spleen. Such alternative promoter usage may increase diversity and flexibility in gene expression depending on developmental stage and tissue or cell type.
Sequence analysis of the human pituitary-specific sst5 promoter revealed no TATA or CAAT boxes, YY motifs, or an inr initiator element. These features are reminiscent of the mouse sst5 promoter (22). However, the proximal promoter region of the human sst5 gene was highly GC rich and contained several SP1-binding sites that may function in transcriptional initiation. Putative binding sites for other transcription factors were found that might allow for tissue-specific expression. The POU domain transcription factor Pit-1 plays a key role in terminal pituitary differentiation (27) and was recently shown to dually regulate the rat sst1 (28). Krox-24 (nerve growth factor-1A, early growth response factor-1) is implicated in somatotrope and gonadotrope function (29). The basic-helix-loop-helic protein pancreas specific transcription factor 1-p48 is essential for formation of the exocrine pancreas and correct spatial organization of the endocrine pancreas (30). Interestingly, two CpG islands were identified in the sst5 promoter, defined by a G+C content greater than 0.5 and an observed/expected presence of CpG greater than 0.6 in a DNA region larger than 200 nt. In vertebrate genomes, CpG islands are often correlated with a promoter position (31).
Whereas only low activity of the 1.7-kb sst5 promoter was found in a monkey kidney cell line, we demonstrated significant activity in a rat pituitary cell line, a human uterine sarcoma cell line, and a chorion carcinoma cell line. A 101-nt proximal promoter fragment is sufficient to direct specific expression of sst5 in such tissues. Studies of a deletion constructs demonstrated that promoter activity depends on a highly GC-rich sequence between nt -51 and -14, which includes SP1-binding sites. Such GC boxes are paramount in increasing transcriptional rates of a core promoter sequence to significant levels (17) and have been proposed to direct gene transcription primarily in housekeeping genes (32). Therefore, additional enhancers binding to the -101 minimal sst5 promoter may allow for more cell type-specific activation, but their nature is currently unknown. A decreased activity is observed for larger promoter sequences. The CpG islands may function as silencers, as the methylation state of such sequences has been correlated with transcriptional inactivity (33, 34). Although a Pit-1-binding site is located at -457, cotransfection studies do not show any significant role for Pit-1-dependent transcriptional activation. In contrast to the specific expression of exon 1a in pituitary tissue, we did not observe a significant difference in promoter activity in GH4 pituitary-derived cells and Skut-1B endometrial cells. Important transcription factor binding sites may be located upstream of nt -1742 to direct cell type-specific activity. Alternatively, expression of transcription factors may vary between immortalized cell lines and normal tissue. The physiological relevance of the promoter studies remains to be investigated.
Results from several systems suggest that sst5 may be under regulatory control. In the rat pituitary cell line GH3, treatment with SRIF increased the steady state levels of sst5 mRNA (35). Administration of GHRH both in vivo and in vitro decreased sst5 mRNA levels, as did forskolin in vitro (36). In contrast, we observed inhibition of the sst5 promoter activity in rat pituitary GH4 cells by SRIF and stimulation by forskolin. The effects of GHRH on transcriptional activity could not be tested, because GH4 pituitary cells do not possess any endogenous GHRH receptor. Responsive elements were localized to the minimal -101 promoter. Although homology regions for the transcription factor cAMP response element-binding protein were identified at nt -331 and -156, no such elements are present in the -101 promoter. However, the activating protein-2 transcription factor-binding site present at nt -60 may mediate regulation of the sst5 promoter by the cAMP cascade (37). The changes in mRNA levels observed by Bruno et al. (35) and Park et al. (36) may represent not transcriptional regulation, but posttranscriptional modification, e.g. RNA degradation. Alternatively, cAMP-responsive elements 5' of the analyzed promoter region or in the intronic sequence may function as silencer to regulate sst5 mRNA levels. Furthermore, the differences may lie in species specificity, as we expressed the human promoter in a rat cell line. Glucocorticoids down-regulate ssts on pituitary cells (38) and pancreatic acinar cells (39) in vitro, but specific subtypes have not been identified. Our search of 1.7 kb of the human sst5 promoter vs. TFMATRIX identified several putative GREs. However, studies of a transient expression system do not suggest regulation of the human sst5 gene through the identified promoter region. In TtT-97 murine thyrotrope tumor cells, sst5 mRNA levels were increased by incubation with T4 (40). We observed a significant stimulation of the -1741 sst5 promoter by thyroid hormone. Analysis of variant promoter constructs localized thyroid hormone response elements to sequences between nt -1741 and -1269 and between nt -317 and -101. In primary rat pituitary cells, E2 induced a discrete inhibition of sst5 mRNA levels, as demonstrated by quantitative RT-PCR analysis (41). However, in a transient expression system we observed no significant regulation of the -1741 sst5 promoter by E2.
In summary, we provide evidence for an upstream promoter of the human sst5 with tissue-specific activity. A formerly unknown 6.1-kb intron in the 5'-utr of the sst5 gene is identified. The newly identified upstream promoter directs cell-specific activity in the transient expression analysis that does not depend on the pituitary-specific transcription factor Pit-1. By deletion constructs a 38-nt sequence located immediately upstream of the transcription start site is identified that is necessary for transcriptional activity. We observed regulation of the pituitary-active sst5 promoter by the cAMP-dependent signal cascade as well as transcriptional activation by thyroid hormone. Future studies will provide further insight into the complex regulation of the human sst5.
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Footnotes |
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Abbreviations: nt, Nucleotide; RNase, ribonuclease; SRIF, somatostatin; sst5, somatostatin (SRIF) receptor subtype 5; utr, untranslated region.
Received October 30, 2001.
Accepted for publication March 13, 2002.
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, The 5'-utr. The ATG start codon is indicated. B, Sst5 transcripts were reverse amplified from various tissues using the above primers. Amplified fragments were detected by ethidium bromide staining (upper panel) and by Southern blotting using an sst5-specific probe (lower panel). The upper arrow indicates the 625-nt promoter-specific PCR product, and the lower arrow points to the 500-nt coding sequence-specific fragment. Tissues analyzed include pituitary (pit), small intestine (int), lung (lun), spleen (spl), and placenta (plc). M, The 100-nt ladder plus digoxigenin-labeled DNA molecular weight marker II; N, negative control (H2O).
