The acid-labile subunit (ALS) of the ternary insulin-like growth factor-binding protein complex has a central role in controlling the bioavailability of circulating ...
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Cloning and characterization of the rat gene for the acid-labile subunit of the insulin-like growth factor binding protein complex P J D Delhanty and R C Baxter The Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia (Requests for offprints should be addressed to P J D Delhanty, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, NSW 2065, Australia)
ABSTRACT
The acid-labile subunit (ALS) of the ternary insulin-like growth factor-binding protein complex has a central role in controlling the bioavailability of circulating insulin-like growth factors. We have shown that gene expression of ALS is regulated by a number of factors, particularly growth hormone. Our aim was to characterize the ALS gene in order to define the mechanism of its regulation. Southern analysis suggests a single copy of the ALS gene in the rat genome. The protein-coding and 3*untranslated regions span 23·5 kilobases of rat genome and are divided into two exons. The 5* flanking region of the gene lacks a consensus
TATA-box or Inr sequence, and primer extension and reverse transcriptase PCR experiments locate multiple transcriptional initiation sites between "505 and "385 nucleotides relative to the translational initiation codon. This putative promoter region, when inserted upstream of the luciferase reporter gene, directs luciferase expression when transfected into H4-II-E cells. Our data demonstrate the uncomplicated structure of the rat ALS gene, and the promoter function and presence of potential regulatory elements in the region upstream of the protein-coding sequence.
INTRODUCTION
Previously, it has been shown by Northern analysis that ALS mRNA is expressed as a single 22 kb major transcript in the adult rat liver (Dai & Baxter 1994, Dai et al. 1994). Using in situ hybridization Chin et al. (1994) showed that this hepatic gene expression was confined to hepatocytes, the main source of another component of the ternary complex, IGF-I. In addition, Chin et al. (1994) demonstrated ALS gene expression in the epithelial cells of the straight proximal tubules of the renal cortex and in cartilaginous bone of the developing fetus. Serum concentrations of the components of the ternary complex are, to varying extents, dependent on growth hormone (GH) status (Baxter & Dai 1994, Baxter & Martin 1989a). The developmental regulation of ALS serum concentrations may be linked to the ontogeny of circulating GH concentrations and the appearance of GH receptors in the liver (Baxter 1990, Tiong & Herington 1992, Walker et al. 1992, Baxter & Dai 1994). IGF-I and ALS appear to be particularly sensitive to serum
The acid-labile subunit (ALS), an 85 kDa glycoprotein, is one of a family of proteins and glycoproteins that commonly form specific protein–protein complexes (Leong et al. 1992, Kobe & Deisenhofer 1994, 1995). A feature of these proteins is a tandemly repeated leucine-rich motif that can occur up to 30 times, for example in the Drosophila cell adhesion molecule, chaoptin (Krantz & Zipursky 1990). ALS contains 20 such repeats, constituting 275% of the protein, and interacts with the binary insulin-like growth factor-binding protein-3 (IGFBP-3)–insulin-like growth factor (IGF) complex forming a ternary complex in the circulation (Baxter & Martin 1989a,b, Baxter et al. 1989, Delhanty & Baxter 1996) This 2140 kDa complex prolongs the systemic half-life of IGF-I and IGF-II and may buffer the insulin-like effects of these peptides by regulating their release from the blood (Binoux & Hossenlopp 1988, Zapf et al. 1991).
Journal of Molecular Endocrinology (1997) 19, 267–277
Journal of Molecular Endocrinology (1997) 19, 267–277 ? 1997 Journal of Endocrinology Ltd Printed in Great Britain 0952–5041/97/019–267 $08.00/0
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GH concentrations, being suppressed in GH deficiency and restored with GH replacement therapy (Baxter 1993). In relation to this, a 20- to 30-fold increase in serum ALS can be induced in hypophysectomized rats by treating them with GH (Fielder et al. 1996). IGF-I has been shown to be regulated primarily at the level of gene transcription (Rotwein et al. 1993); however, the mechanism by which ALS is regulated has not yet been determined. Recently, we showed that steady state concentrations of ALS mRNA in primary rat hepatocyte cultures are markedly upregulated by GH treatment (Dai et al. 1994). Conversely, hypophysectomized rats have less than 10% of basal hepatic ALS mRNA concentrations, and transcript concentrations were also reduced in the kidneys (Chin et al. 1994). In isolated hepatocytes, ALS mRNA concentrations relate to ALS secretion, suggesting that ALS regulation has a major transcriptional component (Dai et al. 1994). GH is not the only regulator of ALS secretion and mRNA concentrations. For example, dexamethasone inhibits ALS secretion and gene expression in primary hepatocytes, and ALS serum concentrations and hepatic mRNA concentrations in in vivo experiments (Dai & Baxter 1994, Dai et al. 1994). Epidermal growth factor decreases ALS mRNA concentrations in primary hepatocytes and, in common with the effect of dexamethasone, this inhibition of gene expression correlates strongly with ALS secretion (Dai et al. 1994). Although the genes that encode human and rat IGF-I and IGFBP-3 have been isolated and well characterized (Rotwein et al. 1986, Shimatsu & Rotwein 1987, Cubbage et al. 1990, Albiston et al. 1995), and the structure of the mouse ALS gene has recently been described (Boisclair et al. 1996), little is known about the organization and regulation of the rat ALS gene. The rat has been used extensively to examine the developmental, hormonal and tissue-specific regulation of ALS mRNA and serum concentrations (Scott & Baxter 1991, Chin et al. 1994, Dai & Baxter 1994, Dai et al. 1994); therefore, to define in more detail the mechanisms of gene transcription in this model of ALS regulation, we have cloned the rat ALS gene and identified its promoter. MATERIALS AND METHODS
Reagents All chemicals were obtained from Sigma Chemicals (St Louis, MO, USA). Most enzymes were obtained from Promega (Madison, WI, USA). The T7 sequencing kit, nucleotides and radioJournal of Molecular Endocrinology (1997) 19, 267–277
labelled nucleotides were obtained from AMRAD (Melbourne, VIC, Australia). The rat liver genomic library and Pfu DNA polymerase were obtained from Stratagene (La Jolla, CA, USA). Deoxyoligonucleotide primers were prepared on an Oligo 1000 DNA synthesizer (Beckman, Fullerton, CA, USA). Sequencing of the 5* end of the rat ALS cDNA The Rals.2 cDNA clone (Dai & Baxter 1992), which had been isolated from a random-primed plasmid cDNA library cloned into pUEX1 with a BamHI/ NcoI oligodeoxynucleotide linker, was sequenced using the pUC/M13 reverse primer (5*-GTTT TCCCAGTCACGAC-3*) and a T7 sequencing kit. Isolation and mapping of genomic clones The Rals.2 cDNA, which extends from nucleotide (nt) "385 to nt 1601 relative to the A+1TG of the published rat ALS cDNA sequence, was digested with BamHI and NheI (nt 907) to create 5*- and 3*-specific fragments, which were 32P-labelled by random priming (see Fig. 1). These probes were used to screen a genomic library constructed from Sau3AI partially digested rat chromosomal DNA inserted into the BamHI site of Lambda DASH (Stratagene). The library was plated out at approximately 50 000 plaques/150 mm plate using XL1-Blue MRA(P2) (Stratagene) as a host to prevent growth of wild-type phage. The two rat ALS cDNA probes were used separately to hybridize with duplicate plaque lifts, altogether representing 5#105 clones. Phage containing ALS genomic fragments were isolated by two more rounds of replating and screening. Single plaques of phage from five clones (1A, 4A, 5B, 8A and 9A) were grown in 10 ml lytic cultures in XL1-Blue MRA (Stratagene), precipitated using polyethylene glycol-6000/NaCl, digested with proteinase K, and then the remaining DNA was extracted with phenol–chloroform, as described previously (Sambrook et al. 1989). Southern analysis of phage DNA from the five clones was performed by digestion with restriction endonucleases and sequential screening with 5* and 3* probes derived from the Rals.2 cDNA by digestion with NheI and BamHI. These analyses determined that clones 1A and 8A overlapped and that, together, they would probably cover the entire coding region of the ALS gene. DNA from these clones was digested with EcoRI and XbaI respectively, and separated electrophoretically in a 1% agarose gel. The 2 and 2·3 kb fragments from 1A (1A2 and 1A2·3) and the 6 kb fragment from clone 8A (8A6) were isolated and subcloned into pUC118
Rat ALS gene ·
to give subclones p1A2, p1A2·3 and p8A6·5 (see Fig. 1). Using the original phage clone 1A as a template, PCR was used to generate an 21 kb fragment that conjoined and overlapped inserts 1A2 and 1A2·3. This fragment was ligated into the SmaI site of pUC118, giving subclone p1A1. The genomic fragments were sequenced on both strands by the dideoxy termination method using a T7 DNA polymerase sequencing kit (Pharmacia, Melbourne, VIC, Australia) and gene-specific deoxyoligonucleotide primers. The full sequence of the gene was reconstructed using Sequencher v.3·0 software (Gene Codes, Ann Arbor, MI, USA). Southern blot analysis Genomic DNA was isolated from rat liver as described previously (Sambrook et al. 1989), and dialysed extensively against Tris–EDTA (10 m Tris, 1 m EDTA, pH 8). Three aliquots of 10 µg purified DNA were digested for 12 h at 37 )C with 20 U each of XbaI, EcoRI and BamHI, followed by the addition of another 20 U of enzyme and a further 12 h incubation. These digested DNAs were then separated electrophoretically on a 0·7% agarose gel (0·5#TBE: 44·5 m Tris base, 44·5 m H3BO3, 1 m EDTA) and then transferred to ZetaprobeGT membrane (Bio Rad Laboratories, Hercules, CA, USA) by capillary blotting in 10#SSC. Hybridization of random-primed 32Plabelled probes was carried out at 42 )C. The blots were washed in 0·1#SSC at 65 )C and analysed using a phosphorimager. Before re-screening with a different probe, the blots were stripped in 0·01#SSC at 95–100 )C for 20–30 min. The probes, described in Fig. 1, were derived from the Rals.2 cDNA (probe I) and ë clone 8A (probe II). Primer extension analysis of rat ALS mRNA Total RNA was isolated from rat livers using the guanidinium/CsCl technique (Sambrook et al. 1989). The oligodeoxynucleotide primer pr156 (5*-CAGACAAGGTGAGGGGAGCTCCGGGG TTCCTGG-3*, complementary to nts "344 to "376) was end-labelled with [ã-32P]ATP to a specific activity of approximately 2#105 c.p.m./ng using T4 polynucleotide kinase. Labelled primers were purified by ethanol precipitation. Total RNA (25 µg) from normal rat liver was hybridized with 1#105 c.p.m. of end-labelled primer in 1 NaCl, 0·17 Hepes (pH 7·5) and 300 µ EDTA overnight at 30 )C. The primers were then extended with 200 U of Superscript II RNase H " reverse trancriptase (Life Technologies GIBCO/BRL; Gaithersburg, MD, USA) in the pres-
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ence of 0·55 m dNTPs at 42 )C for 90 min. After treatment with RNase A and phenol–chloroform extraction, the samples were analysed on 6% polyacrylamide sequencing gels. The sizes of reverse transcribed products were determined by direct comparison with sequences of genomic clone p1A2·3, which were generated using primer pr156. Controls were performed using exactly the same protocol with 25 µg of yeast tRNA and no nonspecific extension products were observed. Reverse transcription PCR of rat ALS mRNA 5* end Rat liver total RNA (2 µg) was reverse transcribed as described previously (Delhanty & Baxter 1996). The first strand synthesis was used in three PCR reactions using a reverse primer (pr152, 5*-GA AAGCCAGAAGCACCACCAGGGCTGGG-3*), which is complementary to the sequence immediately downstream of the intron, and one of three primers (pr154, 5*-CCTAAACCTACCTTGAGC ACC-3*; pr159, 5*-GGATTCCCAGGAACCCC GGA-3*; and pr164, 5*-GAAGGCTGAGACTGG GCCTTGGACAAACCC-3*) complementary to the genomic sequence surrounding the transcriptional start points, which had been identified by primer extension. The PCR products were gelisolated and reamplified by PCR using the same primers. Detectable products were isolated and sequenced using a SequiTherm EXCEL cycle sequencing kit (Epicentre Technologies, Madison, WI, USA). Reporter plasmid constructs and transient transfections A fragment spanning base pairs "2 to "1449 relative to the A+1TG was generated by PCR, and cloned in forward (pGL3/6) and reverse (pGL3/12) orientations into the SmaI site of the promoterless luciferase reporter vector, pGL3-Basic (Promega). The insert was sequenced as described above. Plasmid was prepared for transfection using Qiagen plasmid isolation columns (Qiagen GmbH, Hilden, Germany). The rat hepatoma cell line, H4-II-E (obtained from the American Type Culture Collection), was plated at 1#106 cells per well into six-well plates, and incubated for 16 h in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum. The cells were then washed twice with serum free DMEM, and each well was incubated with 1 ml serum-free DMEM containing 9 µl lipofectamine (Life Technologies GIBCO/BRL), 0·5 pmol reporter construct and Journal of Molecular Endocrinology (1997) 19, 267–277
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1. Structure of the rat ALS chromosomal gene. A restriction map of the ALS gene and flanking regions, generated from sequence and Southern analysis data, is represented at the top. The distance in nucleotides 5* (negative) or 3* (positive) of the adenosine residue of the translational initiation codon is shown for some restriction sites and the map is segmented to fit the scale. The positions of exons 1 and 2 are depicted immediately beneath this map. At the bottom are shown the two overlapping genomic clones and the subclones derived from these clones. Fragments generated by EcoRI digestion are p1A2·3 and p1A2 and the fragment generated by XbaI digestion is p8A6. PCR was used to create subclone pCR1A1 directly from ë clone 1A. B, BamHI; E, EcoRI; N, NheI; S, SacI; X, XbaI.
1 µg pSV-â-galactosidase (Promega) control plasmid. After 5 h, an extra 1 ml serum-free DMEM containing 0·4% bovine serum albumin was added to each well, and the cells were incubated for a further 15 h. Cell lysates were prepared using Reporter Lysis Buffer (Promega), and assayed for luciferase and â-galactosidase activity. The luciferase activity in 20 µl lysate was measured using a luciferyl CoA-based reagent system (Promega), as described by the manufacturer, using a Turner TD20/20 luminometer. A delay period of 3 s and an integration time of 30 s were used. The â-galactosidase activity was determined by incubating 100 µl lysate with 150 µl assay 2#buffer (120 m Na2HPO4, 80 m NaH2PO4, 2 m MgCl2, 100 m 2-mercaptoethanol, 1·33 mg/ml o-nitrophenyl-â--galactopyranoside) at 37 )C for 3 h with shaking. The reaction was then terminated with 500 µl 1 Na2CO3 and the absorbance at 420 nm was read with a spectrophotometer. RESULTS
Gene structure and organization Five positive clones were isolated after screening 5#105 clones from a partial Sau3AI rat liver genomic library with restriction fragments derived from the 3* and 5* ends of the rat cDNA. Southern analysis using the same probes identified two Journal of Molecular Endocrinology (1997) 19, 267–277
overlapping ë clones (ë 1A and ë 8A, Fig. 1) of 215 kb and 218 kb in length respectively, which covered at least the entire coding region of rat ALS. Clone ë 9A mapped exactly with clone ë 8A, clone ë 4A overlapped the 5* end of ë 1A and clone ë 5B overlapped both clones ë 1A and ë 8A. Clones ë 4A, 5B and 9A were not examined further. An 2400 bp fragment between the third EcoRI and third XbaI sites of clone ë 1A was determined to be intronic, hybridizing with neither of the cDNA probes. Three genomic fragments, 2·3 kb and 2 kb EcoRI fragments from clone ë 1A and a 6 kb XbaI fragment from clone ë 8A were isolated, subcloned into pUC118 and sequenced using gene-specific oligodeoxynucleotide primers (see Materials and Methods). The polymerase chain reaction was used to generate an 21 kb fragment (pCR1A1, Fig. 1) using clone ë 1A as a template and which overlapped fragments 1A2·3 and 1A2. Fragment 1A1 was subcloned into pUC118 and its sequence demonstrated that p1A2·3 and p1A2 are contiguous. The relative sizes and locations of the exons and intron of the rat ALS chromosomal gene are depicted in Fig. 1. The map is based on complete bidirectional sequencing of 2 kb of 5* flanking region, the exons and intron, and 3* untranslated region (UTR) to the polyadenylation signal (Figs 2 and 3). In addition, approximately 600 nucleotides of the 3* end of p8A6 were sequenced and confirmed the presence of a BamHI site in this region. The rat ALS gene spans 23·1 kb of chromosomal DNA
Rat ALS gene ·
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2. Nucleotide sequence of the rat ALS gene 5* flanking region and exon 1. The numbering of nucleotides represents the distance 5* (negative) or 3* (positive) from the adenosine residue of the translation initiation codon. Potential CAAT-boxes are double-underlined and Sp1 binding sites are underlined. The positions of transcriptional start points indicated by primer extension using pr156 (tsp1-3), and the most 5* nucleotide common to both ALS cDNA and genomic sequences are marked with arrows. Intronic sequence is shown in lower case.
and contains two exons separated by an intron of 1116 bps. The intron–exon splice sites obey the GT–AG rule (Padgett et al. 1986) and correspond closely to the established consensus sequences (Fig. 3). In order to delineate an approximate region in which transcriptional initiation might occur, we sequenced the complete 5* end of the rat ALS cDNA clone Rals.2. The genomic and cDNA sequences in this region agree completely, and the 5* end of the cDNA occurs at "385 bps relative to the translational initiation codon (Fig. 2; see also Fig. 5A). Exon 1 encodes the entire 5* UTR (based on
the sequence of the Rals.2 clone), and the first five codons and the first nucleotide of the sixth codon encoding the N-terminus of the ALS signal peptide. Exon 2 spans 1796 bps and encodes the remaining 22 residues of the signal peptide and the entire mature ALS peptide, including all of the cysteines, N-linked glycosylation sites and repeated leucinerich motifs. In addition, exon 2 encodes a variant (AAUUAAA) polyadenylation signal starting at nt 3169 in the chromosomal DNA. This site is 2053 bps relative to the translational initiation codon in the cDNA and constitutes a divergence from the cDNA sequence starting at nt 2031. This Journal of Molecular Endocrinology (1997) 19, 267–277
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3. Nucleotide sequence of the intron and the region of exon 2 that encodes the 3* untranslated region of the rat ALS mRNA. Numbers represent nucleotide positions relative to the adenosine residue of the translational initiation codon. Sequences that conform to splice donor and acceptor consensus recognition sites are dottedunderlined. The 3* end of the rat ALS cDNA that is divergent from the genomic DNA sequence in this region is shown in lower case, and the polyadenylation site found in the genomic DNA is underlined.
sets the length of the 3* UTR at approximately 250 nts, although the exact site of polyadenylation has not been determined. There is close identity between the genomic nucleotide sequence and the previously reported rat ALS cDNA sequence (Dai & Baxter 1992). However, the sequence differs at seven sites, which may represent strain-specific polymorphisms: i) there is an extra C inserted after nt "59 in the 5* UTR of the cDNA; ii) nt 1894 is a C instead of a G, which alters codon 233 from GGT to CGT, thus changing Gly233 (incorrectly translated as Arg in the original publication) to Arg, making this residue conservative with its homologue in human ALS; iii) nt 2637 is a G instead of a C, but usage of codon 480 is conserved, substituting CGC for CGG (Arg); iv) nt 2638 is a C instead of a G changing codon 481 from GTT to CTT and thus Val481 to Leu, making this residue conservative with its counterpart in human ALS; v) and vi) nts 2966 and 2983 in the 3* UTR coding region are C and G respectively, instead of T; vii) there is complete divergence of the genomic sequence from that of the cDNA after nt 3146, which may represent a cloning artefact in the cDNA library. The genomic sequence encodes a polyadenylation signal starting at nt 3169. None of these changes has introduced translational terminJournal of Molecular Endocrinology (1997) 19, 267–277
ation codons into the coding region and transcription of this gene would be expected to yield a translatable mRNA. Southern analysis of genomic DNA Southern analyses of rat genomic DNA digested with EcoRI, XbaI and BamHI suggest that there is a single copy of the ALS gene in the rat genome. The same Southern blot was screened with an entirely exon-2-derived cDNA (probe I), and a genomic probe derived mostly from the intron (probe II). Probe I hybridized with single 27·5 kb EcoRI, 25·5 kb XbaI and 23·2 kb BamHI fragments. Probe II identified the same EcoRI and XbaI fragments, and two BamHI fragments of 23·2 and 24·5 kb (Fig. 4). These fragment sizes match those predicted from initial Southern analyses of the ë clones, and from the sequence of the coding region of the gene. Analysis of the transcription start points Sequencing of the extreme 5* end of the original rat ALS cDNA demonstrated that one possible cap site of the rat ALS mRNA occurs at least 385 nucleotides upstream of the translation initiation
Rat ALS gene ·
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4. Southern analysis of rat liver genomic DNA using rat ALS cDNA and genomic probes. Genomic DNA was digested with EcoRI, XbaI and BamHI. Ten microgram aliquots were electrophoresed on a 0·7% agarose gel then transferred to Zetaprobe membrane. The blot was sequentially screened with the two probes described in Fig. 1. The blots illustrated were all washed in 0·1#SSC at 60 )C and visualized using a phosphorimager.
codon (Fig. 2). Primer extension experiments, using a primer that originates just downstream of this possible cap site, demonstrated multiple transcriptional start points, with major sites at "447, "472 and "505 nts relative to the translational initiation codon (Fig. 5A). Identical results were obtained in two experiments using liver total RNA, and one experiment with liver poly-A+ RNA (data not shown). To validate these results we used reverse transcriptase PCR of rat liver total RNA using a reverse primer (pr152) downstream of the mRNA splice junction, and three forward primers (pr154, pr159 and pr164) surrounding the transcription start points identified by primer extension. Primers pr154 and pr159, which are complementary to sequence downstream of the putative transcription start points and the 5* end of the Rals.2 cDNA, generated products, whereas pr164, which is complementary to sequence upstream of the transcription start points, did not (Fig. 5B). The sizes of the PCR products generated with pr154 and pr159 (369 bp and 436 bp respectively) exclude the possibility that they were derived from genomic DNA. Cycle sequencing of the pr154 and pr159
PCR products, using internal reverse primers, confirmed their identity with the rALS sequence. Analysis of promoter function We tested whether the 5* flanking region of the gene could function as a promoter by examining the expression of luciferase in H4-II-E cells transiently transfected with equal molar amounts of a reporter vector containing a fragment 1447 bp 5* of the A+1TG in forward (pGL3/6) and reverse (pGL3/12) orientations upstream of the luciferase gene. This fragment of the gene contains the putative cap sites in addition to several consensus response elements. In triplicate experiments, pGL3/6 showed a significant, fourfold greater, luciferase activity (corrected for â-galactosidase activity) over the anti-sense construct, pGL3/12, and a significant fivefold increase over the promoterless construct, pGL3-Basic (Fig. 6). The level of expression of pGL3/6 was consistently lower (260-fold) than that of the control plasmid pGL3-Control, the luciferase gene of which is under the constitutive control of the SV40 promoter Journal of Molecular Endocrinology (1997) 19, 267–277
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5. Analysis of the rat ALS mRNA cap site. (A) Primer extension analysis of liver total RNA performed using a 32P-end-labelled primer (pr156) that is complementary to nucleotides "344 to "376 relative to the A+1TG. Extension products are compared directly with dideoxy sequence, generated using the same primer, of a genomic clone. The three most 5* transcriptional start points (tsp) are shown. Nucleotide "447 appears to be the major site of transcriptional initiation in this region. (B) Reverse transcriptase PCR (RT-PCR) of the 5* end of rat ALS mRNA. RT-PCR products, generated using primers that anneal either side of the rat ALS intron, were isolated, reamplified with the identical primer set and run on a 1·5% agarose gel. Primers pr154 and pr159 generated products of the appropriate size to have been derived from mRNA. Primer pr164 failed to reamplify a product of the correct size, suggesting that the initial RT-PCR generated a non-specific fragment. Sequencing confirmed that fragments pr154 and pr159 are derived from the rat ALS mRNA. This evidence confirms the position of transcription start points at or upstream of nt "385, but probably not beyond nt "505. These sites are 2450 nts upstream of the mouse initiation sites.
(data not shown). The level of expression of the pGL3-Control plasmid indicates that we had obtained a high level of transfection efficiency. An independent set of triplicate experiments gave similar results. DISCUSSION
The rat ALS gene has a relatively simple organization, consisting of two exons separated by an 21·1 kb intron. The gene encodes a variant polyadenylation signal, not observed in the rat cDNA sequence, starting 240 bp downstream of the translational termination codon. Divergence of the genomic DNA sequence from that of the cDNA at nt 3146 may be explained either by the presence of Journal of Molecular Endocrinology (1997) 19, 267–277
a second ALS gene encoding an mRNA with a different 3* UTR, or by a cloning artefact in the cDNA library that generated a chimeric clone. This basic structure appears to be similar to that of the mouse gene (Boisclair et al. 1996). Comparison of Southern analyses of rat liver genomic DNA with a restriction map generated from restriction analyses of four different genomic clones and the sequence of the ALS chromosomal gene suggest that there is only a single copy of the ALS gene. In addition, a cDNA probe derived from exon 2 (probe I), and a genomic probe derived mostly from the intron (probe II) identify appropriately sized restriction fragments of genomic DNA by Southern analysis. Primer extension and reverse transcriptase PCR experiments identify multiple transcriptional start
Rat ALS gene ·
6. ALS gene promoter activity in H4-II-E cells. A fragment containing 1447 bp (from "2 to "1449 bp 5* of the A+1TG) of the putative rat ALS promoter was cloned in forward (pGL3/6) and reverse (pGL3/12) orientations upstream of the luciferase gene in the reporter vector pGL3. Equimolar quantities of pGL3/6, pGL3/12, and the promoterless vector pGL3-Basic were individually cotransfected with 1 µg pSV-â-galactosidase into H4-II-E cells using lipofectamine. Cell lysates were assayed for luciferase activity, and results (relative light units, RLU) are expressed relative to â-galactosidase activity to correct for transfection efficiency. Construct pGL3/6 expressed fourfold greater luciferase activity than pGL3/12 (P=0·0052), and fivefold greater activity than pGL3-Basic (P=0·0033). Expression of luciferase by pGL3/12 was not significantly different from that of pGL3-Basic. P values were calculated by ANOVA (Fisher’s PLSD (protected least significant difference)) using Statview 4·02 (Abacus Concepts, Inc., Berkeley, CA, USA). Error bars indicate +1 ..
points between "385 and "505 nts relative to the A+1TG, and 50–120 nts upstream of the extreme 5* end of the Rals.2 cDNA (Fig. 5). This region is approximately 370 nts upstream of the most 5* transcription start point determined for the mouse gene (Boisclair et al. 1996). These putative cap sites are in a region that lacks consensus TATA-boxes and does not coincide with the consensus initiator sequence PyPyA+1N(T/A)PyPy (Javahery et al. 1994), perhaps accounting for the loose control over the position of transcriptional initiation. The region around the potential cap sites contains no consensus GC-box sequences, which have been found to initiate basal transcription in other TATA-less gene promoters through binding to Sp1, or Sp1-like proteins (Pugh & Tjian 1990, Boisclair et al. 1993, Kollmar et al. 1994). However, there are several CCCTCC elements that are reminiscent of the CCCCTCC elements that bind Sp1-like proteins in the neuropeptide-Y gene promoter and are believed
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to be involved in transcriptional activation of this gene (Minth & Dixon 1990). The rat ALS gene promoter appears to be similar to the promoters of the mouse ALS gene (Boisclair et al. 1996) and the IGF-I gene, which lacks typical TATA, CCAAT and GC boxes in the vicinity of its multiple transcriptional initiation sites (Rotwein et al. 1993). Computer-aided analysis of 2063 bps of sequence upstream of the translation start site has identified several putative cis elements that may be involved in the regulation of ALS expression. The 800 bps upstream of the translational initiation codon showed significant identity with the published sequence of the homologous region in the mouse gene (Boisclair et al. 1996). GH markedly potentiates serum ALS concentrations in man and rodents, and ALS gene expression and secretion both in vivo in the livers of rats and in primary cultures of hepatocytes (Dai et al. 1994, Fielder et al. 1996). One of the possible signalling pathways induced in the liver by GH involves receptor-associated protein kinase tyrosine phosphorylation of members of the signal transducer and activators of transcription (STAT) family of transcriptional regulatory proteins (Gronowski & Rotwein 1995, Ram et al. 1996). Phosphorylation of these proteins leads to their translocation from the cytoplasm into the nucleus, where they bind to specific cis elements related in sequence to the ã-interferon-activated site (GAS, TTNCNNNAA) (Ihle 1995). Activation of gene transcription by GH also can be mediated through other types of cis element. For example, GH rapidly activates transcription of the early response gene c-fos through a sequence known as the serum response element (SRE), and of the serine protease inhibitor (Spi) 2·1 gene through purine-rich sequences (Le Cam et al. 1994, Meyer et al. 1993). The trans acting factors that bind to these cis elements are probably unrelated to the STAT proteins. The IGF-I gene promoters are both GH responsive; however, the factors and cis elements that activate this response have proven elusive, and may represent another class of GH response (Thomas et al. 1995). Several sites related to the GAS are present in the 5* flank of the ALS gene, and two of them (GAS at "541 to "533, and GAS-like element (GLE, TTC(C/T)(C/G)(A/T)GAA) at "621 to "613) are similar in sequence and position to elements found in the mouse gene (Boisclair et al. 1996). In addition, there is a site that has close homology to the STAT-binding site of the somatostatin gene at "698 to "690 (Symes et al. 1994). The 5* flanking region that we have analysed contained none of the purine-rich sequences that may be Journal of Molecular Endocrinology (1997) 19, 267–277
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Rat ALS gene
involved in GH transcriptional induction of the Spi 2·1 gene (Le Cam et al. 1994). However, sequences are present at nts "1806 to "1797 and "1747 to "1738, which resemble the purine-rich sequence (CAGAGAGAGA) that has been reported to mediate GH induction of the somatostatin gene promoter and render a heterologous promoter GH-sensitive (Billestrup et al. 1993). The functionality of these sequences in relation to transcriptional regulation of ALS by GH is currently being examined. Other putative cis elements upstream of the translation initiation codon include a number of AP2 sites, and a single AP1 site. Potentially, these sites could be involved in regulation of ALS by cyclic AMP, phorbol esters and a variety of other factors. A hepatic nuclear factor-5 binding site at nts "655 to "649 is identical to that in the mouse gene promoter, and may co-ordinate the predominantly hepatic expression of ALS. Elements involved in the basal and hormonal regulation of the ALS promoter have not yet been mapped. However, most of the consensus elements described above occur in the ALS gene fragment, which we have shown to direct luciferase expression during transient transfections in hepatoma cells. H4II-E cells do not secrete ALS, even in the presence of recombinant human GH (unpublished observations). Therefore, it was surprising to us that there was any luciferase expression by these constructs in these cells. However, the low level of expression of the pGL3/6 construct relative to that of the control plasmid pGL3-Control, in which the luciferase gene is under the constitutive control of the SV40 promoter, may indicate that H4-II-E cells that we are using lack a component(s) of the signalling pathway that is necessary for full basal and GH-stimulated expression of ALS. In relation to this, and in contrast to the results of Boisclair et al. (1996) with the mouse ALS promoter, we have been unable to induce activity of the rat ALS promoter reporter construct with GH in our line of H4-II-E cells, or in another rat hepatoma line, HTC (unpublished results). The element(s) involved in GH regulation of rat ALS gene transcription remain to be identified. However, Boisclair et al. (1996) used a shorter promoter fragment extending only to "805 nts relative to the A+1TG, and it is thus possible that the region in the rat ALS promoter from "805 to "1447 contains an inhibitory element. The presence of several cis elements with sequence homology to those found in genes of which the transcription is rapidly activated by GH suggests that the regulation of ALS occurs at the transcriptional level. ALS is one of only a few gene products that have been shown to be controlled by Journal of Molecular Endocrinology (1997) 19, 267–277
GH (Rotwein et al. 1994), and therefore examination of the ALS promoter may provide important insights into the general mechanisms involved in GH-regulated transcription. ACKNOWLEDGEMENTS
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