Untranslated Region Of Exon Research Articles

Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene: analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type 1b.

Expression of the PTH/PTH-related peptide (PTHrP) receptor (PTHR) in the mouse is controlled by at least two promoters. The downstream promoter (P2) is ubiquitously expressed, whereas expression of the upstream promoter (P1) is largely restricted to kidney. These observations may provide a genetic basis for a human PTH resistance syndrome, pseudohypoparathyroidism type 1b (PHP1b), in which renal, but not osseous, signaling by PTH is defective. We, therefore, cloned and characterized the 5'-end of the human PTHR gene and found that its organization is very similar to that of the mouse. Transcription initiation sites of human P1 and P2 promoters are in similar, but not identical, positions to those of the mouse gene. The identification of a human P2 promoter is significant because no P2-specific human PTHR complementary DNAs have been isolated to date. Southern analysis of genomic DNA from seven PHP1b patients did not reveal any rearrangements in proximal promoter regions or exons encoding 5'-untranslated region sequences. No significant sequence differences were found in clones of normal and patient DNAs encompassing proximal promoter sequences, and untranslated region and signal sequence exons. Thus, in the seven PHP1b patients analyzed, no defects were identified that would influence initiation site selection, stability, or splicing of renal PTHR transcripts. These data indicate that the genetic defect(s) in PHP1b in these patients lies in distal enhancer elements of the gene, in an essential transcriptional regulator, or in some as yet unidentified cofactor required for renal PTH signaling.

Complex genetic contribution of the Apo AI-CIII-AIV gene cluster to familial combined hyperlipidemia. Identification of different susceptibility haplotypes.

Familial combined hyperlipidemia (FCH) is a common genetic lipid disorder in Western societies. In a recent report (Dallinga-Thie, G.M., X.D. Bu, M. van Linde-Sibenius Trip, J.I. Rotter, A.J. Lusis, and T.W.A. de Bruin. J. Lipid Res., 1996, 36:136-147) we have studied three restriction enzyme polymorphisms: XmnI, and MspI sites 5' of the apo AI gene and SstI site in the 3' untranslated region of exon 4 of the apo CIII gene in 18 FCH pedigrees, including 18 probands, 178 hyperlipidemic relatives, 210 normolipidemic relatives, and 176 spouses. DNA variations in the apo AI-CIII-AIV gene cluster had a modifying effect on plasma triglycerides, LDL cholesterol, and apolipoprotein CIII levels. In this study, combinations of haplotypes were analyzed to further characterize their interactions and effect on the expression of severe hyperlipidemia in FCH subjects. A specific combination of haplotypes with one chromosome carrying the X1M1S2 (1-1-2) haplotype and the other the X2M2S1 haplotype (2-2-1) was significantly more frequent in hyperlipidemic relatives (6%) than in normolipidemic relatives (3%) and spouses (0.5%). Associated with this combination of haplotypes were significantly elevated plasma cholesterol (P < 0.0001), triglycerides (P < 0.0001), and apo CIII (P < 0.001) levels when compared to the wild type combination of haplotypes 1-1-1/1-1-1. The only spouse with this specific combination of haplotypes showed a severe hyperlipidemic phenotype, similar to FCH. Furthermore, nonparametric sibpair linkage analysis revealed significant linkage between these markers in the gene cluster and the FCH phenotype (MspI P = 0.0088, SstI P = 0.044, and XMS haplotype P = 0.037). The present findings confirm that the apo AI-CIII-IV gene cluster contributes to the FCH phenotype, but this contribution is genetically complex. An epistatic interaction between different haplotypes of the gene cluster was demonstrated. The S2 allele on one haplotype was synergistic to the X2M2 allele on the other haplotype in its hyperlipidemic effect. Therefore, two different susceptibility loci exist in the gene cluster, demonstrating the paradigm of complex genetic contribution to FCH.

Genomic organization and polymorphism of human angiotensin II type 2 receptor: no evidence for its gene mutation in two families of human premature ovarian failure syndrome

Angiotensin II type 2 (AT 2) receptor is highly expressed in the fetal tissues and decreases rapidly after birth. AT 2 receptor is re-expressed in the adult atretic ovarian follicles. Recently, it has been reported that AT 2 receptor mediates apoptosis. Primarily, we have cloned human AT 2 receptor cDNA and mapped it to the X-chromosome. To further analyze the organization and function of the AT 2 receptor gene, in this study we cloned the human AT 2 receptor genomic DNA. Human AT 2 receptor gene is composed of three exons and two introns. Primer extension analysis revealed a putative transcription initiation site at 24 bp downstream from TATA box. Furthermore, we identified a polymorphism (C–A) in 3′ untranslated region of exon 3, which may be a useful genetic marker for genetic analysis of human X-linked inherited disease. In this study, we postulated that the patients with premature ovarian failure, which has been reported to be linked with X-chromosome abnormality, have AT 2 receptor mutation that may contribute to the early onset of atresia. We examined the entire coding sequence of this receptor in two different families of sisters with premature ovarian failure (POF) but found no changes in nucleotide sequences.

Essential promoter elements are located within the 5′ untranslated region of human insulin-like growth factor-I exon I

Multiple mechanisms regulate insulin-like growth factor-I (IGF-I) gene expression in mammals, including transcription from two promoters, alternative RNA splicing, and differential RNA polyadenylation. In previous studies we demonstrated that IGF-I promoter 1, the major human promoter, initiated transcription within a dispersed 158 nt segment of exon 1, and showed that full promoter activity required the entire 322 nt 5′ untranslated region (UTR) of exon 1. We now have examined the functional significance of this highly conserved region by testing the activity of hybrid promoter-reporter genes containing various portions of the 5′ UTR after transient transfection into the IGF-I-producing SK-N-MC cell line. Recombinant plasmids containing the entire 322 nt 5′ UTR of exon 1 and a 1630 nt segment of 5′ flanking sequence stimulated luciferase activity nearly 70 times higher than a promoterless control plasmid. Truncation to +198 had little effect on promoter function, while subsequent 3′ deletions (to +111, +51, and +5) led to a stepwise decrease in reporter gene expression. Internal deletions of nt +6 to +50, +52 to +110, and +112 to +197 led to 65, 25, and less than 10% decreases in promoter function, respectively. Removal of the entire segment from +6 to +197 caused a complete loss of activity. Analysis for DNA-protein interactions by in vitro DNase-I footprinting identified a broad region of protection extending from nt −12 to +38. Further characterization by gel mobility shift assays indicated that several specific DNA-protein complexes could be formed in this region with nuclear protein extracts from SK-N-MC cells. Substitution mutations within the footprinted segment had deleterious effects on promoter function, and one mutation involving nt +10 to +20 resulted in a greater than 70% decline in reporter gene expression. Our results demonstrate that the initial portion of the 5′ UTR of human IGF-I exon 1 is required for high level basal transcription of promoter 1 and provide a starting point for defining the nuclear factors regulating this component of IGF-I gene expression.

Molecular analysis of the fructose transporter gene (GLUT5) in isolated fructose malabsorption.

Fructose, a naturally occurring monosaccharide, is increasingly used as an added sweetener in processed foods in the form of high fructose corn syrup. Increased fructose intake combined with the identification of children with clinical evidence of isolated fructose malabsorption (IFM) has stimulated interest in possible disorders of fructose absorption. The intestinal absorption of fructose is carried out by the facilitative hexose transporter, which has been designated as GLUT5. Functional properties and tissue distribution of GLUT5 suggest that IFM might be due to mutations in the GLUT5 gene. To test this hypothesis, we screened the GLUT5 gene for mutations in a group of eight patients with IFM and in one subject with global malabsorption, as compared with 15 healthy parents of subjects and up to 6 unrelated controls. No mutations were found in the protein coding region of this gene in any of the subjects. A single G to A substitution in the 5' untranslated region of exon 1 was identified in the subject with global malabsorption. This subject and her healthy mother were heterozygous for the variant sequence, suggesting that it was unlikely to be clinically significant. In addition, sequence analysis of each of the 12 GLUT5 exons was performed in the index case and confirmed the negative single-strand conformation polymorphism findings. These studies demonstrate that IFM does not result from the expression of mutant GLUT5 protein.

MTS1 gene mutations in archival oral squamous cell carcinomas.

Multiple tumor suppressor gene 1 (MTS1) has been found mutated or deleted in a variety of human cancers. Our purpose was to identify and characterize MTS1 gene mutations in primary oral squamous cell carcinomas (SCCs) in each of the three exons of the MTS1 gene. Seventeen archival samples of oral SCC were evaluated for the presence of MTS1 mutations using single strand conformation polymorphism (SSCP) and DNA sequencing. Three of 17 tumors exhibited MTS1 gene mutations: one tumor exhibited a mutation in exon 2 and two tumors exhibited mutations at the splice site junction of intron 2 and exon 3. Three tumors also exhibited a common base change in the 3' untranslated region of exon 3, which is interpreted as a likely polymorphic variant. An examination of the three tumors exhibiting MTS1 point mutations revealed no unique characteristics relative to p53 immunohistochemical activity, mitotic frequency, or degree of histologic differentiation. This study indicates that MTS1 gene mutations may be involved in at least a minor proportion of oral SCCs.

The human neuronal alpha 1-chimaerin gene contains a position-dependent negative regulatory element in the first exon.

alpha 1-Chimaerin mRNA, which encodes a neuron-specific GTPase-activating protein for the signal transduction molecule p21 Rac, is highly expressed in certain brain regions and neuronal cell lines. The promoter region of human alpha 1-chimaerin transcriptional unit contains no TATA box, Sp1-binding site or initiator motif. However, a CCAAT box located in the proximal promoter region is essential for promoter activity. We now describe a negative regulatory element in the 5' untranslated region of exon 1 of the human alpha 1-chimaerin gene. Deletion of this 70-bp region from the alpha 1-chimaerin minimal promoter increased the promoter activity 5- to 6-fold. The negative element can suppress heterologous thymidine kinase promoter activity in an orientation-independent manner when placed in its native position. However, its function is position-dependent. The presence of a putative factor in rat liver, HepG2 and SK-N-SH cell nuclear extracts but not in rat brain nuclear extract which interacts with this element suggests a possible role of the negative element in controlling the neuron-specific expression of alpha 1-chimaerin in vivo.

The KRAB Zinc Finger Gene ZNF74 Encodes an RNA-binding Protein Tightly Associated with the Nuclear Matrix

We previously cloned ZNF74, a developmentally expressed zinc finger gene commonly deleted in DiGeorge syndrome. Here, the intron/exon organization of the human gene and the functional properties of the expressed protein are presented. This zinc finger gene from the transcription factor IIIA/Kruppel family contains three exons. A truncated Kruppel-associated box (KRAB) located at the N terminus of the predicted 64-kDa zinc finger protein is encoded by exon 2. The remainder of the protein including the zinc finger domain as well as the 3'-untranslated region (UTR) is encoded by exon 3. Both 5'-UTR (exon 1) and 3'-UTR contain repetitive Alu elements. In vitro translation of a cDNA encoding the entire ZNF74 coding region produced a 63-kDa protein as determined on sodium dodecyl sulfate-polyacrylamide gel. A bacterially expressed fusion protein shown to bind tightly to 65zinc was used to test the nucleic acid binding properties of ZNF74. By RNA binding assays, ZNF74 was found to bind specifically to poly(U) and poly(G) RNA homopolymers. The restricted binding to these homopolymers and not to poly(A) and poly(C) suggested that ZNF74 displays RNA sequence preferences. RNA binding was mediated by the zinc finger domain. Immunofluorescence studies on transfected cells revealed ZNF74 nuclear localization. The labeling pattern observed in the nuclei clearly excluded the nucleoli. The zinc finger region lacks a classical nuclear localization signal but was found to be responsible for nuclear targeting. Subcellular and in situ sequential fractionations further showed that ZNF74 is associated with the nuclear matrix. The RNA binding properties of this protein and its tight association with the nuclear matrix, a subnuclear compartment involved in DNA replication as well as RNA synthesis and processing, suggest a role for ZNF74 in RNA metabolism.

Apolipoprotein A-I/C-III/A-IV gene cluster in familial combined hyperlipidemia: effects on LDL-cholesterol and apolipoproteins B and C-III.

The underlying genetic abnormalities in familial combined hyperlipidemia (FCH) have not been elucidated, although previous association and linkage studies have implicated the apoA-I/C-III/A-IV gene cluster. We now report studies of this cluster in 18 probands, 390 family members (hyperlipidemic relatives, n = 179; normolipidemic relatives, n = 211), and 177 spouses. Three restriction enzyme polymorphisms, XmnI and MspI sites 5' of the apoA-I gene and the SstI site in the 3' untranslated region of exon 4 of the apoC-III gene, were examined. In hyperlipidemic relatives and FCH probands, the frequency of each minor allele was significantly higher than in spouses. Associated with the higher frequency of minor alleles were elevated plasma cholesterol, triglycerides, LDL-cholesterol, apoB, and apoC-III levels. Quantitative sib-pair analysis revealed linkage between the MspI minor allele and plasma LDL cholesterol levels (P < 0.04). The present data indicate that, while apoA-I/C-III/A-IV gene cluster is not the primary cause of FCH, this cluster has a specific modifying effect on plasma triglyceride and LDL cholesterol levels.

A new MspIPCR-RFLP in the human LDL receptor gene.

A new two-allele PCR-based MspI restriction fragment length polymorphism (RFLP) was revealed at the human LDL receptor locus. The polymorphic MspI restriction site is located in the 3'-untranslated region of exon 18 and caused by a T to C substitution at nucleotide 3960. This new MspI RFLP may be useful with other informative RFLPs to follow the segregation of the LDL receptor gene in families with hypercholesterolemia.

Retrotransposon insertion induces an isozyme of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.

The insertion of the blood retrotransposon into the untranslated region of exon 7 of the sn-glycerol-3-phosphate dehydrogenase-encoding gene (Gpdh) in Drosophila melanogaster induces a GPDH isozyme-GPDH-4-and alters the pattern of expression of the three normal isozymes-GPDH-1 to GPDH-3. The process of transcript terminus formation inside the retrotransposon insertion reduces the level of the Gpdh transcript that contains exon 8 and increases the level of the transcript that contains exons 1-7. The induced GPDH-4 isozyme is a translation product of the three transcripts that contain fragments of the blood retrotransposon. The mechanism of mutagenesis by the blood insertion is postulated to involve the pause or termination of transcription within the blood sequence, which in turn is caused by the interference of a DNA-binding protein with the RNA polymerase. Thus, we show the formation of a new functional GPDH protein by the insertion of a transposable element and discuss the evolutionary significance of this phenomenon.

Genomic Organization of the Human Gene (CAS) and Pseudogene for Mitochondrial Carbonic Anhydrase V and Their Localization to Chromosomes 16q and 16p

Carbonic anhydrase V (CA V) is expressed in mitochondrial matrix in liver and several other tissues. It is of interest for its putative roles in providing bicarbonate to carbamoyl phosphate synthetase for ureagenesis and to pyruvate carboxylase for gluconeogenesis and its possible importance in explaining certain inherited metabolic disorders with hyperammonemia and hypoglycemia. Following the recent characterization of the cDNA for human CA V, we report the isolation of the human gene from two λ genomic libraries and its characterization. The CA V gene (CA5) is approximately 50 kb long and contains 7 exons and 6 introns. The exon-intron boundaries are found in positions identical to those determined for the previously described CA II, CA III, and CA VII genes. Like the CA VII gene, CA5 does not contain typical TATA and CAAT promoter elements in the 5′ flanking region but does contain a TTTAA sequence 147 nucleotides upstream of the initiation codon. CA5 also contains a 12-bp GT-rich segment beginning 13 bp downstream of the polyadenylation signal in the 3′ untranslated region of exon 7. FISH analysis allowed CA5 to be assigned to chromosome 16q24.3. An unprocessed pseudogene containing sequence homologous to exons 3-7 and introns 3-6 was also isolated and was assigned by FISH analysis to chromosome 16p11.2-p12.

Mutational analysis of the estrogen-receptor gene in endometrial carcinoma

To determine whether mutation in the DNA of the estrogen-receptor gene occurs in endometrial cancer. Polymerase chain reaction amplification and single-stranded conformation polymorphism analysis of the entire coding region (exons 1-8) of the human estrogen-receptor gene, as well as an untranslated region (exon I*) in the gene, were performed on genomic DNA extracted from 56 snap-frozen endometrial cancers. All cancers demonstrating mobility shifts on single-stranded conformation polymorphism suggestive of DNA sequence alteration were subjected to definitive DNA sequencing of the relevant portion of the estrogen-receptor gene. In addition to detecting a frequent, previously described polymorphism in exon 1, single-stranded conformation polymorphism analysis of the 56 endometrial cancers identified seven cancers with mobility shifts. Three cancers shifted in exon 3, one cancer each shifted in exons 4 and 7, and two shifted in exon 8. Deoxyribonucleic acid sequencing revealed sequence alterations in all seven cases demonstrating mobility shifts. In six of these seven cases, these alterations were consistent with infrequent silent polymorphisms; in the seventh cancer, the sequence alteration proved to be a somatic missense mutation at codon 537 in the region of the estrogen-receptor gene encoding the hormone-binding domain of the receptor protein. The infrequent DNA mutation in the estrogen-receptor gene is unlikely to account for the variation in estrogen-receptor expression observed in endometrial cancer.

Transcriptional Regulation of Decorin Gene Expression: INDUCTION BY QUIESCENCE AND REPRESSION BY TUMOR NECROSIS FACTOR-a

Decorin, a leucine-rich proteoglycan with ubiquitous tissue distribution, may play essential biological roles during inflammation and cancer growth through its ability to bind extracellular matrix constituents and growth factors. In this study, we demonstrate that decorin gene expression is greatly enhanced after normal diploid fibroblasts reach confluency and cease to proliferate. Elevation of decorin mRNA steady state levels was maintained for up to 16 days postconfluency. In vitro transcription analyses indicated enhanced transcriptional activity in quiescent fibroblasts when compared to cells harvested in their logarithmic phase of growth. This phenotypic trait was reversed by the exogenous addition of tumor necrosis factor-alpha (TNF-alpha). Furthermore, transforming growth factor-beta (TGF-beta) down-regulated decorin gene expression in an additive manner with TNF-alpha. Transient cell transfection assays using plasmid constructs harboring the decorin promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated a dose-dependent transcriptional repression by TNF-alpha. These findings were further corroborated by in vitro transcription experiments using nuclear extracts from control and TNF-alpha-treated quiescent fibroblasts. In contrast, the decorin promoter constructs failed to respond to TGF-beta, thus suggesting either post-transcriptional regulation by this growth factor or lack of TGF-beta-responsive elements. Further experiments with 5' deletion constructs showed two TNF-alpha response elements, one residing within the 5'-untranslated region (exon Ib), the other one between residues -188 and -140 of the decorin promoter. Collectively, our results indicate that TNF-alpha, through its ability to transcriptionally inhibit decorin gene expression in growth-arrested cells, may be a key modulator of the biological functions of this proteoglycan.

Screening the dopamine D1 receptor gene in 131 schizophrenics and eight alcoholics: identification of polymorphisms but lack of functionally significant sequence changes.

To determine whether mutations in the D1 dopamine receptor (D1 DR) gene are associated with schizophrenia, the coding sequence was examined in 106 Caucasian, 11 African-American, 8 Asian, and 6 Native American patients. Approximately 350 kb of genomic sequence was screened by dideoxy fingerprinting, a method related to single strand conformational polymorphism (SSCP) analysis that detects virtually 100% of sequence changes [Sarkar et al., 1992: Genomics 13:441-443; Liu and Sommer, 1994: PCR Methods and Applications 4:97-108]. One polymorphism was identified in Asians and one in Caucasians, but neither altered the amino acid sequence (Leu66, and Ser421, respectively). In addition, a previously reported polymorphism in the 5' untranslated region of exon 2 at bp -48 was found to be common, with an allele frequency of approximately 40% in Caucasians of Western European descent. Based on the fact that no sequence changes of likely functional significance were identified, these data suggest that mutations affecting the structure of the D1 dopamine receptor protein are uncommon and are unlikely to contribute significantly to the genetic predisposition to schizophrenia. The D1 DR gene also was examined in eight alcoholics, including 3 African-Americans and 1 Native American, but no sequence changes were identified.

Translation in vivo of 5′ untranslated-region splice variants of human surfactant protein-A

Transcripts of human SP-A genes, SP-A1 and SP-A2, undergo alternative splicing of 5' untranslated-region exons. We reverse-transcribed and amplified free cytoplasmic and polysome-bound RNA and showed that (a) all splice variants of both genes are translated in vivo, (b) the relative translatability of splice variants can differ among individuals, and (c) the relative levels of different SP-A splice variants differ among individuals.

CTLA-4 gene polymorphism associated with Graves' disease in a Caucasian population.

Graves' disease (GD) is an autoimmune thyroid disease. Multiple genetic factors are believed to be involved in its pathogenesis, but the factors are largely unknown, except for sex (female disease preponderance) and the role of human leukocyte antigen (HLA) genes on chromosome 6. To understand the mechanisms underlying the development of GD, a search for non-HLA-linked genes is crucial, and we tested several candidate genes, including the CTLA-4 gene on chromosome 2q33. CTLA-4 molecules may either facilitate or down-regulate the second signal to T-cells, which is provided by the interaction between the two accessory molecules CD28 and B7. One hundred and thirty-three Caucasian patients (26 males) with GD and 85 local controls were included in this study. Polymerase chain reaction was used to amplify DNA containing the (AT)n repeat within the 3'-untranslated region of exon 3 of the CTLA-4 gene. The 5'-forward primer was radiolabeled, and amplified products were resolved on 5-7% sequencing gels. All subjects were previously typed for HLA class II alleles. Twenty-one alleles were observed with sizes ranging from 88-134 basepairs. In the association analysis, the genotype frequencies between GD patients and controls differed significantly (P = 0.012), and the difference was attributable to a higher frequency of the 106-basepair allele among patients (relative risk, 2.82). When the patients were subdivided with respect to sex and HLA, the phenotype frequencies of allele 106 was higher in the female patients with protective HLA specificities (DQA1*0201 positive/DQA1*0501 negative) than in those with susceptible HLA specificities (DQA1*0201 negative/DQA1*0501 positive; 81.8% vs. 45.5%; P = 0.026). The CTLA-4 gene or a closely associated gene (including CD28) confers susceptibility to GD. This association may be more important in female patients with protective HLA specificities, who otherwise would be at low risk of developing the disease.

Genetic Variability of the ET-1 and the ETA Receptor Genes in Essential Hypertension

Because evidence suggests that endothelin-1 (ET-1) plays a role in the pathogenesis of hypertension, we examined the variability within the ET-1 and the ET(A) receptor genes in patients with essential hypertension (EH). Genomic DNA was used for amplification of both genes by PCR. Polymorphisms within these genes were identified by single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequencing. Single-base insertions (C at position 121; A at position 138) were identified in the untranslated region of exon 1 of the ET-1 gene. The C insertion was invariant, whereas the A insertion, which abolished a BsiY1 restriction site, occurred only in samples that showed altered mobility profiles on SSCP analysis. This strategy also identified a silent polymorphism in codon 323 (CAC-->CAT) of the ET(A) receptor gene, which created an AflII restriction site. The frequency distribution of these polymorphisms was compared in an EH population [diastolic blood pressure (DBP) > or = 95 mm Hg; n = 100] and a matched normotensive (NT) group (BP < or = 140/85 mm Hg). No significant differences in AflII genotype distribution were found between the EH and NT groups. However, chi 2 analysis suggested a significant difference between the BsiY1+/+, BsiY1-/+, and BsiY1-/- genotype frequencies in the two groups (EH 58:38:4%; NT 47:44:9% (p = 0.045)). In addition, two-way analysis showed a strong correlation between DBP and the BsiY1-/- polymorphism. These results suggest that these polymorphisms act as markers for the level of DBP.

Genomic structure of the mouse apurinic/apyrimidinic endonuclease gene

A mammalian apurinic/apyrimidinic endonuclease (AP endonuclease) is known to have two distinct functional domains. One domain is responsible for regulating the activity of Fos/Jun proto-oncogene products to bind to DNA at specific recognition sites. The other domain, which is highly conserved from bacteria to mammals, is responsible for repairing DNA damage caused by ionizing radiation, oxidative damage, and alkylating agents. This study reports on the isolation and characterization of the genomic structure of the mouse AP endonuclease gene (Apex). The genomic sequence of the Apex gene was 2.14 kb in length and contained four exons. Exon 1 contained a 0.24-kb untranslated 5' region upstream of the initiation codon. Consensus sequences for two CAAT boxes and a GC box were found upstream of the end of exon 1. A polymorphism was noted in the untranslated region of exon 1 in a comparison of a number of mouse strains. These data indicate that the 5' end of the mouse gene (Apex) differs from the previously isolated human gene (Ape), which has five exons and an untranslated region between exons 1 and 2. Data are also presented that suggest the presence of two pseudogenes in the mouse.

Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no alterations in deoxyribonucleic acid-protein interactions within the major promoter.

Insulin-like growth factor-I (IGF-I) is an important mediator of prenatal and postnatal growth, but little is known about the control of IGF-I gene expression. Previously, we demonstrated that GH rapidly stimulates hepatic IGF-I transcription in vivo in hypophysectomized (hypox) rats. In this study, we show that GH induces IGF-I gene transcription through the major promoter, promoter 1, and identify and characterize DNA-protein interactions throughout the promoter. In vitro deoxyribonuclease-I footprinting was used to analyze 1711 nucleotides of promoter 1 and the entire 328-nucleotide 5'-untranslated region of exon 1, using hepatic nuclear protein extracts from male juvenile hypox rats given a single ip injection of GH or saline 60 min before death. Fourteen DNA-protein binding sites were identified, with 6 located in the highly conserved 5'-untranslated region of exon 1. These latter sites were further characterized for specificity and regulation by GH, using gel mobility shift assays. Two of these DNA-protein interactions were also detected by in vivo dimethylsulfate footprinting. All DNA-protein binding was seen using hepatic nuclear protein extracts from hypox rats and did not change within 15, 30, 60, or 120 min after treatment with GH. Our results thus define a series of constitutive DNA-protein interactions within the major rat IGF-I gene promoter that may be involved in mediating GH-activated nuclear signals to initiate IGF-I transcription.