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human chromosome 20. The regulation of growth hormone synthesis and secretion in the anterior pituitary is under complex neural and hormonal control (1).
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 63-67, January 1985 Biochemistry

Gene encoding human growth hormone-releasing factor precursor: Structure, sequence, and chromosomal assignment (peptide hormone/hypothalamus/alternative RNA processing/chromosome sorting)

KELLY E. MAYO*, GAIL M. CERELLI*, ROGER V. LEBOt, BARRY D. BRUCEt, MICHAEL G. ROSENFELDt, AND RONALD M. EVANS* *Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92138; tHoward Hughes Medical Institute, Department of Medicine, University of California, San Francisco, CA 94143; and tEukaryotic Regulatory Biology Program, School of Medicine, University of California, San Diego, CA 92093

Communicated by Helen M. Ranney, August 31, 1984

MATERIALS AND METHODS

We have isolated and characterized overlapABSTRACT ping clones from phage X and cosmid human genomic libraries that predict the entire structure of the gene encoding the precursor to human growth hormone-releasing factor. The gene includes five exons spanning 10 kilobase pairs of human genomic DNA. There appears to be a segregation of distinct functional regions of the GRF precursor and its mRNA into the five exons of the gene. The DNA sequences of all exons, intron/exon boundaries, and 5' and 3' flanking regions are presented. Dot-blot analysis of DNA from high resolution duallaser-sorted human chromosomes indicates that the singlecopy growth hormone-releasing factor gene is located on human chromosome 20.

Isolation and Mapping of Genomic Clones. The 350-basepair (bp) EcoRI/BamHI fragment from a human GRF cDNA clone (7) was nick-translated using [a-32P]dCTP (410 Ci/ mmol; 1 Ci = 37 GBq) to a specific activity of >108 cpm/hg and used as a hybridization probe to screen at high density human genomic libraries constructed in either X Charon 28 (9) or cosmid pHC79 (10). Positive plaques or colonies were rescreened at lower density until pure. A combination of restriction enzyme mapping and Southern DNA blotting was used to locate regions of the clones that hybridized to the GRF cDNA probe. Exon 1 was localized using a kinase-labeled synthetic oligonucleotide predicted from the sequence of a larger human GRF cDNA (8) to be specific for the 5' nontranslated region of the GRF mRNA (the oligonucleotide corresponds to nucleotides 36-67 of Fig. 3). Details of all techniques used to isolate and analyze these genomic clones have been described (11). [a-32P]dCTP was from New England Nuclear; all enzymes were from either New England Biolabs or Bethesda Research Laboratories. DNA Sequencing. Regions of genomic clones that hybridized to the GRF cDNA probe were subcloned in plasmid vectors by standard techniques. Restriction-enzyme-digested DNA was 5' labeled using bacterial alkaline phosphatase, T4 polynucleotide kinase, and [y-32P]ATP (11). Pst I sites were also labeled by use of [a-32P]dCTP and T4 DNA polymerase (11). DNA sequence determination was by the chemical method of Maxam and Gilbert (12). RNA Analysis. Poly(A)+ RNA was prepared by standard techniques from a human thymic carcinoma ectopically producing GRF (kindly provided by M. Thorner, University of Virginia). RNA blot analysis was carried out as described (13), using denaturing formaldehyde/agarose gels. For primer extension of RNA, a synthetic oligonucleotide complementary to a 32-nucleotide sequence from the 5' nontranslated region of the GRF mRNA (see above) was synthesized. Hybridization and primer extension were done as described (14), using avian myeloblastosis virus reverse transcriptase (Bethesda Research Laboratories). For nuclease S1 mapping, the 450-bp Xba I/EcoRV fragment including most of the first exon (see Fig. 1B) was 5'-end-labeled at the EcoRV site by use of polynucleotide kinase, strand-separated on an acrylamide gel (11), and used according to established protocols (15). Chromosome Mapping. Chromosome suspensions were prepared from a lymphocyte cell line and then stained with

The regulation of growth hormone synthesis and secretion in the anterior pituitary is under complex neural and hormonal control (1). This regulation is mediated in part by two hypothalamic releasing factors. The inhibitory peptide, somatostatin, was characterized more than a decade ago (2) and the stimulatory peptide, growth hormone-releasing factor (GRF), was isolated and characterized only recently from two human pancreatic tumors possessing growth hormonereleasing activity (3, 4). Human GRF is a 44-amino acid peptide amidated at the COOH terminus. Nonamidated forms, consisting of 40 and 37 amino acids, that have full biological activity were also characterized (3, 4). Antibodies raised against the GRF-40 peptide specifically stain cell bodies and nerve fibers in the medial basal hypothalamus and median eminence, suggesting that the peptide isolated from pancreatic tumors is the same as that found in the hypothalamus (5, 6). GRF and other hypothalamic releasing factors play a central role in directing endocrine responses to neural stimuli; analysis of their biosynthesis, therefore, provides a unique opportunity to study coordinate neural and hormonal control of gene expression. Toward this goal, we, and others, have isolated cDNA recombinant clones encoding human GRF (7, 8). Analysis of these cDNA clones indicated that mature GRF is proteolytically processed from a 108-amino acid precursor protein. To further studies of the regulation of this physiologically and clinically important protein, we have now isolated and structurally characterized genomic clones containing the entire human GRF gene. In addition, to further the analysis of the potential role of GRF in various heritable human growth disorders, we have mapped the GRF gene to human chromosome 20. The publication costs of this article were defrayed in part by page charge

Abbreviations: GRF, growth hormone-releasing factor; bp, base pair(s); kb, kilobase pairs; DIPI, 4',6-bis(2"-imidazolinyl-4H,5H)-2phenylindole.

payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Human GRF genomic clones and subclones. (A) Structure of three overlapping clones isolated from human genomic libraries. Dark boxes indicate regions of hybridization to GRF cDNA or oligonucleotide probes, as described in the text. Wavy lines indicate the phage or cosmid vector DNA. The scale is broken by the slashes at the 3' ends of the phage clones and at the 5' end of the cosmid clone. A restriction map of sites for seven different enzymes is shown; all sites indicated are present within the insert of one or more of the clones and do not represent sites within the vector DNA. (B) Subclones used in the sequence analysis of the GRF gene. The location of each subclone within the genomic clones can be determined from A. Subclones and their derivation are: exon 1, 3.5-kb BamHI/BamHI fragment from hGRFcos49; exons 2 and 3, 1.7-kb EcoRI/Bgl II fragment from hGRFcos49; exon 4, 4.5-kb Xma I/Xma I fragment from hGFRX101; exon 5, 1.3-kb HindIII/HindIII fragment from hGRFX101. The dark boxes indicate the exons and parallel slashes indicate a break in the scale. Regions sequenced are indicated below each subclone; filled circles at the origins of arrows indicate restriction sites that were radiolabeled, and the arrows represent the direction and extent of sequence analysis. The two sets of arrows from the unique Pst I site in exon 4 indicate sequence reactions carried out after labeling either 5' ends with T4 polynucleotide kinase or 3' ends with T4 DNA polymerase as described in Materials and Methods.

4',6-bis(2"-imidazolinyl-4H,5H)-2-phenylindole (DIPI)/chromomycin A3 stain-pair (16). Thirty thousand chromosomes of each type were sorted onto a single spot of a nitrocellulose filter using a dual-laser custom FACS IV chromosome-sorter (17). The filter-bound chromosomal DNA was denatured, neutralized, prehybridized, and then hybridized in 10% dextran sulfate to nick-translated GRF cDNA probe (see above). Filters were washed and autoradiographed using standard conditions.

phage A library constructed in Charon 28 (9); we isolated two phage clones that hybridized strongly to the human GRF cDNA probe. The structures of these clones (hGRFX101 and hGRFX111) are shown in Fig. 1A. These two overlapping phage clones were found to contain four distinct regions of hybridization to the GRF cDNA probe; however, subsequent analysis revealed that neither contained the entire GRF gene. Because our experience with the phage clones indicated that the GRF gene was substantially larger than expected, we next screened a human cosmid gene bank (10) and isolated a cosmid clone that overlapped substantially with the two phage clones; the structure of this cosmid clone (hGRFcos49) is shown in Fig. 1A. Because our human GRF cDNA clone did not include 5' nontranslated sequences, it was difficult to conclusively show that the cosmid clone included the 5' end of the GRF gene. To establish this, we used a synthetic oligonucleotide specific for the 5' nontranslated region, based on the published sequence of another, larger human GRF cDNA clone (ref. 8; see Materials and Meth-

RESULTS Screening of Human Genomic Libraries. The insert from a previously isolated human GRF cDNA clone was used as a hybridization probe to screen human genomic libraries. This probe includes nearly all of the GRF precursor coding sequences and the 3' nontranslated region, but does not include the 5' nontranslated region. We initially screened 250,000 plaques from a human phage A library constructed in Charon 4A (18) and 400,000 plaques from another human 5'

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FIG. 2. Structure of the human GRF cDNA and gene. Top line shows a schematic of the GRF cDNA. The 5' nontranslated region (5' NT), the-signal-peptide-encoding region, the GRF-encoding region, the 3' nontranslated region (3' NT), and the poly(A) tract are indicated. Shaded regions encode the NH2-terminal and COOH-terminal flanking peptides (7, 8). Bottom line shows a schematic of the GRF gene. Open boxes indicate noncoding exon regions; dark boxes indicate coding exon regions. Connecting lines indicate the relationship between structural regions of the cDNA and

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Biochemistry: Mayo et aL ods). Use of the combined cDNA and oligonucleotide probes showed that the three overlapping genomic clones depicted in Fig. 1A contained the entire human GRF gene. Structure of the GRF Gene. The three genomic clones isolated (Fig. 1A) were analyzed in detail by restriction enzyme mapping, Southern DNA blotting, and partial DNA sequence analysis. The complete structure of the human GRF gene as predicted from these overlapping genomic clones is shown schematically in Fig. 2. The gene includes five exons separated by 4 introns and spans 10 kilobase pairs (kb) of genomic DNA. Fig. 2 also compares the structure of the gene with that of the cDNA, and shows that the five exons of the gene appear to encode functionally discrete domains of the GRF precursor and its mRNA. Exon 1 includes the 5' nontranslated sequences, exon 2 encodes the signal peptide and small NH2-terminal connecting peptide, exon 3 encodes most of the mature GRF peptide (including all of the biologically active portion), exon 4 encodes the COOH-terminal peptide (of unknown function), and exon 5 contains the 3' nontranslated sequences. DNA Sequence of the GRF Gene. To determine more precisely the structure of the GRF gene and to define sequences important for the expression of this gene, we have sequenced all exons, intron/exon boundaries, and 5' and 3' flanking regions of the human GRF gene. For sequencing, regions of GRF genomic clones were first subcloned in plasmid vectors. The strategy used for DNA sequence analysis is shown in Fig. 1B. The sequence determined is presented in Fig. 3. The sequence is numbered from the putative mRNA 5' cap site, determined as described in the following section. The sequences of all exons agree precisely with those previously determined from human GRF cDNA clones (7, 8). All of the 4 introns begin and end with the consensus sequences G-T and A-G, respectively (19). Sequences related to consensus "TATA" and "CAT" boxes, found in the 5' flanking region of many eukaryotic genes (19, 20), are located 30 and 75 nucleotides, respectively, 5' of the putative mRNA cap site. The sequence A-A-T-A-A-A, thought to be important in polyadenylylation (21), is located 31 nucleotides 5' of the poly(A) addition site. It was previously reported (8) that two distinct types of GRF cDNAs exist. These forms differ by the inclusion or exclusion of 3 nucleotides from the coding region and encode either a 107- or a 108-amino acid GRF precursor protein. Our sequence analysis suggests that these two forms result from alternative RNA processing of a single GRF gene primary transcript. As indicated in Fig. 3, differential utilization of two splice-acceptor sites at the beginning of exon 5 would result in inclusion or exclusion of serine-103 and generate either the 107- or the 108-amino acid forms of the precursor. Analysis of the GRF genomic clones revealed a sequence from exon 2 that was highly repeated in the human This repetitive element has been completely sequenced (see Fig. 1B for location and sequencing strategy) and was found to be highly homologous to the consensus sequence for the human alu family of repeats (22). RNA Analysis. Because it was not obvious from sequence analysis of cDNA and genomic clones where the mRNA 5' cap site was located, we attempted to directly determine this by an analysis of GRF mRNA. To do this, we utilized RNA isolated from a human thymic carcinoma ectopically producing GRF (GRF-tumor). Blot hybridization analysis of this RNA, shown in Fig. 4A, revealed that the GRF mRNA is approximately 750 nucleotides long. Assuming that the poly(A) tract contributes -200 residues (23), this indicates that the cap site should be about 550 nucleotides upstream from the poly(A) addition site. To more precisely define the 5' end of the GRF mRNA, we performed primer extension of GRF-tumor poly(A)+ RNA that had been hybridized to a synthetic oligonucleotide corresponding to a cDNA se-

Proc. Natl. Acad. Sci. USA 82 (1985)

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Fig. 3; a minor extension product of slightly larger size was also identified (Fig. 4B). Finally, we performed a nuclease S1 mapping experiment using GRF-tumor RNA (Fig. 4C). A single band is present in the GRF-tumor RNA sample that is not present in the control RNA sample (Fig. 4C). This protected fragment maps to the C residue at position -1 in Fig. 3. The resolution of these techniques localizes the cap site to within a few nucleotides; however, since most eukaryotic mRNAs begin at an adenosine within the consensus sequence Py-C-A-Py, where Py is a pyrimidine (24), we presume that the A at position 1 in Fig. 3 is most likely to correspond to the 5' end of -350

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FIG. 3. DNA sequence of the human GRF gene. Nucleotide numbering is from the proposed mRNA cap site; the intron nucleotides are not numbered. Boxed regions indicate consensus sequences as described in the text; they are the CAT box, TATA box, and A-A-T-A-A-A sequence, in that order. Arrows indicate the beginning of exons or the poly(A) addition site (last arrow). The twotailed arrow at the beginning of exon 5 indicates two alternative splice-acceptor sites, both of which appear to be utilized (see text). The larger letters represent nucleotides found in the mature mRNA. The first and last 10 nucleotides of each intron are shown and the approximate length of each intron is indicated. Amino acids indicated in italic print are those of the mature 44-amino acid GRF peptide.

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9"~ FIG. 4. Analysis of human GRF mRNA. (A) RNA blot analysis. Lane 1, HeLa cell poly(A)+ RNA (5 pg); lane 2, poly(A)+ RNA (5 gg) from a human tumor ectopically producing GRF. The molecular weight markers are HindIII-digested and denatured simian virus 40 DNA fragments; sizes indicated are in nucleotides. (B) Primer-extension analysis. Lane 1, a synthetic oligonucleotide was used to prime synthesis with reverse transcriptase from human GRF-producing tumor poly(A)+ RNA (10 Ag). G, A, T, and C indicate a set of DNA sequence reactions carried out using a fragment labeled at the EcoRV site in exon 1 (see Fig. 1). The sequence ladder cannot be used to directly read the position of the primerextended product, but rather serves as a complete set of accurate size markers. The size of the band indicated is in nucleotides. The material at the bottom of lane 1 is from labeled primer that has not been extended. (C) Nuclease S1 protection analysis. RNA samples were hybridized to a single-stranded probe labeled at the EcoRV site in exon 1 (see Fig. 1), and nuclease-resistant products were analyzed. Lane 1, no RNA; lane 2, HeLa cell poly(A)' RNA (5 ug); lane 3, human GRF-producing tumor poly(A)+ RNA (5 pg). G, A, T, and C indicate a set of DNA sequence reactions carried out on the labeled fragment used as probe; the position of the nuclease-resistant band can therefore be read directly from the sequence ladder (which is of the anti-sense strand). Several faint bands were present in the higher molecular weight region of the gel (not shown); however, all of these bands were also present in the control lanes (1 and 2) and are presumed to be nonspecific.

the GRF mRNA. This would place the transcription initiation site 30 nucleotides downstream from the TATA box, in agreement with the consensus distance (27-33 nucleotides) between these sequences (19, 24). Interestingly, one cDNA clone that contains sequences 5' of this putative cap site has been identified (8). It is thus possible that a small percentage of the GRF transcripts are initiated from an upstream site. Chromosomal Location of the GRF Gene. Previous analysis of human genomic DNA by Southern blotting using GRF cDNA probes had indicated that the GRF gene was most likely single-copy in the human genome (7). To determine the chromosomal location of this gene, we used a dual-laser custom fluorescence-activated cell-sorter to sort mitotic chromosome suspensions stained with DIPI/chromomycin in conjunction with Hoechst 33258/chromomycin. This technique allows separation of the 24 human chromosome types into 22 fractions (16). After the chromosomes were sorted directly onto nitrocellulose, the chromosomal DNA was denatured and hybridized to a human GRF cDNA probe (7) (Fig. 5). In three independent experiments the GRF genespecific probe hybridized to DNA from chromosome 20, but not to DNA from any other chromosomal fraction. We therefore assign the human GRF gene to chromosome 20.

DISCUSSION We have determined the complete structure of the human GRF gene. This small peptide is encoded by a gene that contains 5 exons and spans 10 kb of human genomic DNA. We have noticed that there appears to be a segregation of distinct structural and/or functional regions of the GRF precursor and its mRNA into the 5 exons of the gene. It is interesting that, although the complete GRF peptide is encoded by two exons (exon 3 and part of exon 4), all of the biologically active portion of the peptide (3, 4) is encoded in exon 3. This exon therefore seems to encode a distinct functional domain. Several other examples of genes in which exons encode discrete structural or functional domains of the protein product have been observed, although the significance of this observation is unclear (25).

DNA sequence analysis provided the complete structure of the GRF gene and revealed that this gene contains all recognized consensus sequences thought to be involved in gene transcription and RNA processing. The 5' flanking region contains two unusual variations of the consensus TATA and CAT sequences (24). In the TATA box, believed to be important for transcription initiation, a G substitutes for the T normally found at position 1, generating the sequence G-AT-A-A-A-T. Further evidence that the assignment of the CAT and TATA boxes is correct comes from an analysis of the rat GRF gene. The rat GRF gene is highly homologous to the human gene in this region, and contains consensus CAT and TATA box sequences in positions exactly analogous to those described here for the human gene (unpublished results). All intron/exon boundaries within the human GRF gene conform to previously described consensus splicedonor and splice-acceptor sequences (19). Two forms of the GRF precursor protein, 107 and 108 amino acids long, have been identified by sequence analysis of

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FIG. 5. Chromosome mapping of the human GRF gene. A duallaser custom fluorescence-activated cell-sorter was used to sort DIPI/chromomycin-stained human mitotic chromosome suspensions into 22 fractions representing the 24 human chromosome types (chromosomes 10-12 are pooled). Thirty thousand chromosomes of each type were sorted and the DNA was hybridized to a human GRF cDNA probe (see Materials and Methods). Regions of a nitrocellulose filter onto which DNAs from four of the chromosomal fractions were sorted are shown. Chromosome 20 was positive in each of three independent experiments. All other chromosome fractions were negative (data not shown).

Biochemistry: Mayo et aL cDNA clones from a human pancreatic tumor (8). Southern blot analysis of human genomic DNA reveals that the GRF gene is single-copy (7), suggesting that the multiple protein precursors are derived from a single gene. Analysis of the GRF gene sequence identifies two consensus splice-acceptor sites (19), spaced by three nucleotides, at the 5' end of exon 5. Differential usage of these two possible splice-acceptor sites would result in either inclusion or exclusion of serine-103 and thus generate the 107- or 108-amino acid forms of the precursor. After proteolytic processing of the GRF precursor protein, this difference would be expected to result in either a 30- or a 31-amino acid COOH-terminal peptide of unknown function. Preliminary analysis of the rat GRF gene (unpublished results) indicates that two consensus splice-acceptor sites spaced by three nucleotides also occur at the 5' end of exon 5 in this gene. A similar type of alternative RNA processing occurs in the human growth hormone gene, where differential usage of two splice-acceptor sites generates growth hormone proteins that differ by inclusion or exclusion of 15 amino acids (26). Because GRF is believed to be an important physiological regulator of growth hormone synthesis and secretion (27), it might potentially be involved in some of the described clinical syndromes in which growth hormone production is impaired: for example, familial isolated growth hormone deficiency (IGHD) and pituitary dwarfism (28). We have now determined by a combined chromosome-sorting/dot-blotting technique that the GRF gene is located on human chromosome 20. This observation indicates that the GRF gene is chromosomally linked to the loci for adenosine deaminase, the src proto-oncogene, inosine triphosphatase, and S-adenosylhomocysteine hydrolase (29). It further suggests that, if GRF is involved in any of the described human growth disorders, it is likely to be involved in those that demonstrate an autosomal inheritance (such as IGHD type IB or pituitary dwarfism type I) rather than in those that are X-linked (such as IGHD type III or pituitary dwarfism type II) (28). The availability of human GRF gene probes will allow us to begin to analyze the manner in which expression of this gene is regulated by hormonal and neural factors. In addition to studying regulation in the hypothalamus, it will now be possible to introduce the cloned gene into animal cells or animals in an attempt to define the types of factors that regulate this gene as well as the genomic sequences that mediate this regulation. We thank Michael Thorner for providing the tumor tissue, Phil Leder and John Collins for making human genomic libraries available, Michael Harpold for providing the synthetic oligonucleotide, and Estelita Ong for help in growing phage. We appreciate comments on the manuscript by our colleagues and the secretarial assistance of Marijke terHorst and Connie Meloan. This work was supported by grants from the National Institutes of Health and National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases

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