Gene, 93 (1990) 321-322 Elsevier

321

GENE 03665

The proximal 5'-flanking region of the gene encoding human growth hormone-releasing factor contains an inserted A l u sequence* (Recombinant DNA; molecular cloning; repetitive DNA; differential gene expression; placenta)

Sergio Gonzilez-C'respo, Merc~ Monfar and Albert Boronat Unit of Biochemistry, School of Pharmacy, Univ. of Barcelona, Barcelona (Spain) Received by M. Salas: 30 October 1989 Revised: 30 January 1990 Accepted: 10 May 1990

SUMMARY

An Alu sequence has been identified between nt -276 and -602 in the 5'-flanking region of the gene encoding human growth hormone-releasing factor.

Human and rat GRFs are encoded by single-copy genes which have been previously characterized (Mayo et al., 1985a,b). Although their expression was initially assigned to the hypothalamus and some ectopic tumours, the presence of immunoreactive GRF in non-hypothalamic sites (Gelato and Merriam, 1986) suggested that the gene could also be expressed in other tissues. We present here the sequence of 1221 bp of the 5'flanking region of a new hGRF genomic clone (~-hGRF-12) isolated from a human peripheral blood DNA library. It contains 826 additional nt upstream from the one previously reported (Mayo et al., 1985a). Sequence analysis revealed the presence of an inverted Alu sequence (nt -276 to -602) referred to as Alu-hGRFI in Fig. 1 which shows all the typical features of a consensus Alu sequence (Kariya et al., 1987). A possible second Alu sequence (Aiu-hGRF2), split in the cloning process, has been found at position - 1159 to -1221 (Fig. 1). The human and rat GRF 5'-flanking regions are highly homologous and their alignment is inter-

rupted by Alu-hGRFI which is only present in the human sequence (Fig. 1). This, together with the two 12-bp perfect direct repeats flanking Alu-hGRFI, suggests ~i~at AluhGRFI would be the result of a recent insertion event in the human genome that occurred after the divergence between rodents and primates. Alu sequences and other transposable-like elements have already been reported in the proximal 5'-flanking region of other mammalian genes and their functional role in regulating gene expression has been considered (Oliviero and Monaci, 1988; Tronik et al., 1988). As concerns GRF, the presence of an Aiu insertion in the human gene together with the demonstration ofGRF immunoreactivity in rat placenta but not in human placenta (Meigan et al., 1988) prompted us to study the possible differential expression of GRF between human and rat placenta. In this respect, Northernblot analysis revealed GRF transcription in rat placenta, but not in human placenta or in the human placental cell lines JAR (ATCC HTB 144) and BeWo (ATCC CCL98)

Correspondence to: Dr. A. Boronat, Unit of Biochemistry, School of Pharmacy, University of Barcelona, Plaza Pio XII s/n, 08028-Barcelona (Spain) Tel. (34-3)3307963; Fax (34-3) 3302903. * On request, the authors will supply detailed experimental evidence for the conclusions reached in this brief note.

Abbreviations: bp, base pair(s); GRF, growth-hormone-releasing factor; GRF, gene coding for GRF; hGRF, gene coding for human GRF; kb, kilobase(s) or 1000 bp; nt, nucleotide(s).

0378-1 ! 19/90/$03.50 O 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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REFERENCES

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(not shown). These results suggest that the lack of hGRF expression in placenta could be related to the AIu-hGRFI insertion, although this statement remains to be assessed.

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Fig. I. Sequence alignment of the human (H) and rat (R) GRF 5'-flanking regions. Identical nt are connected by vertical lines. AluhGRF! (nt -276 to -602) is underlined; it contains two monomer units with an A-rich region at their right ends, an internal 18-bp region equivalent to an internal RNA polymerase I11 promoter sequence (boxed) and is flanked by two 12.bp perfect direct repeats (indicated by arrows). The sequence corresponding to Alu.AORF2 (nt - 11$9 to - 122 I) is also underlined. The sequence reported in this paper has been deposited in the EMBL Data Library under accession number X13132,

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Gelato, M.C. and Merriam, G.R.: Growth hormone releasing hormone. Annu. Rev. Physiol. 48 (1986) 569-591. Kariya, Y., Kato, K., Hayashizaki, Y., Himeno, S., Tarui, S. and Matsubara, K.: Revision of consensus sequence ofhuman Alu repeats - - a review. Gene 53 (1987) 1-10. Mayo, K.E., Cerelli, G.M., Lebo, R.V., Bruce, B.D., Rosenfeld, M.G. and Evans, R.M.: Gene encoding haman growth hormone-releasing factor precursor: structure, sequence, and chromosomal assignment. Proc. Natl. Acad. Sci. USA 82 (1985a) 63-67. Mayo, K.E., Cerelli, G.M., Rosenfeld, M.G. m~d Evans, R.M.: Characterization of eDNA and genomic clones encoding the precursor to rat hypothalamic growth hormone-releasing factor. Nature 314 (1985b) 464-467. Meigan, G., Sasaki, A. and Yoshinaga, K.: Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123 (1988) 1098-1102. Oliviero, S. and Monaci, P,: RNA polymerase III promoter elements enhance transcription of RNA polymerase II genes. Nucleic Acids Res. 16 (1988) 1285-1293. Tronik, D., Ekker, M. and Rougeou, F.: Structural analysis of Y-flanking regions of rat, mouse and human renin genes reveals the presence of a transposable-like element in the two mouse genes. Gene 69 (1988) 71-80.