GHF-1

Biochimica et Biophysica Acta, 1129 (1992) 231-234

231

© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

Short Sequence-Paper

BBAEXP 90300

Nucleotide sequence of the complementary DNA for human Pit-1/GHF-1 Ke-ita Tatsumi, Tsugunori Notomi, Nobuyuki Amino and Kiyoshi Miyai Department of Laboratory Medicbr~; Osaka Unicersity Medical School, Osaka (Japan) (Received 29 October 1991)

Key words: Pit-l; GHF-I: cDNA sequence: Pituitary; (Human)

Human eDNA clones encoding Pit-I/GHF.I, a pituitary-specific DNA binding factor, were obtained by PCR following reverse transcription of human pituitary RNA. It is approx. 1.3 kb in size with 0.1 kb 5' non-coding region, 0.9 kb protein-coding region and 0.3 kb 3' non-coding region. The predicted human Pit-I/GHF-I peptide structure has 291 amino acids and is highly conseived among mouse, rat and bovine. In addition, the 5' nov-coding region is highly conserved with rat pit-I/GHF-I sequence to the transcription start site.

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x Fig. I. Restriction enzyme map of human pit-I/GHF-I eDNA. The protein coding region (open box), the cloned PCR products (dashed box) and PCR primers are indicated. PCR primers are pPit-I(0p): GGTCTAGAAGCTTGTGGGAATGAGTTGCCAACC: pPit-l(251p): CTGAATCT(C/G)GAGAAAGAAGTAG; pPit-l(274r): GT(C/T)TTCACCCGTTTTTCTCTCTGCCTTCGGTT(A/G)CA; pPit-l(292r): GGTCTAGATCTATCTGCA(C/T)TC(A/G)AGATGCACCTT: XSH-adaptor: ACTCGAGTCGACAAGCTT: XSH-adaptor+(dT}15: ACTCG-AGTCGACAAGC1TVITI-I'ITITITI-I-FI.

Pit-1/GHF-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of both growth hormone (GH) and prolactin (PRL) genes [1,2]. While its mRNA is expressed in all

The nucleotide sequencing data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number D01114. Correspondence: K.-i. Tatsumi, Department of Laboratory Medicine. Osaka University Medical School, Fukushima-ku, Osaka 553, Japan.

five types of hormone producing cells in the anterior pituitary, its protein is chiefly expressed in either GH, PRL or thyrotropin (TSH) producing cells [3]. Furthermore, disruption in the pit-l/GHF-I gene was observed in Snell dwarf (dw) mice [4,5], in which GH. PRL and TSH producing cells were absent [6]. These data indicate that Pit-1/GHF-1 is necessary for the ontogeny of GH, PRL and TSH producing cells. To date, bovine [2], rat [1,2] and mouse [5] pit-I/ GHF-1 eDNA has been cloned and studied. To further analyse the potential role of Pit-1/GHF-I in various

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CTCAGAGCCTTCCTGATGTATATATGCAGGTAGTGAGAATTGAATC

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GGCCCTTTGAGACAGTAATATAATAAAACTCTGATTTGGGGAGCAGCGGTTCTCCTTATTTTTCTACTCTCTTGTGGGA

125

ATG AGT TGC CAA GCT TTT ACT TCG GCT GAT ACC TTT ATA CCT CTG AAT TCT GAC GCC TCT MeC Set ~ s Gin Ala Phe Thr Set Ala Asp Thr Phe lle Pro Leu Ash Set Asp Ala Set

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GCA ACT CTG CCT CTG ATA ATG CAT CAC AGT GCT GCC GAG TGT CTA CCA GTC TCC AAC CAT

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GCC ACC RAT GTG ATG TCT ACA GCA ACA GGA CTT CAT TAT TCT GTT CCT TCC TGT CAT TAT

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CAG CCA TCA ACC TAT GGA GTG ATG GCA GGT AGT TTA ACC CCT TGT CTT TAT RAA

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~g. 2. Nucleotidesequencea,idthededucedaminoacidsequenceofhuman pit-l/GHf-l~DNA. Polyadenylationsignalsareunderlined.

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Fig. 3. Theaminoacidcomparisonsbetweenthe ~urcloned Pit-I/GHF-lproteins. ldenticalaminoacidsareindicatedbybars. Regi°ns°f identitybetweenall~urproteinsareboxed.

amplify human pit-I/GHF-I eDNA prepared from human pituitary RNA. For internal and 3' end amplification, the first strand eDNA was synthesized with XSH-adaptor + (dT)t5 and M-MLV reverse transcriptase. The product was PCR amplified with Tth DNA polymerase using internal primers (pPit-l(0p) and pPit1(292r)), or an internal primer (pPit-l(251p)) and

inheritable human growth disorders [7-9], in the ontogeny of GH, PRL and TSH producing cells, and in the regulation of the TSH subunit genes, we attempted to isolate human pit-1/GHF.I eDNA. Total RNA was extracted from human pituitaries obtained from either autopsy or surgical operations as described [10]. The PCR technique [11] was used to

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Fig. 4. Comparison of homologous sequences of the 5' non-coding region of pit-l/GHF-i cDNAs between human, bovine [2] and rat [12.13]. Numbering is in relation to the 'cDNA' sequences. Identical bases are indicated by bars. Gaps which have been introduced ior maximunl alignment are indicated by dots. The transcription start sites are indicated by downward arrowheads. The translation initiation site:~,,,are boxed. Sequences were aligned by using GCG software [14].

~4 XgH-adaptor, res,oectively (Fig. 1). For 5' end amplification, the first s~rand cDNA was synthesized with an internal primer (pPit-l(292r)) and M-MLV reverse transcriptase. The product was oligo(dA) tailed with terminal deoxynucleotidyi transferase, and was PCR amplified with Tth DNA polymerase using an internal primer (pPit-l(2740) and XSH-adaptor + (dT)t5 (Fig. 1), ~ e s e internal primers were designed by aligning rat and bovine pit-l/GHF-I cDNA sequences [1,2]. These amplified products were digested with restriction enzymes, gel purified and cloned into pBiuescript !! SK - vector. Positive clones were identified through hybridization with rat GHF-I eDNA probe kindly provided from Dr. L. Therill, More than three independent clones were sequenced by the dideoxy method for each region to ~liminate misincorporation of nucleotides with Tth DNA polymerase. The nucleotide sequence and the deduced amino ~cid sequence of the human pit.!/GHF.I cDNA are shown in Fig. 2. it is approx. 1.3 kb in size with 0.1 kb 5' non-coding region, 0.9 kb protein-coding region and 0,3 kb 3' non-coding region. The predicted human Pit-I/GHF-I peptide structure has 291 amino acids. The amino acid sequences are highly conserved among the four cloned Pit-I/GHF-I proteins (Fig. 3). The identity between human and bovine, rat or mouse are about 96% at the amino acid level and about 90% at the DNA level in the protein-coding region. At the 5' end, one clone started at nucleotide I and two clones at nucleotide 4. As aligned in Fig. 4, the 5' non-coding regit~n is highly conserved (82.4%) with rat pit.l/GHF.! sequence to the transcription start site, which has been characterized recently by primer extension and RNase-A protection analyses [12,13]. Although the human pit-I/GHF.I gene has not been revealed, on the basis of these findings, we assigned nucleotide ! and 4 as the transcription start sites. On the other hand, the human 5' non-coding region is poorly conserved with bovine pit-l/GHF.I sequence as a whole. Within the 350 nucleotides of the bovine 5' non-coding region, the sequence from nueleotide 20 to nucleotide 335 is 97.4% identical with the 3' part of bovine pit-l/GHF.! eDNA in the reverse strand, As shown in Fig. 4, the remainder

region is well conserved with human pit-l/GHF-! sequence, suggesting that an insertion had occurred within the bovine 5' non-coding region. We have defined two polyadenylation sites, in front of which polyadenylation signals lie (Fig. 2). As multiple mRNA sizes were observed in rat pit-!/GHF-! cDNA [1,2], further studies are needed to reveal the complete structure of human pit-!/GHF-! eDNA. We thank Dr. L. Therill for providing us with the rat GttF-I cDNA clone, and Dr. K Matsubara for his generosity to use facilities of his laboratory. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan to K.T. and K.M.

References

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10 II 12 13

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