Presence of the same transcript of pro-opiomelanocortin (POMC) genes in the porcine anterior and intermediate pituitary lobes

~ Molecularand Cellular Endocrinology

ELSEVIER

Molecular and Cellular Endocrinology103 (1994) 101-108

Presence of the same transcript of pro-opiomelanocortin (POMC) genes in the porcine anterior and intermediate pituitary lobes K o i c h i r o G e n , T o s h i a k i Hirai, T a k a k o Kato*, Y u k i o K a t o Institute of Endocrinology, Gunma Universi~, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan

Received 22 December 1993;accepted 11 April 1994

Abstract

The existence of heterogeneous molecular species of pro-opiomelanocortin (POMC) has been reported and it has been inferred that this explains the distinct release patterns of POMC-derived peptide hormones by the anterior and intermediate lobes of the pituitary gland. The aim of this study was to determine the nucleotide sequences of porcine pituitary anterior and intermediate lobar POMC from animals of the same strain. The POMC cDNAs were obtained using immunoscreening (anterior lobe) and the polymerase chain reaction (intermediate lobe), and their nucleotide sequences determined. Comparisons of the coding and the 5'-untranslated regions of the two POMCs demonstrated that their nucleotide sequences were identical and Northern blot analysis showed that both mRNAs were the same length. Therefore, the results of this study confirm that the same POMC transcript is present in both the anterior and intermediate pituitary lobes. The differences between the nucleotide and amino acid sequences of porcine POMC found hitherto may be attributable to strain differences. Comparisons of porcine and several vertebrate POMCs revealed highly conserved amino acid sequences in the regions corresponding to the peptide hormones, but the regions between them show considerable evolutionary divergence. Keywords: Pro-opiomelanocortin; Pituitary hormone; mRNA; Cloning; Pig; DNA sequence

1. I n t r o d u c t i o n

The pituitary anterior and intermediate lobes synthesize and secrete several peptide hormones, such as melanocyte-stimulating hormones (MSH; a-, fl- and yMSH), adenocorticotrophic hormone (ACTH), corticotropin-like intermediary peptide (CLIP), fl-endorphin and lipotropins (LPH; fl- and ),-LPH), some of which possess identical amino acid sequences. Molecular cloning of the pro-opiomelanocortin (POMC) gene has elucidated the structure of the precursor molecule of the peptide hormones and suggested that differential processing of POMC in the two lobes occurs (Nakanishi et al. 1979) . The precursor-processing enzymes were identified recently and the different enzymes found are expected to be candidates for the distinctly different patterns of peptide hormone production by the anterior and intermediate lobes. On the other hand, several investigators reported, hitherto, the presence of heterogeneous molecules of POMC. Variant forms of POMC with different amino acid sequence are known to be present, which suggests * Corresponding author.

that different peptide hormones may be produced from distinct POMC molecules in the two lobes. By cell-free translation experiments of intermediate lobar POMC mRNAs, two distinct primary translation products were obtained and their molecular sizes and amino acid sequences were different (Boileau et al., 1983b). Two POMC mRNAs of different sizes have also been found in the rat pituitary (Oates and Herbert, 1984) and Xenopus laevis (Martens, 1986). Amino acid sequence analyses of POMC-derived peptides demonstrated the presence of variant forms of POMC in the rat intermediate lobe (Crine et al., 1981). Our preliminary study of porcine pituitary anterior lobar POMC (Kato and Hirai, 1988) also revealed the nucleotide sequence which was not concordant with those of porcine intermediate lobar POMC reported previously (Boileau et al., 1983a; Gossard et al., 1986). These observations suggest a possibility that the variant peptide hormones and different processed peptides in the anterior and intermediate lobes have arisen from endopeptidases with differing conformations and differing accessibilities of these enzymes to the variant POMC. Conversely, there are only a few data, i.e. the size of POMC mRNAs in both lobes are the same (Boileau et al.,

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K. Gen et aL /Mol. Cell Endocrinol. 103 (1994) 101-108

1983a) and a single POMC gene (Nakanishi et al. 1979), to suggest another possibility that POMCs are the same in the anterior and intermediate lobes. Owing to the absence of the evidence that the same POMC molecule exists in the anterior and intermediate lobes, these discrepant results have yet to be elucidated. Therefore, the aim of this study was to determine the nucleotide sequences of porcine pituitary anterior and intermediate lobar POMC from animals of the same strain simultaneously. We have sequenced the entire coding and the 5'-untranslated regions of two POMC mRNAs.

2. Materials and methods

2.1. Preparation of mRNAs and construction of a cDNA library Porcine pituitary anterior and intermediate lobes (the latter was collected together with posterior lobe) were obtained from a local slaughterhouse. The total RNAs were prepared, the poly(A) ÷ RNAs were enriched by oligo-dT cellulose column chromatography and the cDNA library of the porcine anterior lobe was constructed in an expression phage vector, 2gtl 1, as described in a previous paper (Kato, 1988).

2.2. Screening of anterior lobar POMC cDNA Immunoscreening of 5000 plaques was carried out as described by Young and Davis (1983) . The fusion protein expression was generated overnight at 37°C on nitrocellulose filters (BA85, Schleicher and Schuell, Keen, NH, USA), which had been pre-soaked in 10 mM isopropyl fl-D-thiogalactopyranoside. The cDNA clones encoding POMC were obtained by immunoscreening us!ng an antiserum against human A C T H (antiserum against synthetic human A C T H 1-39, NIAMDDK, provided by the National Pituitary Agency) at a dilution of 1:1000 and were detected using [125I]-labelled Protein A.

2.3. Reverse transcriptase-directed polymerase chain reaction (RT-PCR) for POMC cDNA The following four oligonucleotide primers were designed f r o m the nucleotide sequence of anterior lobar POMC cDNA (determined in this study) and synthesized using an Applied D N A synthesizer 380A (Foster City, CA, USA): A5' (nucleotide numbers (n.n.) 56-78), 5'AGTVI'CCTGCCTCGGCGCAGCGG3'; A3' (n.n. 664-646), 5 ' C C A G C T C C C T C C T G A A C T C 3 ' ; B5' (n.n. 627-646), 5 ' G G C C G A G G C C T F C C C C C T C G 3 ' and B3' (n.n. 958-936), 5 ' T G A G A G G C G C C T G C C C C C T G A A 3 ' . The anterior and intermediate lobar POMC cDNAs used for the RT-PCR were synthesized using a cDNA synthesis kit (Pharmacia LKB Biotechnology, Uppsala, Sweden). Each cDNA was amplified in a reaction mixture (100/~1) containing two required primers (50 pmol each) and 1 unit of Vent D N A polymerase (New England BioLabs, Beverly, MA, USA) with 30 denaturation (98°C, 30 s),

annealing (50°C, 2 min) and extension reaction (74°C, 3 min) cycles using a programmable thermal cycler (MJ Research Inc., Watertown, MA, USA). The resulting PCR products were separated using a Suprec-02 centrifuge filter (Takara Shuzo, Kyoto, Japan).

2.4. Cloning and sequencing The cDNAs amplified by the RT-PCR were purified by 1% (w/v) agarose gel-electrophoresis followed by electroelution. Each cDNA was cloned at the SmaI site of the Bluescript KS + plasmid vector (Stratagene, San Diego, CA, USA), and its nucleotide sequence was determined using a dideoxy chain-termination method (Sanger et al., 1977) on an Applied Biosystems 370A automated DNA sequencer or a-[32p]deoxythymidine triphosphate (dCTP).

2.5. Northern blot analyses The total poly(A) ÷ mRNAs (1/zg) extracted from the anterior and intermediate lobes were separated on denaturing formamide gel (Lehrach et al., 1977), transferred to a nylon membrane and fixed with ultraviolet light (Kato and Hirai, 1989). The cDNA encoding the anterior lobe POMC was labeled by the random priming method using an oligolabeling kit (Takara Shuzo) and hybridization was carried out at 68°C overnight in a sealed plastic bag containing 6 x SSC (0.9 M NaCI and 0.15 M sodium citrate, pH 7.0), 0.02% (w/v) bovine serum albumin, 0.02% (w/v) Ficoll and 0.02% (w/v) polyvinylpyrrolidone.

2.6. RNA sequencing and primer extension The 5'-untranslated sequences of the anterior and interTable 1 Porcine anterior lobar hormone cDNA content Hormone

Positive clones

Clones screened

cDNA content (%)

POMCa Prolactina GHa Common a a FSHfla'b LHfla TSHflb

220 350 400 31 17 31 3

5 × 104 5 × 104 5 × 104 1 × 105 1 × 106 1.5 × 105 5 x 105

0.44 0.70 0.80 0.03 0.0017 0.021 0.0006

The porcine anterior lobar hormone cDNA content of the cDNA library is described. All the data, except those for POMC, have been reported in our serial studies (Kato, 1988; Kato and Hirai, 1989; Hirai et al., 1989; Kato et al., 1989, 1990) . The original porcine anterior lobar cDNA library comprised 5.6 x 106 independent clones whose content of cDNA insert is 82% (Kato, 1988) . The numbers of positive clones were obtained by re-screening using each cDNA fragment as a probe and the content of each cDNA was estimated from the number of positive clones in the number of plaques screened. Growth hormone, GH; follicle stimulating hormone, FSH; luteinizing hormone, LH and thyroid stimulating hormone, TSH. a cDNA was obtained by immunoscreening. b Obtained using oligonucleotideas a probe.

103

K. Gen et al. / M o l . Cell. Endocrinol. 103 (1994) 101-108

AA

A

A

A 50

c

NNG~NGCGAGAGGGNAGAGAAAGAGG~G~GAGTGACC~GAGACCGCCG

15o i00 G ~ G AGTT~`CCTGCCTCG~AGC~GAGTCGCCCCGAGAGC~CCTCCCCGCGACAGAGCCTCAGCCTGCC~G~GATGCCGAGA~G~CAGCAGTCGc~GGGG~cCTGCTGCTG~C

MPRLCSSRSGALLLA 4

Sign~ptide

c ~200 c T G 250 7 ~GCTGC~CAGGCC~CATGG~GTGCGTGGC~GTGCCTGGjMAAGCAGCcAGTGTCAGGACCTCTCCACGG~GT~C~GCTG~GTGCATCCGG~CT~A~d~cCAGATCTC~T

LLLQASM~VRGWCLESSQCQDLSTESNLLACIRACKPDLS 300

350

400

GCGGAGACGCCCGTG~TCCC~C~C~CGAC~GC~CCGCTGACCGAG~CCCCCGG~GTACGTCATGGGCCACTTCCGCTGGGACCGC~CGGCCGCCGG~TGGcAGCAGCAGC y-MSH

450

500

GGC~CGGT~CGGT~CGGC~CGCG~CCAG~GCGCGAGGAGGAGGAGGTGGCG~GGGCG~GGCCCCGGGCCCCGC~AGAT~CGTC~GCCG~CCCGCGCCAGGAC~GC~

GGGGGGGGAGQ~---~EEEEVAAGEGPGPRGDGVAPGPRQD~--~ 550

600

650

TCCTACTCCATGGAGCACTTCCGCTGG~C~GCCCGTG~C~G~GCGGCGCCCGGTG~GGTGTATCCC~C~CGCCGAGGACGAG~G~CGAG~C~CCCCCTCGAG~CAGG ~

~

~

~

~

~

~

~

~

~

~

~

a-MSH

v~[~ ~

~

~

ACTII

~I~

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~

v

~

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~

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~

~

~

~

~

~

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~[~

~P

700

750

AGGGAGCTG~CGGG~GCC~CCCGAGCcGGCACGGGACCCCGAG~CCCG~CGAG~CGCG~CGCCCGGGCCGAGCTGGAGTAC~GCTGGTGGCCGAGGcCGAGGCG~CGAG~G

~

L a

G a P ~ ~ P a R ~ P E a P A E~ G a[ a ~' R ag ~ L- E Y, G z. v A~ ~ a u ~ a a ~ g-~ll--

800

850

~GGACG~GCCC~AT~GATGGAGCAC~CCGCTGG~CAGCCCGCCC~GGAC~GCGCTACGGC~C~CATGACC~CGAG~GAGCCAGACGCCcCTGGTCAC~TG~CAAA

~IEGPYKMEHFRWGSPPK~--~YGGFMTSEKSQTPLVTLFK ~-MSH

vLp~

~

900

950

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~

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N

A

I

V

K

N

A

H

~--~

G

Q

S-endo~hin --

~

-~H

Fig. 1. Porcine POMC cDNA nucleotide sequence. The anterior and intermediate lobar POMC cDNA nucleotide and deduced amino acid sequences are shown (intermediate lobar POMC cDNA nucleotide sequence was determined independently). The sequence of intermediate lobar POMC cDNA (corresponding to nucleotide numbers 1-985) was identical to that of anterior lobar POMC cDNA. Dibasic amino acid segments are shown enclosed in boxes and the predicted peptide hormones are indicated by arrows, These data were compared with the intermediate lobar POMC cDNA nucleotide sequence determined previously (Boileau et al., 1983a). The different and absent nucleotides determined by the latter compared with our sequences are indicated by a line above the relevant nucleotide and the amino acid differences are shown in parentheses above the relevant nucleotide sequences.

mediate lobar mRNAs (10/zg each) were determined using the primer extension reaction. The reaction mixture comprised of 34 mM Tris-HC1 (pH 8.3), 5 mM MgC12, 50 mM NaC1, 5 units of avian myeloma virus (AMV) reverse transcriptase, a-[32p]dCTP and dideoxynucleotide triphosphate (ddNTP)/deoxynucleotide triphosphate (dNTP) supplied in a K/RT sequencing system (Promega, Madison, WI, USA) and was incubated for 15 min at 42°C with a primer complimentary to n.n. 88-120. Reactions were also carried out in the absence of dideoxynucleotide to determine the transcription initiation sites of the two POMC mRNAs.

3. Results 3.1. Nucleotide sequence of anterior lobar POMC cDNA Five positive clones were obtained from 5000 plaques using immunoscreening. The content of positive clone was estimated to 0.1%. This number is relevant to 0.6%

POMC cDNA content in the cDNA library considering the possibility of cDNA insert to be expressed as the POMC, because one of the six inserts differing in the orientation and reading frame can be transcribed and translated to POMC. Using plaque hybridization independently, a similar value for the POMC cDNA content was estimated to be about 0.44% (Table 1). The insert lengths were assayed on agarose gel and the clone with the longest insert was used for nucleotide sequence determination (Fig. 1) after the insert had been subcloned using a plasmid vector. The sequence contained 766 bp from the 3' end and contained neither the 5'-untranslated region nor the N-terminal region part. Therefore, the 5' part sequence was determined using the primer extension reaction (Fig. 2). The entire sequence determined comprised 995 bases and the coding sequence (271 amino acids) contained intact peptide hormones that resembled those of man, cow, rat and mouse. When the nucleotide sequence was compared with that of porcine intermediate lobar

104

K. Gen et al. /Mol. Cell. Endocrinol. 103 (1994) 101-108

Anterior

Intermediate

Anterior Intermediate

M

5' 3'

5'

3'

1.2 Kb'-"~

560

Fig. 2. Northern blot analysis and reverse transcriptase-directed polymerase chain reaction (RT-PCR) of porcine anterior and intermediate lobar mRNAs. (a) The mRNAs (1/.tg) from the anterior and intermediate lobes were subjected to Northern blot analysis using a 32p-labeled porcine anterior lobar POMC cDNA insert as a probe. The position of POMC mRNA is indicated in kilobases (kb). (b) The anterior and intermediate lobar mRNA RTPCR products were analyzed on agarose gel. The 5' and 3' parts of both POMC mRNAs, which were expected to be amplified as 608 and 333 bp, respectively, were subjected to the RT-PCR. The molecular size marker (M) is indicated on the left-hand side.

P O M C determined by Boileau et al. (1983a), several differences were found including nine silents and three amino acid substitutions (Fig. 1). 3.2. Intermediate lobar P O M C mRNA Differences between the primary transcripts of anterior and intermediate lobar P O M C m R N A s were investigated by comparing their sizes and nucleotide sequences using Northern blot analysis. The results show that both m R N A s were the same length, about 1.2 kb (Fig. 2a). Next, the RT-PCR was carried out by dividing the POMC m R N A into two parts, because the reaction for the full sequence could not be performed, due to its high GC content (about 72%). The 5' and 3' parts were amplified using two pairs o f primers A 5 ' and A 3 ' and B5' and B3', respectively. The 5' and 3" parts of the anterior and intermediate lobar P O M C m R N A s RT-PCR products were each the same length (Fig. 2b). The amplified cDNAs of the intermediate lobar P O M C were subcloned separately using a plasmid vector and the nucleotide sequences determined. The results showed that its sequence was identical to that of the anterior lobar P O M C (Fig. 1.).

Reverse transcriptase-directed R N A sequencing was also carried out to compare the transcription initiation sites and 5'-untranslated region sequences of the m R N A s of the two lobes (Fig. 3). Reactions with both m R N A s in the absence of dideoxynucleotides generated primary products of the same length, although a few shorter products that may have been due to an immature extension were present. In the presence of dideoxynucleotides, the termination reaction resulted in identical nucleotide sequences for the two POMC mRNAs. Therefore, these results confirm that the same POMC transcripts are present in the porcine anterior and intermediate pituitary lobes. 3.3. Divergence of the P O M C gene Porcine POMC was compared with that of other vertebrates (man, rat and Xenopus). Harr plot analysis demonstrated that three regions in the nucleotide sequences, those encoding ~-MSH, A C T H ( a - M S H and CLIP) and flMSH/fl-endorphin, are well conserved across these species (Fig. 4). The intervening regions between ),-MSH and ACTH, and between A C T H and fl-MSH, however, are poorly conserved across these species, whereas the

Fig. 3. Primer extension analysis of POMC mRNA. Primer extension reactions of the anterior and intermediate lobar mRNAs were carried out in the presence (A, C, G and T) and absence (N) of dideoxynucleotides. The complementary identified and unidentified (X) nucleotides are indicated to the right of the sequence ladder.

K, Gen et al. / Mol. Cell. Endocrinol. 103 (1994)101-108

105

Anterior Intermediate

1¢4~

'~e e P

J

0

o P

106

K. Gen et al. /Mol. Cell. Endocrinol. 103 (1994) 101-108

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Rat 80(1

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L

11 -,

6()() •. \

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81)1) bp

Fig. 4. Harr plot analysis. Harr plot analysis was performed by comparing the porcine POMC cDNA with those of man (Takahashi et al., 1981), rat (Drouin and Goodman, 1980) and Xenopus laevis (Martens, 1986). The position where 15 of 20 nucleotides matched (12 of 20 between pig and Xenopus) is shown by a dotted line. The regions corresponding to 7-MSH (a), ACTH (a-MSH and CLIP (b), and fl-MSH and fl-endorphin (c) are indicated by boxes. signal peptide regions show extremely high homologies. The sequences b e t w e e n 7 - M S H and A C T H and A C T H and f l - M S H show that deletions and/or insertions have occurred d u r i n g vertebrate evolution (Fig. 5), with the exception of that b e t w e e n 7 - M S H and A C T H a m o n g closely related u n g u l a t e species (pig and cow) and rodents (rat and mouse).

4. Discussion The results of this study provide e v i d e n c e that the same P O M C transcripts are present in the pituitary anterior and intermediate lobes of pigs o f the same strain. Northern blot analysis demonstrated that the lengths of the P O M C m R N A s from the anterior and intermediate

Pig Cow

, MPRL~R~GALLLALLLQA~MEVRGW~LE~Q~QDL~TE~NLLA~IRACKPDL~AETPvFPGNGDAQPLTEN~YVMGHFRWDRF~ GSSSGGGGGGGGA ***************************************************************************************** ****S'V** A* Rat ****************************************************************************************** ***A**S* Mouse **************************************************************************************** ***A*SA* Gulnea-pigs * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** ** Man : ***************************************************************************************** S****SS ** Xenopus A : * F * P L W G C F L * I * G I C I F H I G * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Xenopus B : *F*PT*GC*L*I*GVFIFHIG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

signal peptide ~

.4--- 7- MSH.-~

Pig

: GQKR EEEVAAGE GPGPRGDG V A P G P R Q ~ Z S Y S M E H F R W G K P V Q ~ V K V Y P N G A E D E L A E A F P L E ~ .. * . . . . . . . . . . . . . . . . . D ~***'*W .............. i ............ s*Q . . . . . , , ~ *a* A*** T** ************************************************* .ouse .~. A.... vw ***s~.s**,~ .............. i ....... v**~.s ....... *B Guinea-pig: S . . . . . . A**AD **FH . . . . E**L*E* i .............. i ..... A .... E*S ....... * i Man . . . . . D*S***DCGPLPEG ..... S** AK .... E G ~ .............. i ............ S ........ Xenopus A : Y E D I S S Y P V F S L F P L S D QNAPGDNM***PLDRQD ************************************* Xenopus B , Y • *E D I S N Y P V L N L F P S S D N Q N A Q G D N M *** PMDRQ * ************************************* co,, Rat

a- MSH---I~

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: : , : Guinea-pig, Man : Xenopus A : Xenopus B :

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CUP

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13-MSH y~ LPH

~

~

~

~- LPH

~endo*phin

Fig. 5. Sequence homology of porcine POMC with those of other species. The predicted amino acid sequence of porcine POMC was compared with those of man (Takahashi et al., 1981), cow (Nakanishi et al., 1979), rat (Drouin and Goodman, 1980), mouse (Uhter et al., 1983), guinea-pig (Keightley et al., 1991) and Xenopus (Martens, 1986). The amino acid sequences are aligned to achieve maximal numbers of matches. Amino acids identical to those of porcine POMC are indicated by (*) and spaces to achieve maximal matching are denoted by open spaces. Dibasic and tetrabasic amino acid residues are indicated by reversed letters and peptide hormone sequences are indicated by arrows.

K. Gen et al. / Mol. Cell. Endocrinol. 103 (1994) 101-108

lobes were identical, the sequence analyses of these mRNAs demonstrated that their coding region sequences were the same and the primer extension reaction results confirmed that they had identical transcription initiation sites and 5'-untranslated sequences. Our data are not consistent with those of previous studies (Boileau et al., 1983a; Gossard et al., 1986). The former differs in 21 nucleotides (shown in Fig. 1), resulting in three amino acid substitutions. The latter differs in 9 nucleotides (five, three and one nucleotides in the 5'-, coding and 3'-untranslated regions, respectively) without amino acid substitution. Cell-free translation of the porcine intermediate lobar POMC mRNA indicated the presence of two types of POMC molecule with different amino acid sequences (Crine et al., 1981; Boileau et al., 1983). However, our sequence analysis and primer extension data indicate clearly that POMC mRNAs of the anterior and intermediate pituitary lobes of the same porcine strain are identical. These discrepancies may be attributable to strain differences or polymorphism among individual animals used for experiments. Similar discrepant observations, which may have occurred for the same reasons, were recorded in our serial studies of porcine (Kato, 1988; Kato and Hirai, 1989; Hirai et al., 1989; Kato et al., 1989, 1990b, 1992) and rat (Kato et al. 1990a) pituitary hormones. Another explanation is that in vitro transcripts contained a small amount of the molecule, as in the present result, which is probably caused by using mixed animals with individual polymorphism, and this was missed by the quantity and limited technology available at that time. Besides the sequence differences described above, several workers have reported the presence of plural genes and transcription and processing differences. Two non-allelic POMC genes were found in the mouse genome (Uhler et al., 1983), one of which is a pseudogene of low homology with the active POMC gene. Xenopus laevis (Martens, 1986) and rainbow trout (Salbert et al, 1992) have two highly homologous functional POMC genes, which may arise from gene duplication. Oates and Herbert (1984) reported an alternative splicing of rat intermediate lobar POMC mRNA and ectopic ACTH-secreting tumors express a mRNA of abnormal length, which differs from the normal in the 5'and/or 3'-untranslated regions (Clark et al., 1989). However, there are few references to POMC gene expression in the anterior and intermediate pituitary lobes. When the POMCs of various species (man, cow, rat, mouse, guinea-pig and Xenopus) were aligned, it was evident that 7-MSH, ACTH, a-MSH, fl-MSH and flendorphin are highly homologous across these species (Fig. 5). The alignment represents considerable insertions into and/or deletions from the intervening sequences between 7-MSH and ACTH and between ACTH and flMSH. The homologies of the first intervening regions across mammalian species are rather low, the second in-

107

tervening regions show distinctively low homologies, whereas only closely related species (pig and cow) are homologous. As only the second of two introns (first intron is present in the 5'-untranslated region) disrupts the coding sequence between the signal peptide and v-MSH (Takahashi et al., 1981), it is interesting that the rearrangements and insertions/deletions have occurred without disruption and/or shifting the POMC gene reading frame during vertebrate evolution. Cloning of the entire porcine POMC sequence was difficult, due to the presence of long stretches of G and/or C. A similar high GC content has been described in guinea-pig POMC cDNA (Keightley et al., 1991) . In order to amplify the intermediate lobar POMC, we divided it into two parts and carried out successive PCRs. When the coding sequence nucleotide compositions of various species are compared, porcine POMC mRNA has the highest GC content (70.3%), followed by guinea-pig (69.6%), man (67.7%), cow (67.0%), mouse and rat (63.0%) and Xenopus laevis (45.3%). We have accomplished serial cloning of porcine pituitary anterior lobar hormones in this study and the porcine pituitary hormone cDNA contents determined during our serial cloning studies are summarized in Table 1 together with the results of this study. It is noteworthy that the thyroid stimulating hormone (TSH)fl cDNA content was extremely low (0.0006% in the cDNA library), but we do not know why. The a-subunit and luteinizing hormone (LH)fl cDNAs were present in almost comparable amounts and there was one-tenth as much as follicle stimulating hormone (FSH)fl cDNA as its complementary a-subunit cDNA. High contents of growth hormone (GH), prolactin and POMC cDNAs were present. Northern blot analysis of POMC mRNA showed that the amount in the anterior lobe was somewhat higher than that in the intermediate lobe (Fig. 2a), but we do not know the reason for this.

Acknowledgments We wish to thank Dr. A.F. Parlow and the National Pituitary Program for supplying the pituitary hormone antibody. We are grateful to Ms K. Tomizawa for her technical assistance and Dr. T. Ezashi for carrying out the sequence analysis.

References Boileau, G., Barbeau, C., Jeannone, L., Chretien, M. and Drouin, J. (1983a) Nucleic Acids Res. I 1, 8063-8071. Boileau, G., Gossard, F., Seidah, N.G. and Chretien, M. (1983b) Can. J. Biochem. Cell Biol. 61,333-339. Clark, A.J.L., Lavender, P.M., Besser, G.M. and Rees, L.H. (1988) J. Mol. Endocrinol. 2, 3-9. Crine, P., Lemieux, E., Fortin, S., Seidah, N.G., Lis, M. and Chretien, M. ( 1981) Biochemistry20, 2475-2481. Drouin, J. and Goodman. H.M. (1980) Nature 288,610-613.

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