Vol. 122, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

August 16, 1984 Pages 1076-1082

ISOLATION AND NUCLEOTIDE SEQUENCE OF A CLONED CAROIONATRIN cDNA

B.P. Kennedy*, J.J. Marsden*, T.G. Flynn*, A.J. de Bold+ and P.L. Davies*

Departments of Biochemist;"* and Pathology+, Queen's University, and

Hotel Dieu Hospital , Kingston, Ontario, Canada K7L 3N6

Received June 21, 1984

Summary. A cloned cDNA which codes for the C-terminal 62 residues of the precursor molecule for the atria1 natriuretic factor, cardionatrin, has been isolated and sequenced. The nucleotide sequence confirms the amino acid sequence of cardionatrin 1-28 previously determined, and positions this peptide at the C-terminal end of the precursor just two residues away from the termination codon.

It is now well established that the secretory-like morphological

features of mammalian atria1 cardiocytes are related to the capacity of

these cells to synthesize and store peptides which are potent natriuretic

and diuretic agents. Early purification attempts aimed at isolating

these peptides from rat atria1 muscle produced a mixture of biologically

active peptides differing in molecular weights and collectively referred

to as atria1 natriuretic factor (1,Z). The name cardionatrin was

proposed to differentiate the individual peptides (3). One of these peptides,

rat cardionatrin l-28, was recently sequenced by us (4). Subsequently,

Currie et al - -' (5) reported the sequence of two rat atria1 peptides

which were truncated versions of cardionatrin l-28. A similar result was

obtained by Misono et al (6). More recently, the amino acid sequence --

of longer peptides, containing the sequence of cardionatrin l-28 at their

C-termini has been reported (7,8) and Kangawa and Matsuo (9) have determined

the sequence of atria1 natriuretic factor from human heart.

The presence of larger peptides containing biological activity isolated

from heart muscle suggests that the bio-active peptide is produced by

Please send correspondence to: Dr. P.L. Davies.

Vol. 122, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

processing from a precursor molecule. In the course of determining the

complete amino acid sequence of the sequence of the precursor to cardionatrin

by recombinant DNA technoloqy we have isolated and sequenced a cloned cDNA

which codes for the C-terminal portion of the precursor molecule. This

work shows that cardionatrin is located at the C-terminus of pro-cardionatrin.

Materials and Methods

Collection of tissue

Atria and ventricles were obtained from 225-2509 male Sprague-Dawley

fats, rinsed in ice-cold, phosphate buffered saline (pH 7.4) and frozen immediately in liquid N,. The frozen tissue was mixed with solid CO, and pulverized in a pre-cooled coffee grinder.

Preparation of RNA

RNA was extracted from powdered atria and ventricles usinq quanidine thiocyanate in combination with phenol accordinq to the procedure of Feramisco et al. (10). -- 120,000 x g for lh.

Glycyqen was removed by centrifugation at

Poly(A) RNA was prepared from total atria1 RNA by chromatography on oligo(dT)-cellulose (11).

Synthesis and cloninq of Double-Stranded cDNA

Double-stranded cDNA was synthesized from atria1 poly(A)+ RNA using the method of Wickens et al. (12). After treatment with Sl nuclease, the -- 3' end of the double-stranded cDNA was extended with dGMP residues using terminal transferase and dGTP. The cDNA was inserted into the Pst 1 site

of pUC9 which had been previously tailed with 20-30 dCMP residues. The recombinant plasmids were used to transform E. coli JM83 to ampicillin resistance. Colonies which remained white in the presence of x-gal were plated Tut in duplicate. One set was hybridized to cDNA made to atria1

poly(A) RNA. The other set was hybridized to cDNA made to total ventricle RNA. Those clones that hybridized preferentially to atria1 cDNA were further characterized.

DNA sequence determination

DNA was end-labelled using the Klenow fragment of E. coli DNA polymerase I and sequenced according to the procedure OT Maxam and Gilbert

(13).

Results

Isolation of atrial-specific cDNA clones

To identify sequences that are expressed in atria but not in

ventricles, 80D clones from the atria1 cDNA library were screened by

differential colony hybridization (Fig. 1). Clones, such as car 1 and

car 3, which hybridized to atria1 cDNA but not to ventricle cDNA, (Fiq. 1)

were screened a second time with the same probes. Only clones car 1 and

car 3 hybridized specifically to atria1 cDNA in both screening tests.

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Fig. 1 Identification of atrial-specific cDNA clones by colony hybridization

The top filter, bearing DNA from 70 colonies, was probed with "'P-labelled ventricle cDNA. The duplicate filter below was probed with atria1 cDNA. The positions of car 1 and car 3, and the control clone (AtV) which hybridized well to both probes, are identified on both filters.

Northern blot analysis

Equal quantities of total RNAs from atria and ventricles were

electrophoresed in adjacent lanes on an agarose gel in the presence of

methyl mercury hydroxide (14) and then blotted onto DBM paper (15). Plasmic

preparations were made from car 1 and car 3, and from a control clone

which hybridized strongly to both atria1 and ventricular cDNAs. The

plasmids were nick-translated and hybridized separately to the Northern

blots. The control plasmid hybridized with equal intensity to a 900

nucleotide-long sequence in the atria1 and ventricular lanes, whereas both

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a

A v A v AV

O- O-

I a c

L’

\

F-

Fig. 2 Northern blot analysis

Total RNA (15ug) from atria (A) and ventricles (V) was separated by methyl mercury agarose gel electrophoresis (14) and transfered to DBM paper (15). Identical blots were probed with nick-translated car 1

(a), car 3 (b), and a clone which hybridized strongly to both atria1 and ventricular cDNAs (c). The positions of migration of the large rRNA (L), small rRNA (S) and 5.8s rRNA (F) are shown relative to the origin of the gel (0).

car 1 and car 3 hybridized specifically to a 1,000 nucleotide-long sequence

which was present only in the atria1 lane (Fig. 2).

DNA sequence analysis

The inserts in car 1 and car 3 were released from their plasmid

vectors by double-digestion with Barn Hl and Hind III and were estimated

to be 1OObp and 300bp long respectively by agarose gel electrophoresis.

DNA sequence analysis subsequently showed that car 1 and car 3 contained

74bp and 249bp of cDNA sequence respectively, flanked by the appropriate

homopolymeric tails (Fig. 3). When all possible reading frames of the

car 3 insert were analysed, one was found to match the amino acid sequence

of cardionatrin l-28 (4) and part of its N-terminal extension (7). With

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20 ArgAspGlyGlyAlaLeuGlyArgGlyProTrp

Car 3 (C)nGAGAGATGGAGGTGCTCTCGGGCGCGGCCCCTGG

30 40 AspProSerAspArgSerAlaLeuLeuLysSerLysLeuArgAlaLeuLeuAlaGlyPro GACCCCTCCGATAGATCTGCCCTCTTGAAAAGCAAACTGAGGGCTCTGCTCGCTGGCCCT

4 50 60 ArgSerLeuArgArgSerSerCysPheGlyGlyArgIleAspArgIleGlyAlaGlnSer CGGAGCCTGCGAAGGTCAAGCTGCTTCGGGGGTAGGATTGACAGGATTGGAGCCCAGAGC

70 1 GlyLeuGlyCysAsnSerPheArgTyrArgArg GGACTAGGCTGCAACAGCTTCCGGTACCGAAGATAACAGCCAAATCTGCTCGAGCAGATC

GCAAAAG TCCCAAGCCCTTT GGTGTGTCACACA(G)

Car 1 (C)n Ir --

TCCTCACCCCTTT AGAAAGCAGTTGGAAA:AAATAAATCCGAATAAACT

Fig. 3 DNA sequence of atrial-specific cDNA clones car 1 and car 3

The upper DNA sequence is that of car 3. It is shown in register with the cardionatrin amino acid sequence which is numbered according to Thibault et a1.(7).

-7 The lower DNA sequence is that of car 1. Regions of partial homology are enclosed in a box and putative poly- adenylation signals are underlined. Cardionatrin 1-28 is flanked by arrows.

respect to the latter 73-residue sequence the car 3 clone encodes from amino

acid 14 to 73 in which residues 46 to 73 correspond to cardionatrin 1-28.

The DNA sequence shows there are two arginine codons after residue 73

followed by a termination codon. This is followed in turn by 59 residues

of 3' untranslated region.

The cDNA insert in car 1 does not overlap the car 3 sequence though

it does contain a region of partial homolo9.y to the 3' untranslated

portion of car 3 (Fig. 3). The car 1 sequence contains two putative poly-

adenylation signals, (underlined in Fig. 3), one of which is approximately

20bp upstream of a run of A's which marks one end of that clone.

Discussion

The presence in atria1 muscle of peptides that differ considerably in

molecular weight but share natriuretic properties suggest that there exists

precursor-product relationships amonqst the peptides (1,2 and 5). Usinq

extraction conditions which, from the outset, inactivate proteolytic en-

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Vol. 122, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

z.vws (3), the most abundant low molecular weight peptide in rat atria1

muscle is cardionatrin l-28. The sequence of this peptide (4), shows

complete homology with the truncated sequences isolated in other

laboratories (5,6), and with the 28 C-terminal residues of longer sequences

Inore recently published (6,7). The nucleotide sequence of car 3 reported

here confirms the accuracy of the amino acid assignments in the protein

sequences (4,7). All of the peptides sequenced to date have the

characteristic disulfide-bonded peptide at the C-terminus. The reason for

this is now clear from the results presented in this paper. The nucleotide

sequence of car 3 shows that cardionatrin l-28 occupies the C-terminal end

of the precursor (pro-cardionatrin). Only two arginine codons lie between

the DNA sequence for cardionatrin l-28 and the termination codon.

Although the primary translation product would contain these two arginine

residues they do not appear in any of the cardionatrins or cardionatrin-like

peptides that have been characterized to date. It is not yet known whether

the removal of these two basic residues serves to activate or modulate

any of the physiological effects of cardionatrin.

The Northern blot analysis in Fig. 2, confirmed that cardionatrin

is produced in the atria1 but not the ventricular chambers of rat hearts,

thus validating the differential screeninq procedure used to isolate

cardionatrin cDNA clones. The Northern blots also showed that cardionatrin

I~RNA was 1000 nucleotides long. This mRNA is, therefore, long enough to

encode the 22,000 dalton primary translation product, which we see as

the major atrial-specific protein in comparisons of translation products

derived from atria and ventricles (P.L. Davies et al., unpublished --

work). A protein of this size could be coded by 600 nucleotides, leaving

approximately 400 nucleotides for the untranslated reqions of the mRNA

and the poly(A) tail.

Identification of the car 1 clone is not as clear-cut. Like car 3 it

is an atrial-specific sequence which hybridizes to the same-sized mRNA.

The partial homoloqy shown in Fig. 3 also suggests that it is related to

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car 3. These features, and the presence of putative polyadenylation siqnals,

makes it likely that the car 1 sequence represents the 3' end of the

cardionatrin mRNA.

The isolation of a cardionatrin cDNA clone is an important step in

providing access to the chromosomal gene and a reagent for quantitating

cardionatrin mRNA levels.

Acknowledgements

We would like to acknowledge financial support from the IDEA

Corporation of Ontario and the Natural Sciences and Engineering Research

Council of Canada. We thank Tom Abbot and Carol Hegadorn for excellent

technical assistance, Theresa Brennan for typing the manuscript and

Hans Metz for help with the preparation of figures.

1.

2. 3. 4.

5.

6.

7.

8.

9.

10.

11. 12.

13. 14. 15.

References

de Bold, A.J., Borenstein, H.B., Veress, A.T. and Sonnenberq, H. (1981) Life Sci. 28, 89-94. de Bold, A.J. (1982) Proc. Sot. Exp. Biol. Med. 170, 133-138. de Bold, A.J. and Flynn, T.G. (1983) Life Sci. 33, 297-302. Flynn, T.G., de Bold, M. and de Bold, A.J. (1983 Biochem. Biophys. Res. Commun. 117, 859-865. Currie, M.G.,xller, D.M., Cole, B.R., Siegel, N.R., Fok, K.F., Adams, S.P., Eubanks, S.R., Galluppi, G.R. and Needleman, P. (1984) Science 223, 67-69. Misono, K.S., Fukoni, H., Crammer, R.T. and Inaqani, T. (1984) Biochem. Biophys. Res. Commun. 119, 524-529. Thibault, G., Garcia, R., CanticM., Genest, J., Lazure, C.,

Seidah, N.G. and Chretien, M. (1984) FEBS Lett. 167, 352-356.

Kangawa, K., Fukuda, A., Minanino, N. and Matsuo, H. (1984) Biochem. Biophys. Res. Commun. 119, 933-940. Kangawa, K. and Matsuo, H. (198TBiochem. Biophys. Res. Commun. 118, 131-139. Feramisco, J.R., Helfman, D.M., Smart, J.E., Burridge, K. and Thomas,

G.P. (1982) J. Biol. Chem., 257, 11024-11031. Aviv, H. and Leder, P. (1972)roc. Natl. Acad. Sci. USA 69, 1408-1412. Wickens, M.P., Buell, G.N. and Schimke, R.T. (1978) J. BiT. Chem. 253, 2483-2495. Maxam, A. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560.

Bailey, J.M. and Davidson, N. (1976) Anal. Biochem. 70, 75-85. Christophe, D., Brocas, H. and Vassar, G. (1982) Anal. Biochem. lZJ, 259-261.

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