Identification and Characterization of Cdna Clones Encoding Two

MOLECULAR AND CELLULAR BIOLOGY, May 1987, p. 1841-1847 Vol. 7, No. 5 0270-7306/87/051841-07$02.00/0 Copyright © 1987, American Society for Microbiology Identification and Characterization of cDNA Clones Encoding Two Homologous Proteins That Are Part of the Asialoglycoprotein Receptor MICHAEL McPHAULt* AND PAUL BERG Department ofBiochemistry, Stanford University Medical Center, Stanford, California 94305 Received 16 October 1986/Accepted 11 February 1987 The asialoglycoprotein receptor (ASGP-R) from rat liver contains the following three distinct protein species when it is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: RHL1 (42 kilodaltons), RHL2 (49 kilodaltons), and RHL3 (54 kilodaltons). In this paper we describe the isolation of cDNA clones encoding RHL1 and RHL2 from a cDNA library constructed from rat liver mRNA. A comparison of the predicted coding sequence for RHL2 with that for RHL1 showed that these sequences are highly homologous. The library also contained numerous cDNA clones for both RHL1 and RHL2 that were derived from unspliced precursor mRNAs. Differential splicing at the 5' end of the RHL1 transcript was inferred from the finding that two different types of RHL1 cDNA were identified, each having a different 5' terminus. The rat asialoglycoprotein receptor (ASGP-R) binds ter- MATERIALS AND METHODS minal galactose residues in the oligosaccharide chains of numerous serum glycoproteins (1). Electrophoresis of recep- Materials. All restriction enzymes were purchased from tor preparations on sodium dodecyl sulfate (SDS)- New England BioLabs, Inc., Beverly, Mass. Escherichia polyacrylamide gels has indicated that the receptor is com- coli DNA ligase, terminal deoxynucleotide transferase, and posed of three protein chains with apparent molecular RNase H were obtained from P-L Biochemicals, Inc., Mil- weights of 42,000 (RHL1), 49,000 (RHL2), and 54,000 waukee, Wis. E. coli DNA polymerase I was obtained from (RHL3) (8). Whereas RHL1 appears to be unique on the Boehringer Mannheim Biochemicals, Indianapolis, Ind., and basis of its amino acid sequence, RHL2 and RHL3 share a avian myeloblastosis virus reverse transcriptase was ob- common 101-amino acid sequence at their carboxy termini tained from Seikageiku America. The vector-primer carrying (6). The latter finding suggests that RHL2 and RHL3 may polythymidylate tails at one end (14) was supplied by differ at their N-terminal amino acid sequences or they may Takashi Yokota (DNAX, Palo Alto, Calif.). Oligo(dT)- be identical but differ in their glycosylation patterns. To cellulose was purchased from Collaborative Research, Inc., examine this question and to explore the mechanism of Waltham, Mass. E. coli MC1061 was used for all bacterial ASGP-R synthesis and assembly, we set out to isolate the transformations (3). cDNAs encoding these proteins from rat liver RNA. Total rat liver RNA was obtained by homogenizing 6 g of In this paper, we describe the isolation and characteriza- fresh rat liver from a 200-g female Sprague Dawley rat in 60 tion of cDNA clones encoding RHL1 and RHL2. A compar- ml of a 5 M guanidinium isothiocyanate solution in a ison of the amino acid sequences predicted from the cDNA Polytron homogenizer (Brinkman Instruments) for 1 min. sequence indicated that there is extensive homology be- This preparation was then layered over a 5.7 M cesium tween RHL1 and RHL2. In addition, a comparison of the chloride-0.1 mM EDTA solution and centrifuged at 27,000 amino acid sequences of RHL1 and RHL2 with their human rpm in a Beckman type SW28 rotor for 18 h (5). The resulting homologs (abbreviated Hi and H2) revealed conserved pellets were solubilized and precipitated twice from 0.25 M regions among the related pairs, as well as among all four sodium acetate (pH 5.2) by adding 2.5 volumes of ethanol. polypeptides, that may be important for ASGP-R formation Poly(A)+ RNA was isolated by oligo(dT)-cellulose chroma- or function. Numerous RHL1 and RHL2 cDNAs were tography (2) and stored as a precipitate at -70°C until use. isolated from the library that contained one or more introns. cDNA library construction. The cDNA library was pre- Furthermore, we isolated a cDNA clone containing the pared with the pcD expression plasmid of Okayama and coding sequence for RHL1 that possessed a 5' terminus Berg, as described previously (14). Briefly, 4.5 ,ug of the different from that of the major class of RHL1 cDNAs. deoxyribosylthymine-tailed plasmid was annealed with 10 Judging from the sequence at the 5' end of the RHL1 gene, ,ug of poly(A)+ RNA. Sufficient buffer (10 mM Tris, pH 8.35, it appears that the odd RHL1 cDNA arose from an alter- 6 mM MgCl2, 30 mM KCl) was added to achieve a final nately spliced first intron. This alternatively spliced mRNA volume of 60 ,ul. The first cDNA strand was synthesized by may encode an RHL1 protein whose amino terminus is adding 40 U of avian myeloblastosis virus reverse transcrip- different from the termini which are most frequently iso- tase and incubating the preparation at 37°C for 90 min; the lated. reaction was terminated by adding 1 ,ul of 20% SDS and 1 p.l of 0.5 M EDTA. The reaction mixture was extracted once * Corresponding author. with phenol-CHCl3 (1:1), and the aqueous phase was precip- t Present address: Department of Intemnal Medicine, University itated twice from 2 M ammonium acetate by adding 2.5 of Texas Health Science Center at Dallas, Southwestem Medical volumes of ethanol at room temperature. The product was School, Dallas, TX 75235. then precipitated once from 0.25 M sodium acetate and 2.5 1841 1842 McPHAUL AND BERG MOL. CELL. BIOL. volumes of ethanol at -20°C. Deoxycytidylate tails were for 12 h on LB agar plates containing ampicillin (50 pug/ml) added to the first cDNA strand with terminal deoxynucleo- and chloramphenicol (15 ,ug/ml). In screening experiments tide tranferase. The product was cleaved with HindIll en- with both oligonucleotides, the lifts were processed sequen- donuclease, and the vector fragment was annealed with the tially for 15 min each on the following solutions: (i) 10% linker fragment tailed with deoxyguanidylate tails (kindly SDS, (ii) 0.5 N NaOH-1.5 M NaCl, (iii) 1 M Tris (pH 8)-1.5 provided by R. F. Margolskee, Stanford University). After M NaCl, and (iv) 6x SSC (lx SSC is 0.15 M NaCl plus 0.15 the annealing, cyclization, and synthesis of the second M sodium citrate, pH 7.0). The filters were dried and baked cDNA strand, portions of the reaction mixture were used to for 2 h at 80°C in vacuo. The baked filters were washed for transform competent E. coli MC1061. The library, selected 2 h in 1 M NaCl-5 mM Tris (pH 8)-i mM EDTA (pH 8.0) and on ampicillin medium, contained 2.3 x 106 independent in 0.1% SDS at 42°C for 1 h. The filters were then prehybrid- transformants. A portion was grown to saturating density ized at 37°C in a solution containing 5 x SSC, 50 mM sodium (overnight), and plasmid DNA representing the entire library phosphate (pH 6.8), 7 x Denhardt solution (lx Denhardt was prepared by cesium chloride gradient centrifugation solution contains 50 mg of bovine serum albumin per ml, 50 (12). Samples (10 ,ug) ofthe plasmid DNA were digested with mg of Ficoll per ml, and 50 mg of polyvinylpyrrolidone per either SalI, PvuI, or ClaI restriction endonuclease, and after ml), 0.1% SDS, and 150 ,ug of tRNA per ml at 37°C for 12 h. the three digests were pooled, the DNA was fractionated by Hybridization was performed at 37°C in fresh portions of the electrophoresis on a 0.8% low-melting-temperature agarose same solution to which we added approximately 105 cpm of gel. The gel was sliced so as to separate regions correspond- each degenerate oligonucleotide mixture per ml. Washes ing to plasmids containing cDNA inserts of 0 to 800 base were performed once at room temperature and twice (5 min pairs, 800 base pairs to 1.2 kilobases (kb), 1.2 to 2.2 kb, 2.2 each) at the washing temperature in 4x SSC. The wash to 2.8 kb, 2.8 to 5.8 kb, and more than 5.8 kb. Then the DNA temperature was determined empirically for each oligonucle- was isolated from the slices by melting the agarose, cyclized otide (42°C for RHL1 and 40°C for RHL2-RHL3). Each by ligation, and introduced into bacteria. Each sublibrary positive colony was purified twice by replating and contained 2 x 105 to 5 x 105 independent transformants with rehybridization. cDNA inserts corresponding to the size classes indicated Screening the genomic library. The genomic libraries de- above. scribed above were screened by hybridization with cDNAs Genomic library construction. A DNA blot analysis of known to encode RHL1 and RHL2 (p2AA and pllAl, restriction enzyme digests of rat genomic DNA showed that respectively). Two clones from library A hybridized to the the entire coding region was contained on a 15-kb EcoRI RHL1 cDNA and were subsequently found to contain the fragment. A 40-p.g portion of genomic DNA prepared from entire gene. One clone, which hybridized to the RHL2 isolated rat liver nuclei (19) was digested with EcoRI endo- cDNA, was identified from library B and was found to nuclease and loaded onto a 0.6% low-melting-temperature contain the entire coding sequence for RHL2. agarose gel. The fragments migrating in the region corre- DNA sequencing. Clones were sequenced after subcloning sponding to 15 to 18 kb were isolated, and 400 ng was ligated in appropriate M13 vectors (either M13mpl8 or M13mpl9 with 250 ng of purified arms of EcoRI endonuclease-digested A EMBL-3.

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