Complete coding nucleotide sequence of cDNA for the class II RT1.B βI chain of the Lewis rat

Biochimica et Biophysica Acta, 1089 (1991) 414-416 © 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 016747819100187H

414

BBAEXP 90251

Short Sequence-paper

Complete coding nucleotide sequence of cDNA for the class II RT1.B fll chain of the Lewis rat Jessica S y h a - J e d e l h a u s e r , U w e W e n d l i n g a n d K o n r a d R e s k e lnstitut ~ r lmmunologie, Unirersitiit Mainz, Mainz (F.R.G.) (Received 24 May 1991)

Key words: MHC class 11 molecule; eDNA; RTI.B/3 chain; (Rat)

We have established the first full length cDNA clone for the /3 light chain of the MHC class II a, 18 heterodimer (isotype RTI.B) of the rat. Clone pLR/HI8 was obtained from a self-primed Agtl0 cDNA library of IFN.,r treated bone marrow-derived macrcphages of thc Lewis rat. Subcloning of pLR~BII8 into a transcription vector with subsequent in vitro transcription and translation using the reticulocyte lysate system in the presence of microsomes followed by immunoprecipitation with mAb OX6 and two-dimensional gel electrophoresis revealed the intact RTI.B iBt-chain.

The rat major histocompatibility complex encodes three isotypic class II a, fl heterodimers referred to as RT1.B, RT1.D and RT1.H. As to the RT1.B antigen two serologically discrete a,/3 heterodimers have been identified at the cell surface of rat splenocytes by the non-crossreactive mAb OX3 and OX6. Both forms were shown to originate from a common OX6-reactive biosynthetic intermediate containing terminally glycosylated a,/3 and invariant z-chains. The possibility for generation of functional diversity of RT1.B dependent class II molecules at the posttranslational level with implications for antigen presentation has been discussed [1]. To examine this concept by means of transfection experiments we decided to establish full length eDNA clones of the respective class II subunits. While full length clones of the RT1.Ba [2] and invariant ~" chain [3] have been derived from a Agtll eDNA library prepared from Lewis rat splenocytes, attempts to detect RT1.B/3-specific clones were unsuccessful Therefore, a eDNA library was constructed in Agtl0 using IFN-¢ treated bone marrow derived macrophages because these cells express significantly higher quantities of class II molecules as compared to

The sequence data in this paper have been submitted to the EMBL/Genebank Data Libraries under accession number X56596. Correspondence: K. Reske, lnstitut fiir Immunologie, Obere Zahlbacher Str. 67, W-6500 Mainz, F.R.G.

the heterogeneous spleen cell population [4]. In this library approx. 100000 pfu were screened with a mouse I-A/3 eDNA probe [5] and 295 positive clones were further investigated. 70% of the clones analyzed fell 108 bp short at the 5' end of the coding region of the RT1.B/3 chain, while the remaining 30% of the clones were substantially shorter. We then prepared a selfprimed eDNA library from the same cell source starting 70 bp downstream the TGA stop codon of the /3I chain nucleotide sequence. The sequence of the primer oligonucleotide (broken underlined in Fig. 1) was determined using sequence data obtained from the incomplete RT1.B/3 clones. Within 15 positive clones analyzed of this library full length clone pLR/3118 was detected. Fig. 1 depicts the combined nucleotide sequence data obtained. The deduced amino acid sequence with an open reading frame of 263 amino acids from the ATG start codon at nucleotide position 8 to the termination codon at position 797 is indicated in the three-letter code. The overlapping region between full length clone pLR/3118 and a representative incomplete clone of the first Agtl0 library shows 100% sequence identity (marked by the two triangles in Fig. 1). The entire sequence of 1374 bp consists of a 7 bp 5' non-coding segment, a 789 bp coding region and a remarkably long 3' non-coding part. The occurrence of a typical splice donor site at nucleotide position 817/818 (marked by a rhomb in Fig. 2) is consistent with the notion that part of the 3' non-coding region encompasses intron related se-

415

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18 61

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Val Leb Set Ser Pro Gly Thr Glu GlytArg Asp Set Pro Arg Asp Phe Val T y / G 1 n

37

&~C CCA GGG ACT GAG G~C AGA GAC TCC CC.%&GG G&T '2'TC OTG T&C CAG

128

Phe Lys G1y Leu Cys Tyr ryr ~ h r Ash Oly Thr Gin Arg lie Arg Asp Val Zle Arg "LTCJU~GGGC C T ~ T ( ~ T&C T&C &CO ]t4C ~ &C~ CAO CGC &TJ& C ~ G&T OT~ & T C A G A

175

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~tC GT~ (~C GAG TAC ~

56

75 232

57 Ala Leu Thr G1u Le. G1y Arg Pro ~ G C G C T G ACC G A G CTG G ~

Ala Glu Tyr Phe Asn £ys Gln Tyr Leu G1u

94

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289

Gln Thr Arg Ala G1u Leu Asp Thr val Cys Arg His ASh Tyr G1u G1y Set G1u Yal

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CAG ACG CGG GCC GAG C'JL'G O&¢ ACG GTC TGC A G A CAC AA¢ T&C ~

GGG TO~ GAG GTC

346

Arg Thr Set Leu Arg Arg Leu G1u Gln Pro Asn Val Ala Ile S e t Leu Set Azg Thr &CA

132 ~03

G1u Ala Leu Asn His His ASh £eu Leu Val Cys Set Val Thr Asp Phe Tyr Pro Ala GAG GCC CTC R~C CA¢ CAC R&C ~ CT~ GTC ~ TCIt GT~ &CA G&T TTC TJtC CCA GCC

151 460

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Gin I1e Lys Val Arg Trp Phe Arg Ash G1y Gin Glu G1u Thr Ala G1y Val Yal Set

170

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527

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208

Pro V a l T h r va2 G2u T.,'p Arg A.la 0 I n S e t Glu S e t A / a Gln S e t

227

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A

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ss2 1027 2202 2277 2252 1327 237t

Fig. I. Nucleotide sequence of the RTI.B ,8 t chain specific clone pLR/3118 and its deduced amino acid sequence, The sequence has been determined using the dideoxy chain termination method [9]. The stretch of sequence shared by full length clone pLR/3118 and a number of incomplete/3 z clones is indicated by the two triangles ( • ). The transmembrane region is ztnderlined: the potential N-glycosylation site is marked by an asterisk (*); the cleavage position of the signal peptide is indicated by an arrow ( J, ); a potential sp',ice donor site at nucleotide position 817/818 is marked by a rhomb (<>). The segment of 3' untranslated sequence that served for initiation of self-primed DNA synthesis is marked by a broken line.

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Fig. 2. Two-dimensional analysis of in vitro transcribed and translated RTI.B fll chain following immunoprecipitation with the ,8 chain-reactive mAb OX6. The retieulocyte lysate system in the presence of microsomal membranes was used for in vitro translation. (A) RTI.B fll chain precipitated with mAb OX6 from an NP40 extract of the in vitro translation mixture; differentially glycosylated forms are marked by arrowheads. (B) Control immunoprecipitate obtained with mAb OX6 from [~SS]methionine labelled Lewis rat splenocytes.

quence information that has not been removed due to a possible loss of the corresponding splice acceptor site during evolution. The deduced amino acid sequence exhibits typical transmembrane protein characteristics such as a cleavage position for the signal peptide between amino acid 27 and 28 (arrow), a potential N-glycosylation site at amino acid 46 (asterisk) and the transmembrane region ranging from amino acid 216 to 252 (underlined). in the course of studies to define diabetogenic characteristics within the /31 domain of several RT1.B /3 chains Chap et al. [6] established an incomplete RT1.B /31 clone by PCR. As the homologous I-A/3 chain of the NOD mouse and the class II /3 chains from insulin-dependent diabetes meUitus (IDDM) sensitive and resistant strains of BB rats (haplotype u) the RTI.B/31 chain of the diabetes resistant Lewis strain has a Ser residue at position 57. The conclusion was reached that in contrast to the human and mouse system amino acid position 57 appears not to represent

a disease susceptibility marker for autoimmune diabetes. Sequence comparison between the incomplete PCR-derived RT1.B fit clone and our sequence information revealed identity at all positions except nucleotide 164 and 237, where we obtained G instead of A and T instead of A, respectively. The nucleotide sequence of the RT1.B fit chain has an average sequence identity of 97% with rat RT1.B/3 chains, 85% with mouse I-A/3 and 71% with human HLA-DQ fl chains. Subcloning of pLR/3118 into the plasmid pUHE 25 [7] and in vitro transcription using E. coli RNA polymerase resulted in substantial quantities of RT1.B/3 I specific mRNA. By use of the reticulocyte lysate system in the presence of microsomal membranes translation of the RT1.B /3~ transcripts yielded intact /3 chains. This was evidenced by specific immunoprecipitation with mAb OX6 and two-dimensional gel analysis as indicated in Fig. 2. OX6 in contrast to OX3 has earlier been shown to recognize a sequence determinant on the RT1.B fit chain [1,8]. We thank Prof. H. Bujard (Center for Molecular Biology (ZMBH), University of Heidelberg) for the generous gift of vector pUHE 25. I-A/3 cDNA containing vector pKCR3 was kindly donated by Dr. D. Mathis, Laboratoire de G6n6tique Mol6culaire des Eucaryotes du CNRS, Strasbourg, France). The expert technical assistance of Ms. Sabine Neis is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 311, C2. References 1 Reske, K. and Weitzel, R. (1985) Eur. J. lmmunol. 15, 1299-1239. 2 Syha, J., Henkes, W. and Reske, K. (1989) Nucleic Acids Res. 17, 3985. 3 Henkes, W., Syha, J. and Reske, K. (1988) Nucleic Acids Res. 16, 11822. 4 Schneider, F.J., Opel, B., Ballhausen, W., Henkes, W., Steinlein, P. and Reske, K. (1987) Eur. J. lmmunol. 17, 1235-1242. 5 Landais, D., Beck, B.N., Buerstedde, J.-M., Degraw, S., Klein, D., Koch, N., Murphy, D., Pierres, M., Tada, T., Yamamoto, K., Benoist, Ch. and Mathis, D. (1986) J. Immunol. 137, 3002-3005. 6 Chap, N.J., Timmerman, L., McDevitt, H.O. and Jacob, C.O. (1989) lmmunogenetics 29, 231-234. 7 Bujard, H., Gentz, R., Lanzer, M., Stueber, D., Mueller, M., Ibrahimi, i., Haeuptle, M.-T. and Dobberstein, B. (1987) Methods En~mol. 155, 416-433. 8 McMaster, W.R. (1981) lmmunogenetics 13, 347-350. 9 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5643-5647.