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Exon 2 DNA sequence of the HLA-DRw13b allele obtained from genomes of five different individuals
oI6I-sxYo/Yo $3.01) + 0.00 Pergamon Prr.9~ plc
PRELIMINARY
COMMUNICATION
BBOW 2 DNASBQDENCB OFTEB BW-DBwl3bALLBIBOBTAEBD FBGWGBWOWBSOFPIVBDIFFBBBWT IWDIVIDDALS.
ALFRBDo -, VICABIO*,
DBPARTNSNT
WADBID,
and
PILAB vABlm4, J.WAWDBL WARTIN-VILLA, JOBGBWMTIWBX-LAS0 andMTOWIOABWAIX-VILLBWB.
J.LUIS
HOSPITAL -DDCB DE OGTDBBB-, OF EEWOIGGY, * LABOBBTGBIO DE HISTOCONP ATIBILIDAD, W TR?iNSFlJSION C.&X. XADRID, SPAIN.
28041DE
(First received 12 December 1989: accepted in revked form
18 Jmuary
1990)
Bxon 2 nucleotide seguence of the DE1 gene encoding the BLA-Dml3b allele defined by DWA-BFLP (Restriction Fragumnt Length Polymorphi88) typi8g, haa been obtained by using five heteroxygoue individual6 WC DWA Its cavpariaon with and a non isotopic autcmated 'dideoxi' methodology. other know8 homologous DBBl eeguencee suggeete that two different mechauiexm which generate WLB allele variability may have occurred in this particular exon 2: a gene convereion between DBwll or DBw13 as acceptors and DR4-Dw15 or DBwS.1 as donors and in addition, a non-conservative point rmrtation at base 221, The relationship between this HLA sequence characterietice and certain diseases euaceptibility ie dimcuemed. INTRODUCTION The HW-DBwl3b alloepecificity defined by BFLP amlyeie (Bidmll et to type and easily mieaeeigned al. 1988) ie difficult aa DBwl2 by serological typing (see in Betuel et al 1984 and Schreud8r et al 1984, Bawxygoue Typing Cell (EC] 9W1802=BAG). Several review papers containing the exon 2 seguencee from different EL&DRB gene vaxiante have recently bee8 published (Sodm8r et al. 1989, Warsh andBodumr, 1989); BTGe Wer8 ueed a8 source material. However, none of them included the DBwl3b subtype, mostly due to the lack of BTG defining this specificity in the 10th Interuatioual Hw Workshop and aleo to its poorly defined serology. In the present work, w8 have obtaiued the Corresponding 8x08 2 Hw-DBB g888 DNA eeguence W-8 specific polymorphism of each HL&DR allele is determined became it containe information to translate the first dcmain of the EL?+DR j3 chain. MATERIAL AND METHODS Polymerame ChainReaction (PCB)method (Saiki et al. 1985) waa umed to amplify DBB exon 2 eeguencee fran 5 different Spanish individuals sharing TABGEl the DBw13b allele. Hw-DR and -DQ phenotypes of the 5 individuals wed in this at&y.
MRB
:sc nGA JR9
vZ,v3 Vl,V3 v3 Vl,V3 vZ,v3
6,7 v6 5,d 2;r"7
133.7.2 13b,13a3 v11,13b v15,13b 13b.7.1
(see + Bidvell J. 1988 and Arnais-Villena l Martinez-Las0 et al, 1989)
313
3,3b* lb,3bf g,;g 3,ib*
2b,3b la,3b 3b,3b lb,3b 2b.3b
et al, 1989 ;
314
previouely described: primer GE46 (S*-tXGGAmTDaedprimerewerethoae (S'AGlTG~-3‘), GH50 G-a&3') and primer (8charf et al. 1988). DNA warn prepared from petripheral blood leukocytes of to standard methcde (Wyman and White, 1980). PCR individuals accordiug mixtures (loo@) coneieted of gencmic DNA (1 pg) in a solution containing 50 mN KCl, 10 mN Trie-Cl pa 8.3, 1.5 mN wpCl2, 0.01% (w/v) gelatin, 200 jd4 each dGTP, and dTTP, l@ of each oligonucleotide primer and 2.5 ofdATP, dCTP, unite Taq polymaraee (Perkin Bluer Cetus, U.S.A.). Amplification of targeted DR81 sequences was carried out by denaturation of DNA (7 min at 94-C followed by rapid cooling on ice) followed by 30 cycles of ccmbined primer annealing and extension at 68-C (2 min) and denaturation at 94-C (1 min). Amplification Products were purified and ineerted into 8ma-I digested Nl3pml8 RF DNA and the resulting recanbinante were used to traneform coli TGl cells. After a screening for DR8 inserts, 20 different canpetent E. colourless reccmbinant Wl3 plaques from each individual were amplified in B. coli TGl by overnight culture. Culture supernatanta were incubated with PBG/NaCl to precipitate Xl3 particles (30 min at 4-C) and then were purified phenol-chloroform and ether extractione and, ethanol finally, by Single precipitation. stranded DNA templates were sequenced using the Sanger*8 dideoxy chain termination method and an Applipd Bioeysteam 370A automated DNA sequencer (Gocayue et al. 1987) am follows: Dye-labeled Wl3 primers were added to annealing reaction mixturee:A) 25Ong of template/l ~1 of JOB/l.8 711 of 5x buffer;C) 250 ng of template/l ~1 of FAN/l.8 ~1 of 5x buffer;(;) 500 ng of template/2 ~1 of TANRA/3.6 ~1 of 5x buffer;T) 500 ng of template/2 ~1 of ROX/3.6 ~1 of 5x buffer. Mixes were heated to 65-C for 5 min and cooled to room temperature over 30 min. After a brief dideoxynucleotide t exmination/Taq polymeraee mixtures centrifugation, were added to each tube (A, 3~1; C, 3~1; G, 6~1; T, 6~1) and the reaction mixtures were incubated at 70-C for 10 min. A new dome of Taq polymerase wae added (1 711 A and C; 2 ~1 G and T) and the mixtures were incubated for 10 additional min at 7O'C, pooled, and precipitated by addition of 6 711 of 31 NaDAc (pH 5.2) and 150 ~1 of ethanol. After a 70% ethauol wash, the aaaplea were briefly dried under vacuum and redissolved in 1 ~1 50 mW BDTA and 5 pl of formamide. Bach eample (6 ~1) was electrophoresed at a constant 30 W in an 6% polyacrylaiaide gel in the sequencer. RESULTS AND DISCUSSION All but one of the axon 2 sequences obtained from each individual were assigned to the different variant8 of DR81, DR82, DR83 or DR84 genes according to their 8LA phenotype, when ccmpared with those previouely deecribed (Bodrer et al. 1989, Wareh and Hodmer 1989). The remaining sequence, identical in all the individuals, wae assigned to the exon 2 DRwl3b sequence.
FIGURE 1. Exon 2 nucleotide sequence and deduced amino acid sequence of the first domain of HLA-DRw13b ( positions not traduced due to PCR primers modifications). RN8L Data Bank Acceeion Number: X16649/HL&DR81. 1 55 109 163 217
CGGATC
CTT CGT GTC CCCACAGCACGT
TTC TTGGAGTAC TCTACGTCT GAGTGT R F E Y 8 T 8 E C CAT TTC TTCAAT GGGACGGAGCGGGTGCGGTkC CTGGACAGATAC TTC TATAAC H F F N G T E R V R Y Y N CAAGAGGAGTAC GTGCGC TTC GACAGC C& Gk&&TAC CGGSGGTGACG Q E E Y V R F D 8 T G Y R GAGCTGGGGCGGCCTAGC GCC GAGTAC T&A AGC &MGGP.CAk C&AA &AkGC&$C $GGk&A& DKRAAVDTYCRHNY
T~T&A&&~T&&&&G&
Exon 2 DNA
scquencc of the HLA-DKwl3h
315
aIIclc
Ccmparieone with other DR5 aud DRw6 allele eeguencee (figure 2) suggest DRwl3b eight have originated ftaz DRwll (1102) or DRul3 (1301 or 1302) that donation fram DR4-Dw15 (0405) or DRw8.1 (0801) and a gene convereion regions ; this donation would at least involve baeee 109-207 and would be been reflected in changes in aminoacid 57. Gene conversion has previously implicated as a mechaniee to generate allelic variations in human claee-II MHC genes (Gregereen et al. 1987). ?IGDRS 2. Deducad amino acid mquanceS of thm tint dDuin of dlffernt DR5 (DlW11,w12) ud DE‘"6 (Dl?wlJ,wl4) aabty~m and of possibh gene donorm (DR4.S,DS"6.1) for a gorim coarrrmion mvent. Ilot= that DR4(0405) could give to DRwl3b, a &ortoe region {ramidamm 34-66) than 03~6.1 (0601) {remidueS 17-66) If origiml amxptor DRwll(llO2)i DRwl3 (1301 01 1302) could have also beOn acceptor and DRw6.1tlm dcnmr {remidu.m 17-66). (Sam reference Warah et al. 1989 for nomenclature).
DRBl*llOl DRB1*1102 DRB1*1103 DRSl*llOl DRB1'1201 rmS1=1301 DREl*1302 DRS1+1401 DRBl*1402
fXTRPW&
Dml."13b
sE=gi _-
DRB1*0405 DRB1*0601
-Q
10
20
30
40
50
60
I
I
I
I
I
I
STS(grmo
TSRVWLDRi
-1-
--Gc-8-~
lW8WMli
70
80
90
I
I
DSDVGIURh~TXUllPDXKiWNSQKD?LSiIRRMvDRtiEUYQVCS83T --I-~-"-----v-"-*-
-vA-S-I----k--I-_-V-*_~I_-'-..---.
-p--le-----a-~-T--*--~ ff-+-T------Akfl--bR-le----v-
PII--_
-T-----_8A-----_I-S~-,=--_ _X-
-------I--SA--L-g -T--Sk_
-M
(where DBBlfllO2, *1301 or l1302 may have been However, in this case, acceptors) the appearance at residue 71 of a Lyeine instead of Glutaeic Acid point mutation; suggests the existence of au additional non-coneervative this and the residue 57 eubetitution (Aepartic Acid to Seriue) may be the most important 13b/ezon 2 features which may cause both an overall eolecular conforeational change (and thus a serological epitope variation) and disease susceptibility which is postulated to be liuked to neutral residues at the position of the B-DR chain (Erlich B.A., eerine in 13b 57 1989):i.e. allele. Therefore, individuals carrying the DRwl3b allele should be searched for auto izmuue diseases (i.e.: pemphigue, associated to HLA-DR4) in contrast to other DR5 and DRw6 positive subjects. To our knowledge, this is the first BLA-DNA sequence which has been obtained by a non isotopic automated method; also, genomic DNA from several heterozygoue individuals ie used instead of the usual cDNA extracted from hoewzygoue cell lines.
REFERENCES Arnaiz-Villeua A., Vicario J.L., Martinez-Laeo J., Core11 A., Regueiro J.R., Galmz-Rein0 J.F. aud Gcmez-Reino J.J. (1989). An Eco RI polymorphic site in the human complement C4 gene distinguishes Juvenil Rheumatoid Arthritis (JBA) susceptibility-bearing haplotypee. Mol. -01. 26: 427-430. EKituel H., Gebuhre4.rL., Schereuder G.&T., Goldmann S.F., Arnaiz-Villena A. and Layrieee Z. (1984). BLA-DRw6 and Its Subgroups HLA-DRwl3 and HLA-DRwl4 in Hiet ocompatibility Testing 1984 pp 190-192, Springer-Verlag, Berlin. Bidwell J-(1988). DNA-RFLP analysis antigene. Immunology Today 9: 18-23
and
genotyping
of
HLA-DR
and
DQ
Boduer J.G., Rareh S.G.E., Parham P., Erlich B.A., Albert E., Bodmer W.F., Saeazuki T., Schreuder G.&T., Dupont B., Mach B., Mayr W.R., Strcminger J.L., Svejgaard A. and Teraeaki P-1.(1989). Nomenclature for Factors of HLA System, 1989. Izzaunology Today, in press.
Erlich H.A. 415.
(1989). HLA and Ineulin-dependent
diabetes mellitue. Nature 337:
Gocayne J., Robinson D.A., Fitzgerald H.G., mung F-Z., Kerlavage A-R., Lentee K.U., Lai J., Wang C.D., Fraser C.H. and Venter J.G. (1987). Primary structure of rat cardiac /%adrenergic and muecarinic cholinergic receptors obtained by automated DNA sequence analyeie: Further evidence for a multigene family. P.N.A.S. 84:8296-8300. Gregereen P-K., Silver J and Winchester R.J. Hypothesis. Arth. & Rheum. 30: 1205-1213.
(1987).
The Shared Rpitope
and Rodmar J.G. (1989). HLA-DR and -DQ epitopee and monoclonal Warsh S.G.E. antibody specificity. Imnunology Today 10: 305-312. Xartinez-Laso J., Vicario J-L., Core11 A., Worales P., Regueiro J.R. and Arnaiz-Villena A. (1989). A new HLA-DQAl RFLP allele [DQc3b] distinguiehes between DQa genes of DQw2-DR3 and DQw3-DR5 haplotypes. Nut. Acid Rem. 17 (12): 4911.
Saiki R.K., Scharf S., Paloona F, Xullie K-B., Horn G-T., Erlich H-A. and Arnheim N. (1985). Enzymatic Amplification of j%Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia. Science 230: 1350-1354 Scharf S-J., Long C.H. and Erlich H.A. (1988). Sequence Analysis of the HLADR/j and ELA-DQB loci from Three Pemphigus vulgarie Patients. HumanImmunol 22:61-69 Schreuder G.&T., Kennedy L.J., Gebuhrer L., Awad J., Rdtuel H., Degoe L. and Jeannet M. (1984). HLA-DR5 and Its Subtypee RLA-DRwll and DRwl2 in Hiatocompatibility Testing 1984 pp 192-197. Springer-Verlag, Berlin. Wyman A. and White R. (1980). A highly polymorphic A, S. USA. 77: 6754-6758.
locus in human DNA. P. N.
PRELIMINARY
COMMUNICATION
BBOW 2 DNASBQDENCB OFTEB BW-DBwl3bALLBIBOBTAEBD FBGWGBWOWBSOFPIVBDIFFBBBWT IWDIVIDDALS.
ALFRBDo -, VICABIO*,
DBPARTNSNT
WADBID,
and
PILAB vABlm4, J.WAWDBL WARTIN-VILLA, JOBGBWMTIWBX-LAS0 andMTOWIOABWAIX-VILLBWB.
J.LUIS
HOSPITAL -DDCB DE OGTDBBB-, OF EEWOIGGY, * LABOBBTGBIO DE HISTOCONP ATIBILIDAD, W TR?iNSFlJSION C.&X. XADRID, SPAIN.
28041DE
(First received 12 December 1989: accepted in revked form
18 Jmuary
1990)
Bxon 2 nucleotide seguence of the DE1 gene encoding the BLA-Dml3b allele defined by DWA-BFLP (Restriction Fragumnt Length Polymorphi88) typi8g, haa been obtained by using five heteroxygoue individual6 WC DWA Its cavpariaon with and a non isotopic autcmated 'dideoxi' methodology. other know8 homologous DBBl eeguencee suggeete that two different mechauiexm which generate WLB allele variability may have occurred in this particular exon 2: a gene convereion between DBwll or DBw13 as acceptors and DR4-Dw15 or DBwS.1 as donors and in addition, a non-conservative point rmrtation at base 221, The relationship between this HLA sequence characterietice and certain diseases euaceptibility ie dimcuemed. INTRODUCTION The HW-DBwl3b alloepecificity defined by BFLP amlyeie (Bidmll et to type and easily mieaeeigned al. 1988) ie difficult aa DBwl2 by serological typing (see in Betuel et al 1984 and Schreud8r et al 1984, Bawxygoue Typing Cell (EC] 9W1802=BAG). Several review papers containing the exon 2 seguencee from different EL&DRB gene vaxiante have recently bee8 published (Sodm8r et al. 1989, Warsh andBodumr, 1989); BTGe Wer8 ueed a8 source material. However, none of them included the DBwl3b subtype, mostly due to the lack of BTG defining this specificity in the 10th Interuatioual Hw Workshop and aleo to its poorly defined serology. In the present work, w8 have obtaiued the Corresponding 8x08 2 Hw-DBB g888 DNA eeguence W-8 specific polymorphism of each HL&DR allele is determined became it containe information to translate the first dcmain of the EL?+DR j3 chain. MATERIAL AND METHODS Polymerame ChainReaction (PCB)method (Saiki et al. 1985) waa umed to amplify DBB exon 2 eeguencee fran 5 different Spanish individuals sharing TABGEl the DBw13b allele. Hw-DR and -DQ phenotypes of the 5 individuals wed in this at&y.
MRB
:sc nGA JR9
vZ,v3 Vl,V3 v3 Vl,V3 vZ,v3
6,7 v6 5,d 2;r"7
133.7.2 13b,13a3 v11,13b v15,13b 13b.7.1
(see + Bidvell J. 1988 and Arnais-Villena l Martinez-Las0 et al, 1989)
313
3,3b* lb,3bf g,;g 3,ib*
2b,3b la,3b 3b,3b lb,3b 2b.3b
et al, 1989 ;
314
previouely described: primer GE46 (S*-tXGGAmTDaedprimerewerethoae (S'AGlTG~-3‘), GH50 G-a&3') and primer (8charf et al. 1988). DNA warn prepared from petripheral blood leukocytes of to standard methcde (Wyman and White, 1980). PCR individuals accordiug mixtures (loo@) coneieted of gencmic DNA (1 pg) in a solution containing 50 mN KCl, 10 mN Trie-Cl pa 8.3, 1.5 mN wpCl2, 0.01% (w/v) gelatin, 200 jd4 each dGTP, and dTTP, l@ of each oligonucleotide primer and 2.5 ofdATP, dCTP, unite Taq polymaraee (Perkin Bluer Cetus, U.S.A.). Amplification of targeted DR81 sequences was carried out by denaturation of DNA (7 min at 94-C followed by rapid cooling on ice) followed by 30 cycles of ccmbined primer annealing and extension at 68-C (2 min) and denaturation at 94-C (1 min). Amplification Products were purified and ineerted into 8ma-I digested Nl3pml8 RF DNA and the resulting recanbinante were used to traneform coli TGl cells. After a screening for DR8 inserts, 20 different canpetent E. colourless reccmbinant Wl3 plaques from each individual were amplified in B. coli TGl by overnight culture. Culture supernatanta were incubated with PBG/NaCl to precipitate Xl3 particles (30 min at 4-C) and then were purified phenol-chloroform and ether extractione and, ethanol finally, by Single precipitation. stranded DNA templates were sequenced using the Sanger*8 dideoxy chain termination method and an Applipd Bioeysteam 370A automated DNA sequencer (Gocayue et al. 1987) am follows: Dye-labeled Wl3 primers were added to annealing reaction mixturee:A) 25Ong of template/l ~1 of JOB/l.8 711 of 5x buffer;C) 250 ng of template/l ~1 of FAN/l.8 ~1 of 5x buffer;(;) 500 ng of template/2 ~1 of TANRA/3.6 ~1 of 5x buffer;T) 500 ng of template/2 ~1 of ROX/3.6 ~1 of 5x buffer. Mixes were heated to 65-C for 5 min and cooled to room temperature over 30 min. After a brief dideoxynucleotide t exmination/Taq polymeraee mixtures centrifugation, were added to each tube (A, 3~1; C, 3~1; G, 6~1; T, 6~1) and the reaction mixtures were incubated at 70-C for 10 min. A new dome of Taq polymerase wae added (1 711 A and C; 2 ~1 G and T) and the mixtures were incubated for 10 additional min at 7O'C, pooled, and precipitated by addition of 6 711 of 31 NaDAc (pH 5.2) and 150 ~1 of ethanol. After a 70% ethauol wash, the aaaplea were briefly dried under vacuum and redissolved in 1 ~1 50 mW BDTA and 5 pl of formamide. Bach eample (6 ~1) was electrophoresed at a constant 30 W in an 6% polyacrylaiaide gel in the sequencer. RESULTS AND DISCUSSION All but one of the axon 2 sequences obtained from each individual were assigned to the different variant8 of DR81, DR82, DR83 or DR84 genes according to their 8LA phenotype, when ccmpared with those previouely deecribed (Bodrer et al. 1989, Wareh and Hodmer 1989). The remaining sequence, identical in all the individuals, wae assigned to the exon 2 DRwl3b sequence.
FIGURE 1. Exon 2 nucleotide sequence and deduced amino acid sequence of the first domain of HLA-DRw13b ( positions not traduced due to PCR primers modifications). RN8L Data Bank Acceeion Number: X16649/HL&DR81. 1 55 109 163 217
CGGATC
CTT CGT GTC CCCACAGCACGT
TTC TTGGAGTAC TCTACGTCT GAGTGT R F E Y 8 T 8 E C CAT TTC TTCAAT GGGACGGAGCGGGTGCGGTkC CTGGACAGATAC TTC TATAAC H F F N G T E R V R Y Y N CAAGAGGAGTAC GTGCGC TTC GACAGC C& Gk&&TAC CGGSGGTGACG Q E E Y V R F D 8 T G Y R GAGCTGGGGCGGCCTAGC GCC GAGTAC T&A AGC &MGGP.CAk C&AA &AkGC&$C $GGk&A& DKRAAVDTYCRHNY
T~T&A&&~T&&&&G&
Exon 2 DNA
scquencc of the HLA-DKwl3h
315
aIIclc
Ccmparieone with other DR5 aud DRw6 allele eeguencee (figure 2) suggest DRwl3b eight have originated ftaz DRwll (1102) or DRul3 (1301 or 1302) that donation fram DR4-Dw15 (0405) or DRw8.1 (0801) and a gene convereion regions ; this donation would at least involve baeee 109-207 and would be been reflected in changes in aminoacid 57. Gene conversion has previously implicated as a mechaniee to generate allelic variations in human claee-II MHC genes (Gregereen et al. 1987). ?IGDRS 2. Deducad amino acid mquanceS of thm tint dDuin of dlffernt DR5 (DlW11,w12) ud DE‘"6 (Dl?wlJ,wl4) aabty~m and of possibh gene donorm (DR4.S,DS"6.1) for a gorim coarrrmion mvent. Ilot= that DR4(0405) could give to DRwl3b, a &ortoe region {ramidamm 34-66) than 03~6.1 (0601) {remidueS 17-66) If origiml amxptor DRwll(llO2)i DRwl3 (1301 01 1302) could have also beOn acceptor and DRw6.1tlm dcnmr {remidu.m 17-66). (Sam reference Warah et al. 1989 for nomenclature).
DRBl*llOl DRB1*1102 DRB1*1103 DRSl*llOl DRB1'1201 rmS1=1301 DREl*1302 DRS1+1401 DRBl*1402
fXTRPW&
Dml."13b
sE=gi _-
DRB1*0405 DRB1*0601
-Q
10
20
30
40
50
60
I
I
I
I
I
I
STS(grmo
TSRVWLDRi
-1-
--Gc-8-~
lW8WMli
70
80
90
I
I
DSDVGIURh~TXUllPDXKiWNSQKD?LSiIRRMvDRtiEUYQVCS83T --I-~-"-----v-"-*-
-vA-S-I----k--I-_-V-*_~I_-'-..---.
-p--le-----a-~-T--*--~ ff-+-T------Akfl--bR-le----v-
PII--_
-T-----_8A-----_I-S~-,=--_ _X-
-------I--SA--L-g -T--Sk_
-M
(where DBBlfllO2, *1301 or l1302 may have been However, in this case, acceptors) the appearance at residue 71 of a Lyeine instead of Glutaeic Acid point mutation; suggests the existence of au additional non-coneervative this and the residue 57 eubetitution (Aepartic Acid to Seriue) may be the most important 13b/ezon 2 features which may cause both an overall eolecular conforeational change (and thus a serological epitope variation) and disease susceptibility which is postulated to be liuked to neutral residues at the position of the B-DR chain (Erlich B.A., eerine in 13b 57 1989):i.e. allele. Therefore, individuals carrying the DRwl3b allele should be searched for auto izmuue diseases (i.e.: pemphigue, associated to HLA-DR4) in contrast to other DR5 and DRw6 positive subjects. To our knowledge, this is the first BLA-DNA sequence which has been obtained by a non isotopic automated method; also, genomic DNA from several heterozygoue individuals ie used instead of the usual cDNA extracted from hoewzygoue cell lines.
REFERENCES Arnaiz-Villeua A., Vicario J.L., Martinez-Laeo J., Core11 A., Regueiro J.R., Galmz-Rein0 J.F. aud Gcmez-Reino J.J. (1989). An Eco RI polymorphic site in the human complement C4 gene distinguishes Juvenil Rheumatoid Arthritis (JBA) susceptibility-bearing haplotypee. Mol. -01. 26: 427-430. EKituel H., Gebuhre4.rL., Schereuder G.&T., Goldmann S.F., Arnaiz-Villena A. and Layrieee Z. (1984). BLA-DRw6 and Its Subgroups HLA-DRwl3 and HLA-DRwl4 in Hiet ocompatibility Testing 1984 pp 190-192, Springer-Verlag, Berlin. Bidwell J-(1988). DNA-RFLP analysis antigene. Immunology Today 9: 18-23
and
genotyping
of
HLA-DR
and
DQ
Boduer J.G., Rareh S.G.E., Parham P., Erlich B.A., Albert E., Bodmer W.F., Saeazuki T., Schreuder G.&T., Dupont B., Mach B., Mayr W.R., Strcminger J.L., Svejgaard A. and Teraeaki P-1.(1989). Nomenclature for Factors of HLA System, 1989. Izzaunology Today, in press.
Erlich H.A. 415.
(1989). HLA and Ineulin-dependent
diabetes mellitue. Nature 337:
Gocayne J., Robinson D.A., Fitzgerald H.G., mung F-Z., Kerlavage A-R., Lentee K.U., Lai J., Wang C.D., Fraser C.H. and Venter J.G. (1987). Primary structure of rat cardiac /%adrenergic and muecarinic cholinergic receptors obtained by automated DNA sequence analyeie: Further evidence for a multigene family. P.N.A.S. 84:8296-8300. Gregereen P-K., Silver J and Winchester R.J. Hypothesis. Arth. & Rheum. 30: 1205-1213.
(1987).
The Shared Rpitope
and Rodmar J.G. (1989). HLA-DR and -DQ epitopee and monoclonal Warsh S.G.E. antibody specificity. Imnunology Today 10: 305-312. Xartinez-Laso J., Vicario J-L., Core11 A., Worales P., Regueiro J.R. and Arnaiz-Villena A. (1989). A new HLA-DQAl RFLP allele [DQc3b] distinguiehes between DQa genes of DQw2-DR3 and DQw3-DR5 haplotypes. Nut. Acid Rem. 17 (12): 4911.
Saiki R.K., Scharf S., Paloona F, Xullie K-B., Horn G-T., Erlich H-A. and Arnheim N. (1985). Enzymatic Amplification of j%Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia. Science 230: 1350-1354 Scharf S-J., Long C.H. and Erlich H.A. (1988). Sequence Analysis of the HLADR/j and ELA-DQB loci from Three Pemphigus vulgarie Patients. HumanImmunol 22:61-69 Schreuder G.&T., Kennedy L.J., Gebuhrer L., Awad J., Rdtuel H., Degoe L. and Jeannet M. (1984). HLA-DR5 and Its Subtypee RLA-DRwll and DRwl2 in Hiatocompatibility Testing 1984 pp 192-197. Springer-Verlag, Berlin. Wyman A. and White R. (1980). A highly polymorphic A, S. USA. 77: 6754-6758.
locus in human DNA. P. N.