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DR typing of rhesus monkeys by restriction fragment length polymorphism (RFLP)
18
Abstracts
B 2.3.23
DRwll SERVES AS A RECIPIENT GENE TO CREATE A NEW DRw6 AI,LEI.E IN THE KOREAN POPULATION. KWLee, andES Choi, Department of Clinical Pathology, Kangdong Sacred Heart Hospital, College of Medicine, Hallym University, Seoul, KOREA. DRw6 is a complex a l l e l e family composed of at l e a s t 13 d i f f e r e n t a l l e l e s (5 a l l e l e s of DBwl3 and 8 a l l e l e s of DRwl4). Although 26~ of Koreans express DBw6 a l l e l e s , t h i s a l l e l e family has not been well studied in the population. DNA samples obtained from 257 unrelated individuals were screened by in v i t r o gene a m p l i f i cat i o n using Taq DNA polymerase and a DRB1 g r o u p s p e c i f i c PCR primer s e t which amplifies the polymorphic second exon of DR3, DRwll and DRw6 DRB1 a l l e l e s (except DRBI*1404). To i d e n t i f y the DBw6 a l l e l i c frequencies in t h i s population, PCR p o s i t i v e samples (102 samples) were further analyzed by dot blot hybridization using chemiluminescent-labeled sequence s p e c i f i c o l i g o n u c l e o t i d e probes and DRB1 a l l e l e s assigned by comparison of t h e i r h y b r i d i zat i o n patterns to that of HTCs. In t h i s process, a new DRB1 a l l e l e was i d e n t i f i e d by i t s unexpected h y b r i d i z a t i o n p a t te r n and was f u r t h er ch ar act er i zed by d i r e c t sequencing a f t e r PCR. The new DRB1 sequence i s s i m i l a r to DRBl*1101 d i f f e r i n g at codon 47 (TAC(Tyr)/TrC(Phe)) and at cedon 58 (GCC(Ala)/GAG(Glu)). Based on sequence comparisons, the new a l l e l e may have a r i s e n by a gene conversion event between DRBI*llO1 and DRBI*OIO1 a l l e l e s . The r e s u l t a n t DR molecule bears DRw6 s e r o l o g i c determinants as determined by s e r o l o g i c typing and, based on sequence, is probably a DRw13 and not a DRw14 a l l e l e . These data suggest that the DRwll a l l e l e has frequently acted as a r e c i p i e n t gene in the gsne conversion events which created the subfamily of ORw13 a l l e l e s , DRBI*1303, .1304..1305, and the new a l l e l e described here.
B 2.3.24
DR T Y P I N G OF R H E S U S M O N K E Y S BY R E S T R I C T I O N F R A G M E N T L E N G T H P O L Y M O R P H I S M (RFLP) J. K a s t e n - J o l l y , D. Taylor, J.M. Thomas, D e p a r t m e n t of Surgery, E a s t C a r o l i n a U n i v e r s i t y , G r e e n v i l l e , NC. T r a n s p l a n t a t i o n of n o n h u m a n p r i m a t e s is i m p o r t a n t for p r e c l i n i c a l e v a l u a t i o n of new i m m u n o s u p p r e s s i o n and t o l e r a n c e strategies. A l i m i t a t i o n of o u t b r e d p r i m a t e s m o d e l s is the lack of u n d e r s t a n d i n g of the role of shared M H C Class II alloantigens, w h i c h can i n f l u e n c e graft survival results. Serologic t y p i n g for rhesus m o n k e y (RM) D R has been developed, but the r e a g e n t s are limited by supply and by crossreactions, often r e n d e r i n g t y p i n g a s s i g n m e n t s uncertain. For this reason, we e x a m i n e d the u t i l i t y of RFLP to i d e n t i f y R M DR r e g i o n genes. 12 m o n k e y s were t y p e d s e r o l o g i c a l l y for RM DR a n t i g e n s w h i c h had the d i s t r i b u t i o n : DRI(n=5); DR3(n=4); DR4(n=4); DR8(n=5); DR2(n=2), and DRX (n=4). S o u t h e r n s w e r e p r o b e d with a human cDNA to DR~ c o n t a i n i n g both d o m a i n s 1 and 2, the m e m b r a n e anchor region, and the 3 ' - u n t r a n s l a t e d region. After digestion with Eco-R1, RFLP were calculated. C o r r e l a t i o n b e t w e e n RFLP m o t i f s and DR type was e x a m i n e d by chi square a n a l y s i s and found to be highly significant. Of the DR1 ÷ cells, all 5 had a 20.2 ~ 0.5 and a 15.0 ± 0.4 kb fragment and 4 of the 5 had a 12.0 ± 0.2 kb fragment (p<0.025 vs. D R 1 cells). All DR3 ÷ cells, had a 13.8 0.5 kb fragment in c o m b i n a t i o n w i t h an 18.2 ~ 0.5 kb fragment, a 6.3 0.3 kb fragment (1/4) or b o t h (3/4) (p<0.025 vs. D R ) . All DR4+cells (4/4) had fragments of 9.0 ± 0.5 kb, 10.5 ! 0.5 kb and 6.0 ± 0.3 kb (p<0.05 vs D R 4 ) . Of DR8 + cells, 4/5 had a 21.4 ± 0.3 kb fragments in c o m b i n a t i o n w i t h either a 7.3 ! 0.5 kb fragment (1/4), or b o t h a 7.3 kb and a 6.0 Z 0.3 kb fragment (3/4); one had only the 6.0 and 7.3 f r a g m e n t s (p<0.06 vs D R 8 ) . DR2 + cells had f r a g m e n t s of 18.5 ± 0.i kb, 5.2 kb and 4.7 ~ 0.2 kb (p<0.10). Of DRX types, 2 had a DR8 RFLP pattern. The v a r i a t i o n s w i t h i n RFLP m o t i f s suggest DR subtypes. A d d i t i o n a l RFLPs could not be a s s i g n e d to d e f i n e d antigens, s u g g e s t i n g that the m o n k e y DR region is complex. T h e s e studies p r o v i d e new i n f o r m a t i o n on D N A t y p i n g of the rhesus m o n k e y D R r e g i o n and indicate that RFLP w i t h Eco-R1 digest is useful for a n a l y s i s of M H C Class II r e g i o n in t h e s e primates. RLFP w i t h other e n z y m e s is u n d e r study.
Abstracts
B 2.3.23
DRwll SERVES AS A RECIPIENT GENE TO CREATE A NEW DRw6 AI,LEI.E IN THE KOREAN POPULATION. KWLee, andES Choi, Department of Clinical Pathology, Kangdong Sacred Heart Hospital, College of Medicine, Hallym University, Seoul, KOREA. DRw6 is a complex a l l e l e family composed of at l e a s t 13 d i f f e r e n t a l l e l e s (5 a l l e l e s of DBwl3 and 8 a l l e l e s of DRwl4). Although 26~ of Koreans express DBw6 a l l e l e s , t h i s a l l e l e family has not been well studied in the population. DNA samples obtained from 257 unrelated individuals were screened by in v i t r o gene a m p l i f i cat i o n using Taq DNA polymerase and a DRB1 g r o u p s p e c i f i c PCR primer s e t which amplifies the polymorphic second exon of DR3, DRwll and DRw6 DRB1 a l l e l e s (except DRBI*1404). To i d e n t i f y the DBw6 a l l e l i c frequencies in t h i s population, PCR p o s i t i v e samples (102 samples) were further analyzed by dot blot hybridization using chemiluminescent-labeled sequence s p e c i f i c o l i g o n u c l e o t i d e probes and DRB1 a l l e l e s assigned by comparison of t h e i r h y b r i d i zat i o n patterns to that of HTCs. In t h i s process, a new DRB1 a l l e l e was i d e n t i f i e d by i t s unexpected h y b r i d i z a t i o n p a t te r n and was f u r t h er ch ar act er i zed by d i r e c t sequencing a f t e r PCR. The new DRB1 sequence i s s i m i l a r to DRBl*1101 d i f f e r i n g at codon 47 (TAC(Tyr)/TrC(Phe)) and at cedon 58 (GCC(Ala)/GAG(Glu)). Based on sequence comparisons, the new a l l e l e may have a r i s e n by a gene conversion event between DRBI*llO1 and DRBI*OIO1 a l l e l e s . The r e s u l t a n t DR molecule bears DRw6 s e r o l o g i c determinants as determined by s e r o l o g i c typing and, based on sequence, is probably a DRw13 and not a DRw14 a l l e l e . These data suggest that the DRwll a l l e l e has frequently acted as a r e c i p i e n t gene in the gsne conversion events which created the subfamily of ORw13 a l l e l e s , DRBI*1303, .1304..1305, and the new a l l e l e described here.
B 2.3.24
DR T Y P I N G OF R H E S U S M O N K E Y S BY R E S T R I C T I O N F R A G M E N T L E N G T H P O L Y M O R P H I S M (RFLP) J. K a s t e n - J o l l y , D. Taylor, J.M. Thomas, D e p a r t m e n t of Surgery, E a s t C a r o l i n a U n i v e r s i t y , G r e e n v i l l e , NC. T r a n s p l a n t a t i o n of n o n h u m a n p r i m a t e s is i m p o r t a n t for p r e c l i n i c a l e v a l u a t i o n of new i m m u n o s u p p r e s s i o n and t o l e r a n c e strategies. A l i m i t a t i o n of o u t b r e d p r i m a t e s m o d e l s is the lack of u n d e r s t a n d i n g of the role of shared M H C Class II alloantigens, w h i c h can i n f l u e n c e graft survival results. Serologic t y p i n g for rhesus m o n k e y (RM) D R has been developed, but the r e a g e n t s are limited by supply and by crossreactions, often r e n d e r i n g t y p i n g a s s i g n m e n t s uncertain. For this reason, we e x a m i n e d the u t i l i t y of RFLP to i d e n t i f y R M DR r e g i o n genes. 12 m o n k e y s were t y p e d s e r o l o g i c a l l y for RM DR a n t i g e n s w h i c h had the d i s t r i b u t i o n : DRI(n=5); DR3(n=4); DR4(n=4); DR8(n=5); DR2(n=2), and DRX (n=4). S o u t h e r n s w e r e p r o b e d with a human cDNA to DR~ c o n t a i n i n g both d o m a i n s 1 and 2, the m e m b r a n e anchor region, and the 3 ' - u n t r a n s l a t e d region. After digestion with Eco-R1, RFLP were calculated. C o r r e l a t i o n b e t w e e n RFLP m o t i f s and DR type was e x a m i n e d by chi square a n a l y s i s and found to be highly significant. Of the DR1 ÷ cells, all 5 had a 20.2 ~ 0.5 and a 15.0 ± 0.4 kb fragment and 4 of the 5 had a 12.0 ± 0.2 kb fragment (p<0.025 vs. D R 1 cells). All DR3 ÷ cells, had a 13.8 0.5 kb fragment in c o m b i n a t i o n w i t h an 18.2 ~ 0.5 kb fragment, a 6.3 0.3 kb fragment (1/4) or b o t h (3/4) (p<0.025 vs. D R ) . All DR4+cells (4/4) had fragments of 9.0 ± 0.5 kb, 10.5 ! 0.5 kb and 6.0 ± 0.3 kb (p<0.05 vs D R 4 ) . Of DR8 + cells, 4/5 had a 21.4 ± 0.3 kb fragments in c o m b i n a t i o n w i t h either a 7.3 ! 0.5 kb fragment (1/4), or b o t h a 7.3 kb and a 6.0 Z 0.3 kb fragment (3/4); one had only the 6.0 and 7.3 f r a g m e n t s (p<0.06 vs D R 8 ) . DR2 + cells had f r a g m e n t s of 18.5 ± 0.i kb, 5.2 kb and 4.7 ~ 0.2 kb (p<0.10). Of DRX types, 2 had a DR8 RFLP pattern. The v a r i a t i o n s w i t h i n RFLP m o t i f s suggest DR subtypes. A d d i t i o n a l RFLPs could not be a s s i g n e d to d e f i n e d antigens, s u g g e s t i n g that the m o n k e y DR region is complex. T h e s e studies p r o v i d e new i n f o r m a t i o n on D N A t y p i n g of the rhesus m o n k e y D R r e g i o n and indicate that RFLP w i t h Eco-R1 digest is useful for a n a l y s i s of M H C Class II r e g i o n in t h e s e primates. RLFP w i t h other e n z y m e s is u n d e r study.