Analysis of MHC genomic structure and gene content between HLA-B and TNF using yeast artificial chromosomes.
To characterize the genomic structure and gene content between HLA-B and TNF we studied three overlapping yeast artificial chromosome (YAC) clones derived from the Boleth cell line carrying the 62.1 MHC ancestral haplotype (AH). In particular, we have evaluated the stability of haplospecific sequence motifs (haplospecific geometric elements) in YACs. By PCR and gene scanning we show that the two larger YAC clones (318G11 and 8F2) exhibit features characteristic of the 62.1 AH carried by the parental cell line as well as those of a separate cell line (BSM) which also carries 62.1. It is concluded that a highly polymorphic duplicated region is represented faithfully in spite of the fact that it contains sequences that could be interpreted as simple repeats liable to mutations. In contrast, microsatellites AFM031 and AFM158, which are also located in the MHC, showed greater instability and the structure in the YACs was not consistent with genomic structure. Likewise, different examples of the same ancestral haplotype show differences at these microsatellite loci. We conclude that polymorphic repeat sequences may exhibit a range of stabilities in vivo and that this may be reflected in their stability in YACs. Northern blotting using the largest YAC clone revealed several novel transcripts which await localization.
Marshall
B
B
Department of Clinical Immunology, Royal Perth Hospital, Western Australia.
Tay
G
G
Marley
J
J
Abraham
L J
LJ
Dawkins
R L
RL
D015152:Blotting, Northern; D002460:Cell Line; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004276:DNA, Satellite / Q000235:genetics; D005805:Genes, MHC Class I; D015235:HLA-B Antigens / Q000235:genetics*; D006801:Humans; D008285:Major Histocompatibility Complex; D010179:Pancreas / Q000276:immunology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D004276:DNA, Satellite; D015235:HLA-B Antigens; D014409:Tumor Necrosis Factor-alpha; D012313:RNA