Cloning of the HLA class II region in yeast artificial chromosomes.

Abstract

Yeast artificial chromosomes (YACs) have been applied to clone the entire class II region of the human major histocompatibility complex (MHC), including its flanking regions, in a contig over 1.5 million base pairs (bp) long. The human DNA inserts in the YACs have a size between 60 and 1300 kbp and were isolated from two EcoRI partial digest libraries. The gaps between DRA and DRB, DRB and DQA, and DOB and DPA, which had not been cloned by other means, have been bridged with YAC clones. The contig extends through the 400 kbp of DNA between the DRA and C4 genes, thus linking the class II region with the complement gene cluster in the class III region. The cloning in YACs has been supported by a conventional cosmid walk of 290 kbp in the C4-DRA region. Restriction enzyme sites in the YAC clones were compared to the sites in the cosmid walk, to published cosmid clones, and to the already existing physical maps, leading to a detailed characterization of a region of the human genome over 1500 kbp. The YAC clones will be valuable for functional analysis of the MHC.The human major histocompatibility complex (MHC) is situated on the short arm of chromosome 6 in the 6p21.3 band. It contains more than 50 genes in about 4 million base pairs (bp) of DNA (1). The organization of the genes has been determined by a combination of techniques including recombination analysis, cosmid walking, and pulsed-field gel electrophoresis (PFGE) (2-5). The telomeric end of the MHC contains the class I genes (6) and the centromeric end the class II genes (7). The class I and class II gene products play a central role in immune responses by presenting antigens to T lymphocytes. The class III region, between class I and class II, contains a large number of genes encoding proteins of diverse functions such as the complement components C2 and C4 (8), steroid 21-hydroxylase (9), tumor necrosis factors a and /, and the heat shock protein HSP70 (3, 10, 11).The class II region is divided into the DR, DQ, and DP subregions, each of which contains at least one A and one B gene, encoding one a and one f class II protein chain, respectively. The organization of the class II genes has been analyzed in detail by PFGE, leading to the construction of physical maps (12-15). Differences in the maps between individual haplotypes have been revealed (16,17), in particular in the segment of DNA between the DQA and DRA genes (14). Differences have also been observed in the class III region due to the number and size of C4 genes present (18,19). Cosmids have been isolated from each of the class II subregions, but large uncloned gaps remain. In the DR subregion different groups have isolated cosmids containing DRA or DRB genes, but it was not possible to link all DRB genes with each other (20) or to DRA (21-23). The DQ subregion has been cloned (24), but it was not linked to DR or to the DNA gene (25). The DNA gene has not been linked to the DP subregion by cosmid walking (25). Similarly, the 400 kilobase pair (kbp) region between class II and the complemen...

Report of the committee on the genetic constitution of chromosome 6

“…This region is included within a 2.5 Mb map characterized by jumping clones, YAC and cosmid clones Ragoussis et al, 1991;Glynne et al, 1991;Spies et al, 1990;Ragoussis et al, 1986), and is in good agreement with pulsed field gel electrophoresis maps. The order of loci is unambiguous.…”

Genes in one megabase of the HLA class I region.

“…Human thymus, spleen, liver, and adult testes were obtained from the National Disease Research Interchange (Philadelphia). Spontaneously aborted total fetus (9 weeks of gestation) and fetal brain (11 (25). The cDNA inserts were amplified by PCR with the SK and KS primers (25).…”

“…In humans it is composed, from centromere to telomere, of the class II region spanning about 850 kb, the class III region spanning about 1100 kb, and the class I region spanning about 1500 kb (8,9). The class II and class III regions have been extensively investigated, and the density of genes in this area is in the range of one gene or pseudogene every 15-20 kb (8,(10)(11)(12)(13)(14)(15). Although a number of class I genes and non-class-I genes have been identified in the class I region (14,(16)(17)(18)(19), there has been no exhaustive study of the gene content of this portion of the human MHC.…”

Yeast artificial chromosomes for the molecular analysis of the familial polyposis APC gene region.

“…DNA was transferred to Hybond N+ (Amersham), and blots were hybridized with appropriate probes to identify true positives. Large-scale agarose block preparations were then made from these clones by standard methods (20,21). In situ fluorescence hybridization of total yeast DNA to human metaphase spreads was carried out by using a modification ofthe method described by Williams et al (22).…”