A duplicated locus encoding the major heat shock-induced protein HSP70 is located in the major histocompatibility complex (MHC) class III region 92 kilobases (kb) telomeric to the C2 gene. Nucleotide sequence analysis of the two intronless genes, HSP70-1 and HSP70-2, has shown that they encode an identical protein product of 641 amino acids. A third intronless gene, HSP70-Hom, has also been identified 4 kb telomeric to the HSP70-1 gene. This encodes a more basic protein of 641 amino acids which has 90% sequence similarity with HSP70-1. In order to investigate the expression of the three (MHC)-linked HSP70 genes individually by northern blot analysis, we have isolated locus-specific probes from the 3' untranslated regions of the genes. The HSP70-1 and HSP70-2 genes have been shown to be expressed at high levels as a approximately 2.4 kb mRNA in cells heat-shocked at 42 degrees C. HSP70-1 is also expressed constitutively at very low levels. The HSP70-Hom gene, which has no heat shock consensus sequence in its 5' flanking sequence, is expressed as a approximately 3 kb mRNA at low levels both constitutively and following heat shock.
The chemical reactivity of thymine (T), when mismatched with the bases cytosine, guanine, and thymine, and of cytosine (C), when mismatched with thymine, adenine, and cytosine, has been examined. Heteroduplex DNAs containing such mismatched base pairs were first incubated with osmium tetroxide (for T and C mismatches) or hydroxylamine (for C mismatches) and then incubated with piperidine to cleave the DNA at the modified mismatched base. This cleavage was studied with an internally labeled strand containing the mismatched T or C, such that DNA cleavage and thus reactivity could be detected by gel electrophoresis. Cleavage at a total of 13 T and 21 C mismatches isolated (by at least three properly paired bases on both sides) single-base-pair mismatches was identified. All T or C mismatches studied were cleaved. By using end-labeled DNA probes containing T or C single-basepair mismatches and conditions for limited cleavage, we were able to show that cleavage was at the base predicted by sequence analysis and that mismatches in a length of DNA could be readily detected by such an approach. This procedure may enable detection of all single-base-pair mismatches by use of sense and antisense probes and thus may be used to identify the mutated base and its position in a heteroduplex.Definition of the exact single-base change in genes as a result of mutation is an important goal in genetic research. As sequencing complete genes to identify base changes is tedious, attempts have been made to improve the efficiency of the procedure (1-4). (i) Heteroduplexes formed between wild-type and variant DNAs have been treated with the single-strand-specific S1 nuclease to cleave the DNA at the point of the mismatched bases (1) (6) (Fig. 1).Subclones carrying the desired DNA fragment complementary to the probe required were labeled by standard methods (9) by using the M13 universal sequencing primer. All dNTPs were at a concentration of 0.25 mM except dATP, which was added so that the [a-32P]dATP was diluted 1:9. Typically, 2 ng of primer was annealed with 50 ng of M13 DNA in 6.5 /l, by heating at 90'C for 4 min followed by incubation at room temperature for 30 min. dNTPs were then added with 1 pl of [a-32P]dATP (3000 Ci/mmol; 1 Ci = 37 GBq; Radiochemical Centre) and 1 p.1 (7.5 units) of the Klenow fragment of DNA polymerase I (Pharmacia) in a final volume of 17.6 .ld, and the mixture was incubated at 20-240C for 1 hr. All dNTPs were then added at 0.25 mM and incubated 30 min to chase. Samples were then extracted with chloroform/phenol, 1:1 (vol/vol), and the DNA was precipitated with ethanol. Immediately after labeling DNA was digested (in a final volume of 20-50 pL) with restriction enzymes appropriate for the heteroduplex being studied (see Table 1). End-labeled DNA probes (see Table 1) were derived from the appropriate digests of the 3.7-kilobase Taq I fragment of the wild-type or mutant 21-OHase B genes or the 5.5-kilobase Bgl II-BamHI fragment of the 21-OHase A gene, cloned in the Pvu II site of the plasmid pAT153/PvuI...
Here we report the first complete sequence and gene map of a human major histocompatibility complex (MHC), a region on chromosome 6 which is essential to the immune system. When it was discovered over 50 years ago the region was thought to specify histocompatibility genes, but their nature has been resolved only in the last two decades. Although many of the 224 identified gene loci (128 predicted to be expressed) are still of unknown function, we estimate that about 40% of the expressed genes have immune system function. Over 50% of the MHC has been sequenced twice, in different haplotypes, giving insight into the extraordinary polymorphism and evolution of this region. Several genes, particularly of the MHC class II and III regions, can be traced by sequence similarity and synteny to over 700 million years ago, clearly predating the emergence of the adaptive immune system some 400 million years ago. The sequence is expected to be invaluable for the identification of many common disease loci. In the past, the search for these loci has been hampered by the complexity of high gene density and linkage disequilibrium.
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