WONITOR

Chromosome crawling in the MHC JOHN BELt

INSTITUTE FOR MOLECULAR MEDICINE, JOHN RADCLIFFE HOSPITAL,

HEADINOTON, OXmRD OX3 9DU, UK.

Once the large scale physical map- pers have finished their hopping, jumping and walking there is left the difficult task of establishing the content of large segments of genomic DNA. Although there has been much discussion about new methods for large scale mapping, approaches designed to identify new expressed sequences have only recently been considered. The first detailed studies of this kind have recently been published and apply to the major histocompatibility complex (MHC) on the short arm of chromosome 61, 2 . Two groups have now mounted detailed searches along large segments of the MHC, looking for expressed se- quences. Although the approaches they have used to solve this prob- lem will almost certainly fail to detect some expressed sequences, they have already provided evi- dence of a remarkably high density of expressed genes in this region. These data demonstrate the chal- lenges that will be presented throughout the genome to those attempting to distinguish sequences of structural interest.

There is good reason for the interest in defining all the expressed sequences within the MHC. This region encodes the highly polymorphic class I and class II loci responsible for genetic regulation of immune responsive- ness. In addition, susceptibility to over 40 diseases has been associated with MHC haplotypes and although class I and class II alleles may

account for some of these associ- ations3, it is unlikely that they will account for them all. Any new expressed gene in this region is, therefore, a candidate gene for sus- ceptibility to many major common disorders. The MHC has provided the testing ground in human genet- ics for many mapping strategies. It was the first region of the human genome to be pulsed field mapped 4 and has been extensively mapped by cosmid walkersS,6. The recent studies have now provided a careful examination of each of these overlapping cosmids for expressed sequences, a process that has proved remarkably difficult to complete comprehensively but which has turned up a wealth of new information that will keep MHC geneticists working for a long time to come.

Within the MHC, in addition to the class I and class II loci that flank the complex, numerous other expressed genes of interest had already been described. The com- plement compounds C2, C4 and factor B, the tumour necrosis factor

and ~ loci, the 21-hydroxylase genes, the heat-shock protein HSP- 70 genes, and the RD gene had all been identified in the region between DR~ and HLA-B. The two groups attempting to complete the list of expressed sequences began with long stretches of overlapping cosmid clones. Spies et al. obtained 435 kb of cosmids extending from HLA-B (Ref. 1) to beyond the TNF loci, while Sargent et a12 studied

541 kb of cosmids in two clusters, one extending 393 kb from the centromeric end of the com- plement cluster to a site 100 kb from TNE and a second 148 kb long encompassing the TNF loci. Each group applied a slightly dif- ferent strategy to identify the expressed sequences within their cosmids.

Spies et al. probed northern blots directly with cosmids, screen- ing for the presence of transcripts in B cells, T cells, monocytes, epithelial cells and fibroblasts. Using this strategy they identified five new transcribed loci (Fig. 1) and proceeded to obtain cDNA clones for each. All transcripts were constitutively expressed in all the tissues screened.

Sargent et al. used a more thor- ough approach for identifying sequences. They first identified HTF islands within the cloned DNA. These CpG-rich regions were identified by the presence of clus- ters of restriction sites for the enzymes BssHII, EagI and SaclI and the lack of methylation of these sites in genomic DNA. Such sequences are commonly found adjacent to expressed house- keeping genes. Sequences adjacent to these HTF islands were then used to probe northern blots to identify the transcribed sequences. This approach revealed 12 novel genes (Fig. 1) and established a density of such genes of 1 every 23 kb of DNA between the C4 and the TNF loci.

HSP70 TNF 21B C4AG11 RDBF C2 G10 G9 G82 1 G7G6 G5G4G3G2 G1 B144 ~-I]

6 50 160 1i0 260 250 360 350 460 4g0 (kb)

[] I 1 3 D I II [] BAT5 BAT4 BATIBAT2 B144 ~18 BAT1

TNF

FIGIrl A comparison of the genes in the HLA class III region mapped by Spies et al. (below the rule) and Sargent et al. (above the rule). The BAT2, 3 and 4 genes mapped by Spies et al. are probably the same as the G2, G3 and G5 genes mapped by Sargent et al.

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Although both these studies carefully attempted to identify as many expressed loci as possible, it is very likely that many were missed. Genes that show some tissue specificity in their expression are often not associated with HTF islands 7. Several such sequences a r e known to exist already within the MHC (C4, C2, factor B). Tissue- specific genes may also have been missed for the reason that cosmids were screened against RNA from only a small number of cell types in a single stage of differentiation or activation.

Having crawled along these regions of DNA, the mappers may well have left important expressed sequences behind. Such sequences may have to wait until cosmids are screened against large numbers of RNA species or until sequencing of the entire region allows the identi- fication of the expressed products by the presence of particular sequence motifs.

Even without having identified the full complement of expressed loci in this region, these two stud- ies are important because they

provide an impression of high den- sity of sequences in particular genomic regions. The MHC is found in a Giemsa light or GC-rich region of the genome, consistent with this very high frequency of housekeeping genes s. For those embarking on systematic mapping or sequencing studies of the human genome, these studies have major implications. Similar high gene densities can be expected in other genomic regions associated with Giemsa light bands or GC-rich isochores. In contrast, the presence of such densely inhabited regions of genome must be balanced with large regions of DNA from which little is expressed. The prospect of sequencing across such known regions of DNA must raise substan- tial concern and the targeting of these efforts either to Giemsa light genomic regions or to cDNA clones in the first instance must look increasingly appealing.

Do the loci defined by these two papers explain any of the dis- ease susceptibilities mapping to the MHC? The answer is that it is too early to know. The genes must be

characterized, and polymorphism, if present, detected and characterized in the context of what we already know about MHC haplotypes, link- age disequilibrium and disease sus- ceptibility. This work has initiated an exciting new era of MHC genet- ics. It will require much work to characterize these new genes but judging from the pearls already thrown up by the MHC, there will be many more to find.

R e f e r e n c e s

1 Spies, T. eta/. (1989) Science243, 214-217

2 Sargent, C.A., Dunham, I. and Campbell, R.D. (1989) EMBOJ. 8, 2305-2312

3 Todd, J.A. et al. (1988) Science 240, 1003-1009

4 Hardy, D.A. et al. (1986) Nature 323, 453-455

5 Okada, K. etal. (1985) Proc. Natl Acad. Sci. USA 82, 3410-3414

6 Rollini, P., Mach, B. and Gorski, J. (1985) Proc. Natl Acad. Sci. USA 82, 7197-7201

7 Bird, A.P. (1987) Trends Genet. 3, 342-347

8 Holmquist, G., Gray, M., Porter, T. and Jordan, J. (1982) Cell 31, 121-129

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