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The YAC contig was constructed using the CEPH mega YAC library and was characterized and fine mapped with 20 newly generated hybridization markers, 7 STS markers and 40 gene specific markers. Using the STSs, genetic and physical map were integrated. Minimally 2 clones link more than 90 % of the markers. All results were verified by comparison with a long-range restriction map generated by PFGE with genomic DNA of the cell line H2LCL.
Using 4 overlapping YACs covering 5 MBp of the EDC region, cDNAs were selected by hybridisation. The used library contained 184 320 cDNAs constructed (see TIP nr. 1) from cultured keratinocytes. 22 cDNAs have been newly assigned to the EDC.
In order to create a resource to identify novel genes located to 1q21 and expressed during squamous differentiation, a cDNA library has been created from RNA isolated from cultured human keratinocytes, the major cell type of stratified squamous epithelia. To facilitate handling and analysis, the library was spotted on 10 separate filters, containing each 368.640 separate clones. This approach has allowed the identification and cloning of approximately 50 novel genes, expressed in keratinocytes and mapping to 1q21.
Human keratinocytes were cultured in vitro in the presence of 3T3 feeder cells according to the procedures first described by Rheinwald and Green. RNA was isolated from cells grown under various physiological conditions (growth factors, concentration of external calcium, degree of confluence and stratification), pooled and size fractionated on sucrose gradients. Four different fractions were collected, ranging from more than 5000 bp (fraction 1) to less than 1500 bp (fraction 4). Each fraction was converted into cDNA with reverse transcriptase (SuperScript II RNase H-). The four fractions of cDNA were independently cloned in the Uni-ZAP XR lambda vector according to the protocol supplied by the manufacturer (Stratagene, La Jolla, CA). The primary complexity of the libraries varied from 1.3 to 2 million plaques. The libraries from fractions 1/2 and 3/4 were pooled and the inserts were excised in vivo with the Exassist M13 helper phage (Stratagene), generating two pBluescript based bacterial libraries. Each library was spotted on 5 filters, containing each 368.640 individual bacterial clones.
As described in the scientific results, we have generated involucrinless mice by targeted ablation of the gene, but we do not anticipate any commercial use of these mice. However, in the course of the project, we have generated a clone encompassing most of the coding region of mouse involucrin. Dr. Fiona Watt's laboratory has used our clone to produce recombinant involucrin in bacteria and to generate a polyclonal antibody directed against the recombinant protein. This antibody has proved useful for staining the outermost layers of murine stratified squamous epithelia. Previously existing anti-mouse involucrin antibodies were not adequate for immunohistochemical staining.
Terminal differentiation of keratinocytes involves the sequential expression of several major proteins that can be identified in distinct cellular layers within the mammalian epidermis and are characteristic for the maturation state of the keratinocyte. Many of the corresponding genes are clustered in one specific human chromosomal region 1q21. It is rare in the genome to find in such close proximity the genes belonging to at least three structurally different families, yet sharing spatial and temporal expression specificity, as well as inter-dependent functional features. This DNA segment, termed the Epidermal Differentiation Complex, contains 27 genes, 14 of which are specifically expressed during calcium dependent terminal differentiation of keratinocytes (the majority being structural protein precursors of the cornified envelope) and the other 13 belong to the S100 family of calcium binding proteins with possible signal transduction roles in the differentiation of epidermis and other tissues. In order to provide a bacterial clone resource that will enable further studies of genomic structure, transcriptional regulation, function and evolution of the EDC, as well as the identification of novel genes, we have constructed a single 2.45 Mbp long continuum of genomic DNA cloned as 45 PAC, 3 BAC, and 34 cosmid clones. The map encompasses all of the 27 genes so far assigned to the EDC, and integrates the physical localisation of these genes at a high resolution on a complete NotI and SalI, and a partial EcoRI restriction map. This map is, at present, the starting resource for the large-scale genomic sequencing of this region by The Sanger Centre, Hinxton, UK.
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