Feature mapping of the HLA class I region: localization of the POU5F1 and TCF19 genes.

The class I region of the human leukocyte antigen (HLA) complex located on chromosome 6p21.3 is gene dense. To define the gene content of the class I region, we are constructing genomic DNA feature maps. Here we report mapping of the POU5F1 and TCF19 genes to an approximately 0.2-Mb region between the HLA-C and the S genes. Localization of these genes was facilitated by subcloning genomic DNA fragments from the 0.2-Mb region into a transposon gamma delta-based vector, selecting transposon-mediated deletions in vivo in Escherichia coli, and sequencing a nested subset chosen for their uniform distribution of deletion endpoints. The POU5F1 and TCF19 genes are located approximately 130 kb telomeric of the HLA-C locus, approximately 0.6 kb apart from each other. Complete sequencing of a 5.5-kb EcoRI fragment containing the TCF19 gene revealed that it is composed of three exons, bounded by consensus splice signals. These experiments illustrate that the transposon-based nested deletion sequencing method provides an easy and efficient approach to feature mapping genomic fragments and to high-resolution analysis of selected subportions.

Construction of a genomic DNA 'feature map' by sequencing from nested deletions: application to the HLA class I region.

We are applying a transposon-based approach for detecting and mapping features of special interest to construct 'feature maps' of currently uncharacterized portions of the human leukocyte antigen (HLA) complex on chromosome 6. Such feature maps should facilitate identifying regions for high resolution analysis. Here we describe the feature mapping of a 35 kb DNA fragment located between the HLA-C and HLA-E loci. This fragment was cloned into a transposon gamma delta-based cosmid vector designed for generating nested deletions in vivo. Seventy informative nested deletions extending into the cloned fragment were isolated, and DNA adjacent to the deletion endpoints was sequenced by fluorescent automated technology. These islands of DNA sequences constituted the foundation of the feature map, and (i) identified putative exons, (ii) determined the positions of Alu elements, (iii) determined the span of the keratinocyte-specific S gene, and (iv) localized evolutionarily conserved sequences. The construction of feature maps using this in vivo nested deletion-sequencing approach provides a rapid and efficient means to identify DNA regions that merit more detailed analysis.

Molecular linkage of the HLA-DR, HLA-DQ, and HLA-DO genes in yeast artificial chromosomes.

Eight major histocompatibility complex (MHC) class II loci and the newly defined Y3/Ring 4 locus were isolated in overlapping yeast artificial chromosome (YAC) clones defining a 420-kb segment of human chromosome 6p21.3. YAC B1D12 spanning 320 kb contained seven of these loci from HLA-DRA to HLA-DQB2. A 330-kb YAC, A148A7, spanned from the HLA-DQA1 locus through the Y3/Ring 4 locus and extended at least 130 kb centromeric of YAC B1D12. Southern blotting demonstrated that YAC B1D12 derived from the HLA-DR3 haplotype and that YAC A148A7 derived from the HLA-DR7 haplotype of the heterozygous library donor. A third 150-kb YAC, A95C5, lay within this contig and contained only the HLA-DRA locus. A fourth 300-kb YAC, A76F11, was isolated by chromosome walking from the telomeric end of YAC B1D12. Probes isolated from the ends of the YAC genomic inserts have been used to confirm overlaps between the clones. These analyses demonstrated that the centromeric end of YAC A76F11 used the same genomic EcoRI cloning site as the telomeric end of YAC A95C5. YAC B1D12 used an EcoRI site only 2.1 kb telomeric of the aforementioned EcoRI site. These data suggest that certain EcoRI sites are used preferentially during construction of the library. These YACs complete the linkage of the DR and DQ subregions of the HLA complex in cloned DNA and provide the substrate for precise analysis of this portion of the class II region.