Abstract
Polymerase chain reaction-based screening of an arrayed human P1 artificial chromosome (PAC) library using primer pairs specific for the human type I hair keratins hHa3-II or hHa6, led to the isolation of two PAC clones, which covered 190 kilobase pairs (kbp) of genomic DNA and contained nine human type I hair keratin genes, one transcribed hair keratin pseudogene, as well as one orphan exon. The hair keratin genes are 4-7 kbp in size, exhibit intergenic distances of 5-8 kbp, and display the same direction of transcription. With one exception, all hair keratin genes are organized into 7 exons and 6 positionally conserved introns. On the basis of sequence homologies, the genes can be grouped into three subclusters of tandemly arranged genes. One subcluster harbors the highly related genes hHa1, hHa3-I, hHa3-II, and hHa4. A second subcluster of highly related genes comprises the novel genes hHa7 and hHa8, as well as pseudogene PsihHaA, while the structurally less related genes hHa6, hHa5, and hHa2 are constituents of the third subcluster. As shown by reverse transcription-polymerase chain reaction, all hair keratin genes, including the pseudogene, are expressed in the human hair follicle. The transcribed pseudogene PsihHaA contains a premature stop codon in exon 4 and exhibits aberrant pre-mRNA splicing. Evolutionary tree construction reveals an early divergence of hair keratin genes from cytokeratin genes, followed by the segregation of the genes into the three subclusters. We suspect that the 190-kbp domain contains the entire complement of human type I hair keratin genes.
Highlights
The keratin multigene family comprises the cytokeratins or soft ␣-keratins, which are expressed in the various types of epithelia, and the hair keratins or hard ␣-keratins, involved in the formation of hard keratinized structures
Screening of P1 artificial chromosome (PAC) Clones, Identification of 10 Human Type I Hair Keratin Genes and One Orphan Exon—The combination of PAC and cloning allowed the isolation of a 190 kbp contiguous stretch of human genomic DNA harboring 10 human type I hair keratin genes (Fig. 1)
In the present study we have performed a systematic analysis of the genomic region upstream and downstream of a previously described type I human hair keratin locus on chromosome 17q12-q21 [13], which harbored the entire hHa2 hair keratin gene as well as a partial sequence of the hHa5 gene both oriented in the same transcriptional direction [16]
Summary
The keratin multigene family comprises the cytokeratins or soft ␣-keratins, which are expressed in the various types of epithelia, and the hair keratins or hard ␣-keratins, involved in the formation of hard keratinized structures. Both can be divided into type I (acidic) and type II (basic-neutral) proteins that form the 10-nm intermediate filament network of epithelial cells by obligatory association of equimolar amounts of type I and type II keratins [1, 2]. The present study attempts a bidirectional expansion and analysis of the hHa2/ hHa5 gene region by means of combined and P1 artificial chromosome (PAC) cloning in order to learn more about the organization of the human type I hair keratin gene cluster on chromosome 17q12-q21 and to possibly detect novel type I hair keratin genes
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