Abstract
Most models of hereditary hypotrichosis are due to alterations in growth factors and transcription factors, and the examples of causative mutations in hair keratin genes are limited. The Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDRs). In this study, the locus of the responsible gene was examined by linkage analysis and mapped on chromosome 7q36. Because many basic keratin genes are clustered on 7q36, their expression was examined. Reverse transcription-PCR and genomic PCR indicated that the Kb21 (Krt81), -23 (Krt83), and -26 (Krt86) genes encoding basic hair keratins were not expressed and were deleted. Furthermore, 80-kb genomic DNA ranging from exon 9 of Kb25 (Krt85) to exon 9 of Krt2-25 was deleted. The breakpoints of these genes were within a 95-bp portion shared by the two genes, suggesting that deletion due to non-allelic homologous recombination occurred. Proteins identified as Kb21, Kb23, and Krt2-25 in SDR hairs by mass spectrometry were not detected in HHR. Instead, the product of a fusion gene became dominant in HHR. Because fusion occurred between the exons of the two genes with the same sequences, the product was identical to the wild-type Kb25 protein. By using immunohistochemistry, Kb21 was not detected in HHR hair follicles. Kb25 was expressed in the cortex in HHRs, whereas it was in the medulla in SDRs. This study clearly illustrates the importance of hair keratin genes in hair growth.
Highlights
The large keratin multigene family comprises cytokeratins, which are differentially expressed in the various types of epithelia, and hair keratins, expressed in hard keratinized structures such as hairs, nails, and claws
To purify basic hair keratins, Sprague-Dawley rat (SDR) 51-kDa proteins, and Hirosaki hairless rat (HHR) 56-kDa protein resolved by SDSPAGE, the gel portions containing the respective proteins were cut with a razor, homogenized in 1% SDS, 20 mM Tris-HCl, and rotated overnight
Sp1 Hoxc8, Hoxc13 tel Mapping of the hhr—To map the hhr locus, the backcross N2 progeny were produced from the (BN ϫ HHR) F1 mated with HHR
Summary
Rats—HHRs were maintained by brother-sister mating of hhr/ϩ heterozygous females and hhr/hhr homozygous males. Location of the Deletion Breakpoints—PCR was performed on genomic DNA derived from the livers of HHRs and SDRs. Amplification was carried out for 35 cycles, each consisting of 30 s at 94 °C, 30 s at 60 °C, and 2 min at 72 °C. To purify basic hair keratins, SDR 51-kDa proteins, and HHR 56-kDa protein resolved by SDSPAGE, the gel portions containing the respective proteins were cut with a razor, homogenized in 1% SDS, 20 mM Tris-HCl (pH 8.0), and rotated overnight. To produce anti-Kb21 antibody, the C-terminal peptide of Kb21 was synthesized by a peptide synthesizer (model 432A, Applied Biosystems), based on the amino acid sequence of the rat Kb21 (473SAVSCGRKC481) that was specific to the keratin (4). The peptide was coupled to keyhole limpet hemocyanin with m-maleimidobenzoyl N-hydroxysuccinimide ester (29) This peptide-hemocyanin complex was injected into a rabbit to produce anti-Kb21 antibody, as described above. B, physical map of the marker genes, hair growth-related genes, and the basic keratin gene cluster on rat chromosome 7q36
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