Sequencing Of Partial Hairless Gene Of Yuyi Hairless Mice And Its Detection By PCR In Situ

Objective:In 1990, several hairless mice were found from KM experiment mice' reproducing group in the Lab Animal Center of Henan. 10 years later, the first inbred strain of hairless mice in China was founded. And it was named Yuyi hairless mice (for short: YYHL). These mice develop a normal first pelage up to the age of 2~3 weeks, at which time they begin to lost their fur coat in a cephalocaudal pattern. The hair loss is normally completed within 1 week except of their beard, after which the animals remain naked all their lives accompanied by thymus's premature degeneration, skin's premature aging, shortened life span (about 12 months) and abnormalities in immunology and reproduction. YYHL have the similar appearance to hairless mice reported abroad. According to the analysis of genetic spectrum, it has been confirmed that the hairless mutation is autosomal recessive allelic mutation that maps to mouse Chromosome 14. However, the function of hairless gene has remained largely unexplored. In order to research the mutation of YYHL's hairless gene, the cellular and molecular pathways of hairless gene activity and the role of the hairless gene product in hair follicle biology, we researched the YYHL from the points of molecular biology and genetic mapping for the first time. Methods:We designed several primers based on the available murine cDNA sequence of hairless gene (Genbank accession number Z32675). Then we cloned and sequenced partial hairless gene of YYHL. After that, we compared the partial DNA sequence of YYHL's hairless gene with that of mice, rat and human, respectively. Besides that, hairless gene of YYHL was detected by the method of PCR in situ. Results: 1: Cloning and sequencing of hr gene of YYHL and KM miceThe DNA fragment sequenced of YYHL mice and KM mice both contains 923 bp. Each of them includes 4 exons (exon13, 14, 15, 16) and 3 introns(intron 13, intron14, intron15). And the 3 introns are reported for the first time. 2: Comparison of partial hr gene between YYHL and KM miceThe partial hr gene of YYHL shares 100% identity with that of KM mice. S.The nucleotide homology and amino acid sequence homology of YYHL's hr gene compared with that of mouse, rat and humanThrough the analysis on computer, we obtain that the homology of YYHL hr gene compared with mouse, rat and human is 100.0%, 95.0%, 84.0%, respectively. Analysis reveals that the coding sequence of the partial hr gene of YYHL contains 138 amino acid, sharing 100.0%, 97.8% and 84.0% identity with that of mouse, rat and human, respectively, and confirms that hr gene is highly conserved among different mammalian taxons. We can also deduce that the hr gene of YYHL shoud map to Chromosome 14. 4. PCR in situ for detection of the hr gene of YYHLWe detected hr gene in the slide of metaphase chromosome of YYHL by the method of PCR in situ. The result is positive. We can find obviously amplifying signals. There are 1 ~2 green signals in one metaphase chromosome and interphase nucleus. It not only confirms that the partial hr gene sequenced this time is right, but also paves the way for accurate mapping of hr gene of YYHL. Conclusions:1. Partial hairless gene of YYHL was sequenced. The mutation of YYHL's hairless gene does not happen within the sequence cloned this time, and that it must happen in otherpositions.2. The degree of conservation of the hr gene among different mammalian species is high. The normal hr gene product may be implicated into some basic regulatory pathways. We can also deduce that the hr gene of YYHL maps to Chromosome 14.3. 3 introns of YYHL's hr gene is reported for the first time.4. The method of PCR in situ for detection of hr gene is founded.The result of the research helps us to know more about the mutation of YYHL, the structure, function and product of hr gene, the pathomechanisms of hairless phenotype. And it also helps us. to clarify the pathomechanisms of human papular atrichia.