Smith, Matthijs Johannes; (1992) Molecular analysis of the human pseudoautosomal gene MIC2 and a related locus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Genomic sequences for the human gene were isolated in a chromosome walk spanning 95kb of DNA from the pseudoautosomal region. This walk overlaps with a previously published chromosome walk and completes the isolation of sequences between MIC2 and the human pseudoautosomal boundary. The distance between the 5' end of MIC2 and the pseudoautosomal boundary is 95kb consistent with predictions from previous genetic studies. Analysis of the gene's intron/exon structure showed that it is composed of 10 small exons distributed over 52kb. Many of the exons encode predicted structural domains within the protein product. Sequences related to MIC2 could only be detected in primates using either the cDNA or genomic probes containing individual exons of the gene. However, the genomic probes for exons 1 and 5 of MIC2 both detected a second locus in human DNA. These loci mapped to both the human X and Y chomosome suggesting a pseudoautosomal localisation. Two YAC clones were isolated from the proximal portion of the pseudoautosomal region which contained both MIC2 and the loci related to exons 1 and 5. Isolation of the exon 1 related loci showed that it contained a CpG-rich island that was unmethylated on the human Y chromosome and both the active and inactive X chromosomes. Pulsed field mapping demonstrated that this island was located 130kb distal to the MIC2 CpG island. The sequences of the exon 1- and exon 5-related loci were used to design specific oligonucletide primers. PCR of reverse transcribed RNA with these primers generated four major bands. Sequence analysis of the RT-PCR generated cDNAs showed that they were composed of several copies of sequences related to exons 4 and 5 of MIC2 but did not contain an open reading frame. Genomic DNA from this locus, named MIC2R, was isolated and revealed that, in addition to the exon 1-related sequence, there were four tandem duplications containing sequences related to exons 4 and 5 and the intron that separates them. Homology comparisons and analysis of the shared differences between these four sequences suggest that they arose by individual, sequential duplications of a unit containing exon 4 and 5 of MIC2. In addition, the absence of an open reading frame and the types of mutations found at this locus suggest that MIC2R is a transcribed pseudogene. If this is so, it is unusual in that it is associated with an unmethylated CpG-rich island.

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