|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1990 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1989 : ¥800,000 (Direct Cost : ¥800,000)
1. Isolation of Y-specific DNA : Nine Y-specific DNA clones were isolated from a flow-sorted human Y chromosomal library, and their male specificity was confirmed by Southern blot analysis. of these 9 clones, a 4.6 kb long, Y-specific Pragment was further characterized. The fragment, designated as pY-80, was proven with an in situ hybridization experiment to have originated from the Ypl 1.2-Ypter region. Its 2, 808 bp section was sequenced. The pY-80 clone had no homology with previously sequenced Y specific clones.
2. Sex determination using polymerase chain reaction(PCR) : PCR proceeded with oligonucleotides flanking a 666 bp PstlEcoRl fragment of the sequence as primers and a male genomic DNA as a template, but not with a female genomic DNA. Samples of various sources successfully detected the Y-specific fragment in male-derived samples, including mouth wash, single hair roots, urinary epithelial cells, dried blood spots and Omniotic fluid cells.
3. Identification of small marker chro
mosomes : A 10-year-old girl and a 10-month-old girl, both with ambiguous genitalia, were found to have 45, X/46, X, mar and 45, X/46, X, r(?)mosaicism. The marker chromosomes in both girls were very small. Polymerase chain reaction, with synthetic oligonucleotide primers from Y-specific DNA sequences pY-80 and pY53.3 containing the sex-determining region Y(SRY), proved the marker chromosomes to contain the Y short arm material. In situ hybridization with probe pY-80 comfirmed that the marker chromosomes included the Y short arms. These findings, together with ambiguous genitalia in the girls, indicate that the marker chromosomes include the testis determing factor gene.
4. Appication of pY-80 for fluorescence in situ hybridization : Fluorescence in situ hybridization(FISH)using biotinylated or digoxigenin labeled pY-80 as a probe showed that pY-80 was located at Ypl2. In addition, it was shown that polymerase chain reaction(PCR)products flanking Pstl-EcoRl fragment of pY-80 could be used as a probe for FISH. These results indicated that pY-80 would be useful for not only the identification of Y chromosomal material in various samples including amniotic fluid cells, peripheral blood lymphocytes, buccal mucosal cells and urinary epithelial cells, but also direct visualization of its localization on metaphase cells. For one instance, the PCR and FISH methods were used to identify the origin of marker chromosome in two male infertile patients with 46, X, +mar karyotype and indicated that the marker chromosomes were pseu dic(Y). The origin of small ring chromosome in patients with Kabuki make-up and Turner syndrome was also proven to be Y chromosomal by PCR and FISH methods.
5. Identification of Y chromosomal materials in a 1-year-old boy with 46. XX : PCR using pY-80 and sex determining region Y(SRY)was performed on the patient. The PCR products were not obtained from the patient's DNA. The results indicated that the etiology of 46, XX male with hypospadias is different from that of 46, XX male without hypospadias in whom SRY is usually positive. Less