Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.
Professor, Dept. of Human Genetics, School of International Health, University of Tokyo, JAPAN
Different approaches are needed, including the use of deletion mapping, construction of physical maps, use of YAC libraries, and Noah's ark blots. We have successfully cloned both X and Y counterparts of the amelogenin gene using the Noah's ark approach. Cloning of other genes using deletion mapping is also in progress.
Sex chromosomes show characteristic behaviour in male meiosis where a crossing over between short arms of both X and Y is obligatory. The crossing over is limited within the pseudoautosomal region which spans 2.5Mb. It is distal to the SRY gene. The testis-determining SRY gene remains on the Y chromosome and is not involved in the exchange.
In addition to the SRY gene, the presence of some other genes are proposed on the Y chromosome, including growth control factor Y, a gene or genes related to spermatogenesis and a gene related to the development of gonadoblastoma. Chromosome Y reduces the total ridge count of fingers which is proportional to the water content of the embryonic body and so a gene on the Y chromosome may reduce the water content of the embryonic body. There are also genes related to IQ, and the enamel of teeth.
In cloning the genes or DNA fragments from the Y chromosome, we started by designing a positive cloning vector. The plasmid was basically the PAT153 plasmid. Two copies of DYZ1 repeating units were arranged in a tail-to-tail orientation. When this particular plasmid is used to subclone inserts of a lambda phage library, only plasmids with an insert larger than 0.7kb can survive. Any empty plasmids, those with an insert smaller than 0.7kb, or those with DYZ1 inserts, cannot transform host bacteria. A very efficient cloning is possible (Nakahori et al. 1991a).
Using this particular vector, a total of 31 clones were obtained from an EcoR1 digested library (ATCC). Southern blot analysis against male and female DNA was carried out using these 31 clones. Eleven had single copy fragments and detected 12 loci. They were mapped together with 5 probes obtained from other sources. The latter 5 detected 11 loci on the Y chromosome. Four probes were supplied from Dr. Weissenbach in Paris, and one from Dr. Page in Massachusetts. In addition, DYZ1 repeated DNA family was studied using Southern blotting and PCR. The sequence of a repeating unit consisting of 3564bp was determined in our laboratory. An additional two loci, PABY and SRY were examined by PCR. Either primer sequence or genomic DNA sequences were obtained from the literature.
The present status of this DNA analysis in our laboratory is summarised in Figure 1. We are still trying to increase the number of probes as well as the number of patients, to increase the resoultion of deletion mapping. These 24 cosmid clones were obtained from Dr. Yusuke Nakamura and are still in the process of being mapped. These are DNA probes and each line represents the separately identifiable individual sites.
Deletion mapping on 69 patients with a structurally abnormal Y chromosome has been conducted. See Figure 1 for loci. Locus 50f2D represents the centromere. DYZ1 represents the Q band positive fluorescent segment. The size of this segment is long. The SRY gene was detected in patients who are XX males. 87-4 (AMGL) is the amelogenin-like gene on the Y chromosome. Using this mapping panel, the gonadoblastoma gene was excluded from the distal half of the non-fluorescent part of the long arm.
There is a deletion common to six patients with azoospermia. Under a microscope, these Y chromosomes have a perfectly normal appearance even by high resolution banding. Additionally, one patient had a deletion associated with an apparently normal Y chromosome. The deleted segment was different. In addition several patients of azoospermia showed obvious deletion on the Y chromosome. At the moment, we are thinking that the gene deletion causing azoospermia is likely to be close to here. Table 1 summarises the results on azoospermic patients. Out of 54 patients with apprently normal Y chromosomes, 7 cases, over 10%, showed a small deletion on DNA analysis.
Table 1: Summary of loci deletions in patients with azoospermia
By using such analysis, we can identify clones which show homology to different primate species. The preservation of such fragments in species including mice, rats and bovine clones can also be examined. If the sample shows a stronger signal in the female, then the locus is on the X chromosome. We sequenced both X and Y counterparts and compared the sequences for homology. In both the X and Y sequences, three exon-like structures were identified. In one intron there is a 180 bp deletion in the Y chromosome. The presence of such exon structure on the Y chromosome surprised us, as Y specificity was rather limited in the Noah's ark blots (Nakahori et al. 1991b).
Amino acid sequences were deduced from the three exons of both the chromosome X and Y genes. Corresponding sequences of bovine and murine amelogenin were obtained from sequence databases and compared. The first fifty amino acids were very well preserved. There is a 4 amino acid insertion in the murine gene, and longer insertions in the bovine. In the human gene, the Y sequence shows more substitution compared to the chromosome X counterpart. Substitution of DNA sequences was analysed by Dr. Gojobori in the National Institute of Genetics. Human X and Y sequences may have been separated by some 25 million years. Presumably both sequneces are in the pseudoautosomal region, and at that time, may be by an inversion, both sequences may have started to evolve independently. The Y sequence shows faster evolution, which suggests that the Y sequence is genetically inactive although it preserves exon/intron structure.
We supplied our clone to research groups in Sweden, Wales and San Francisco. The Swedish group found a 5kb deletion in a patient with amelogenesis imperfecta of the sex-linked type. By PCR analysis all three exons were found to have been deleted (Lagerstrom et al. 1991).
To facilitate faster screening and also to screen a YAC library, we have started to subclone some of the probes and obtain partial sequences. This work is ongoing. I would like to thank young scientists who contributed to this research, Yutaka Nakahori, Shigeo Nagafuchi, Takashi Tamura, Hironao Numabe and Satoko Seki. Thank you for your attention.
Lagerstrom, M. et al. (1991) "A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIHI)", Genomics 10: 971-5.
Nakahori, Y. et al. (1991a) "Molecular cloning and mapping of ten new probes on the human Y chromosome", Genomics 9: 765-9.
Nakahori, Y. et al. (1991b) "Human X-Y homologous region encodes 'amelogenin'", Genomics 9: 264-9.