BIMM 110    Lectures 14-15

TEXTBOOK:

SLIDES 14
SLIDES 15

A. Molecular biology of the Y chromosome

1. Morphology and complexity of the Y chromosome

      - large heterochromatic region on long arm (few genes)

2. X and Y chromosomes in meiosis I

      - the pseudoautosomal region of X and Y chromosomes;
      - legitimate and illegitimate recombination

3. Molecular-genetic analysis of the Y chromosome (D. Page at MIT)

       - Y chromosome-specific (unique) cloned DNA sequences; large heterochromatic region on long arm (Yq)
      - lambda, cosmid and YAC libraries
      - ordered sequences and contigs; chromosome walking

4. Search for the TDF (testis determining factor)

- deletion mapping in XX males and XY females:     mapping SRY
              X chromosomes with portions of Y (icluding TDF gene);
              Y chromosomes with portions of X (missing TDF gene)
              delimiting the region for the putative TDF gene (D. Page group at MIT)
- Criteria for identifying pieces of DNA as relevant in the search for the TDF :

a) ORFs - open reading frames; likely protein coding regions;
b) "Zoo blots" - search for homologous sequences (orthologs) in other species; exon sequences are expected to be conserved;
c) amino acid sequence motifs - comparison with known motifs may suggest function (e.g. transcription factor);
d) gene dosage in XY males and XX females - TDF gene was expected to be absent in females

===>    Identification of the genes for the zinc finger proteins ZFY and ZFX:

- initial arguments in favor of ZFY = TDF:
                 ZFY and ZFX are sufficiently different (???)
                 one gene is inactive in females (X-inactivation), while both are active in males
                 increasing doubts about their primary function.

- arguments that eliminated the ZFY gene product as the TDF

5. Identification and cloning of SRY gene

- re-examination of a set of XX males : PCR-based identification and distinction of .PAR-Y boundary region from PAR-X boundary region

- the SRY gene is closer to the boundary than the ZFY gene

- the following diagram illustrates the rationale for the observation of a female with the karyotype

46, X, t(Y;22) (p11.2;q11)

- a re-examination confirmed that both SRY and ZFY had been lost in this female, but what was not anticipated was the finding the loss involved two noncontiguous deletions, one of which was originally not detected with the relatively low density of markers.

- further analysis of XX males (pseudo-hermaphrodites) with even shorter Y chromosome translocations
- the following diagram illustrates how PCR with specific primers flanking the boundary between the pseudoautosomal region and the X or Y chromosomes can be used to verify the presence of the tip of the Y in XX males

- the TDF (SRY) locus and its primary role in male differentiation
- Transgenic mice with Sry gene injected into XX embryos

- the SRY gene product: a transcription factor (?); target genes ? member of HMG family of proteins

 A. Historical

 - Anderson, M., Page, D.C., and De la Chapelle, A. (1986). Chromosome Y-specific DNA is transferred to the short arm of X chromosome in human XX males. Science 233, 786-788.
- Buckle, V., Mondello, C., Darling, S., Craig, I.W., and Goodfellow, P.N. (1985). Homologous expressed genes in the human sex chromosome pairing region. Nature 317, 739-741.
- Cooke, H. (1990). The continuing search for the mammalian sex-determining gene. TIG 6, 273-275.
- Craig, I. (1990). Sex determination: Zinc fingers point in the wrong direction. TIG 6, 135-137.
- Disteche, C.M., Casanova, M., Saal, H., Friedman, C., Sybert, V., Graham, J., Thuline, H., Page, D.C., and Fellous, M. (1986). Small deletions of the short arm of the Y chromosome in 46,XY females. Proc. Natl. Acad. Sci. USA 83, 7841-7844.
- Ferguson-Smith, M.A. (1988). Genes on the X and Y chromosomes controlling sex. Br. Med. J. 297, 635-636.
- Goodfellow, P., Darling, S., and Wolfe, J. (1985). The human Y chromosome. J. Med. Genet. 22, 329-344.
- Goodfellow, P.J., Darling, S., Thomas, N.S., and Goodfellow, P.N. (1986). A pseudoautosomal gene in man. Science 234, 70-743.
- Mardon, G., and Page, D.C. (1989b). The sex-determining region of the mouse Y chromosome encodes a protein with a highly acidic domain and 13 zinc fingers. Cell 56, 765-770.
- Koopman, P., Ashworth, A., and Lovell-Badge, R. (1991a). The ZFY gene family in humans and mice. TIG 7, 132-136.
Koopman, P., Gubbay, J., Vivian, N., Goodfellow, P., and Lovell-Badge, R. (1991b). Male development of chromosomally female mice transgenic for Sry. Nature 351, 117-121.
- Mardon, G., Mosher, R., Disteche, C.M., Nishioka, Y., McLaren, A., and Page, D.C. (1989a). Duplication, deletion, and polymorphism in the sex-determining region of the mouse Y chromosome. Science 243, 78-80.
- McLaren, A. (1990). Sex determination: What makes a man a man. Nature 346, 216-217.
- McLaren, A. (1991). Sex determination: The making of male mice.Nature 351, 96.
- Mittwoch, U. (1988). Y chromosome and sex determination. Lancet 2, 52-53.
- Noethiger, R., and Steinmann-Zwicky, M. (1987). Genetics of Sex Determination in Eukaryotes. In Structure and Function of Eukaryotic Chromosomes, W. Hennig, ed. (Berlin-Heidelberg: Springer-Verlag), pp. 271-300.
- Page, D.C., Brown, L.G., and De la Chapelle, A. (1987a). Exchange of terminal portions of X- and Y- chromosomal short arms in human XX males. Nature 328, 437-440.
- Page, D.C., Fisher, E.M.C., McGillivray, B., and Brown, L.G. (1990). Additional deletion in sex-determining region of human Y chromosome resolves paradox of X,t(Y;22) female. Nature 346, 279-281.
- Page, D.C., Mosher, R., Simpson, E.M., Fisher, E.M.C., Mardon, G., Pollack, J., McGillivray, B., De la Chapelle, A., and Brown, L.G. (1987b). The sex-determining region of the human Y chromosome encodes a finger protein. Cell 51, 1091-1104.
- Palmer, M.S., Berta, P., Sinclair, A.H., Pym, B., and Goodfellow, P.N. (1990). Comparison of human ZFY and ZFX transcripts. Proc. Natl. Acad. Sci. USA 87, 1681-1685.
- Roberts, L. (1988). Zeroing in on the sex switch. Science 239, 21-23.
- Schneider-Gädicke, A., Beer-Romero, P., Brown, L.G., Nussbaum, R., and Page, D.C. (1989). ZFX has a gene structure similar to ZFY, the putative human sex determinant, and escapes X inactivation. Cell 57, 1247-1258.
- Simmler, M.-C., Rouyer, F., Vergnaud, G., Nystrom-Lahti, M., Ngo, K.Y., De la Chapelle, A., and Weissenbach, J. (1985). Pseudoautosomal DNA sequences in the pairing region of the human sex chromosomes. Nature 317, 692-697.
- Thomas, B.J., and Rothstein, R. (1991). Sex, maps, and imprinting. Cell 64, 1-3.
- Vergnaud, G., Page, D.C., Simmler, , M.-C., Brown, L., Royer, F., Noel, B., Botstein, D., De la Chapelle, A., and Weissenbach, J. (1986). A deletion map of the human Y chromosome based on DNA hybridization. Am. J. Hum. Genet. 38, 109-124.
-Berkovitz, G.D., Fechner, P.Y., Marcantonio, S.M., Bland, G., Stetten, G., Goodfellow, P.N., Smith, K.D., and Migeon, C.J. (1992). The role of the sex-determining region of the Y chromosome (SRY) in the etiology of 46,XX true hermaphroditism. Hum. Genet. 88, 411-416.
-Foote, S., Vollrath, D., Hilton, A., and Page, D.C. (1992). The human Y chromosome: Overlapping DNA clones spanning the euchromatic region. Science 258, 60-66.
-Hamer, D.H., Hu, S., Magnuson, V.L., Hu, N., and Pattatucci, A.M.L. (1993). A linkage between DNA markers on the X chromosome and male sexual orientation. Science 261, 321-327.
-Su, H. and Lau, Y.-F.C. (1993). Identification of the transcriptional unit, structural organization, and promoter sequence of the human sex-determining region Y (SRY) gene, using a reverse genetic approach. Am. J. Hum. Genet. 52, 24-38.
-Vollrath, D., Foote, S., Hilton, A., Brown, L.G., Beer-Romero, P., Bogan, J.S., and Page, D.C. (1992). The human Y chromosome: A 43-interval map based on naturally occurring deletions. Science 258, 52-59.

 B. Recent

 - Bogan, J.S. and Page, D.C. (1994). Ovary? Testis?--A mammalian dilemma. Cell 76, 603-607.
- Capel, B. (1995). New bedfellows in the mammalian sex-determination affair. TIG 11, 161-163.
- Clapham, D.E. (1994). Signal transduction: Why testicles are cool. Nature 371, 109-110.
- Fiddler, M., Abdel-Rahman, B., Rappolee, D.A., and Pergament, E. (1995). Expression of SRY transcripts in preimplantation human embryos. Am. J. Med. Genet. 55, 80-84.
- Goodfellow, P. N. and R. Lovell-Badge. 1993. Sry and sex determination in mammals. Annu. Rev. Genet. 27:71-92.
- Haqq, C. M., C.-Y. King, P. K. Donahoe, and M. A. Weiss. 1993. SRY recognizes conserved DNA sites in sex-specific promoters. Proc. Natl. Acad. Sci. USA 90:1097-1101.
- Haqq, C.M., King, C.-Y., Ukiyama, E., Falsafi, S., Haqq, T.N., Donahoe, P.K., and Weiss, M.A. (1994). Molecular basis of mammalian sexual determination: Activation of Müllerian inhibiting substance gene expression by SRY. Science 266, 1494-1500.
Hawkins, J.R. (1994). Sex determination. Hum. Molec. Genet. 3, 1463-1467.
- López, M., Torres, L., Méndez, J.P., Cervantes, A., Alfaro, G., Pérez-Palacios, G., Erickson, R.P., and Kofman-Alfaro, S. (1995). SRY alone can induce normal male sexual differentiation. Am. J. Med. Genet. 55, 356-358.
- McElreavey, K., E. Vilain, N. Abbas, I. Herskowitz, and M. Fellous. 1993. A regulatory cascade hypothesis for mammalian sex determination: SRY represses a negative regulator of male development. Proc. Natl. Acad. Sci. USA 90:3368-3372.
- Schmitt-Ney, M., Thiele, H., Kaltwasser, P., Bardoni, B., Cisternino, M., and Scherer, G. (1995). Two novel SRY missense mutations reducing DNA binding identified in XY females and their mosaic fathers. Am. J. Hum. Genet. 56, 862-869.
- Werner, M.H., Huth, J.R., Gronenborn, A.M., and Clore, G.M. (1995). Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex. Cell 81, 705-714.
-Zinn, A.R., Page, D.C., and Fisher, E.M.C. (1993). Turner syndrome: The case of the missing sex chromosome. Trends Genet. 9, 90-93.

- Palmirotta, R., Verginelli, F., Cama, A., Mariani-Costantini, R., Frati, L., and Battista, P. (1998). Origin and gender determination of dried blood on a statue of the Virgin Mary.  J.Forensic Sci. 43, 431-434.

- Kuroda-Kawaguchi, T., et al., (2001) The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nature Genet. 29: 279-286.