Abstract
The Y chromosome is perhaps the most interesting element of the mammalian genome but comparative analysis of the Y chromosome has been impeded by the difficulty of assembling a shotgun sequence of the Y. BAC-based sequencing has been successful for the human and chimpanzee Y but is difficult to do efficiently for an atypical mammalian model species (Skaletsky et al. 2003, Kuroki et al. 2006). We show how Y-specific sub-libraries can be efficiently constructed using DNA amplified from microdissected or flow-sorted Y chromosomes. A Bacterial Artificial Chromosome (BAC) library was constructed from the model marsupial, the tammar wallaby (Macropus eugenii). We screened this library for Y chromosome-derived BAC clones using DNA from both a microdissected Y chromosome and a flow-sorted Y chromosome in order to create a Y chromosome-specific sub-library. We expected that the tammar wallaby Y chromosome should detect ∼100 clones from the 2.2 times redundant library. The microdissected Y DNA detected 85 clones, 82% of which mapped to the Y chromosome and the flow-sorted Y DNA detected 71 clones, 48% of which mapped to the Y chromosome. Overall, this represented a ∼330-fold enrichment for Y chromosome clones. This presents an ideal method for the creation of highly enriched chromosome-specific sub-libraries suitable for BAC-based sequencing of the Y chromosome of any mammalian species.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alsop A, Miethke P, Rofe R et al. (2005) Characterizing the chromosomes of the Australian model marsupial Macropus eugenii (tammar wallaby). Chromosome Res 13: 627–636.
Charlesworth B, Charlesworth D (2000) The degeneration of Y chromosomes. Phil Trans R Soc Lond B Biol Sci 355: 1563–1572.
Delbridge ML, Lingenfelter PA, Disteche CM, Graves JAM (1999) The candidate spermatogenesis gene Rbmy has a homologue on the human X chromosome. Nat Genet 22: 223–224.
Delbridge ML, Longepied G, Depetris D et al. (2004) Tspy, the candidate gonadoblastoma gene on the human Y chromosome, has a widely expressed homologue on the X – implications for Y chromosome evolution. Chromosome Res 12: 345–356.
Elkahloun AG, Meltzer PS, Guan XY, McNinch JS, Trent JM, de Jong PJ (1996) Isolation of a cosmid sublibrary for a region of chromosome 12 frequently amplified in human cancers using a complex chromosome microdissection probe. Genomics 31: 343–347.
Foster JW, Graves JAM (1994) An SRY-related sequence on the marsupial X chromosome: implications for the evolution of the mammalian testis-determining gene. Proc Natl Acad Sci USA 91: 1927–1931.
Graves JAM (1995) The origin and function of the mammalian Y chromosome and Y-borne genes – an evolving understanding. Bioessays 17: 311–320.
Graves JAM (2006) Sex chromosome dynamics: degeneration of the Y chromosome. Cell 124: 901–914.
Graves JAM, Wakefield M, Toder R (1998) The origin and evolution of the pseudoautosomal regions of the human sex chromosome. Hum Mol Genet 7: 1991–1996.
Grützner F, Crollius HR, Lütjens G et al. (2002) Four-hundred million years of conserved synteny of human Xp and Xq genes on three tetraodon chromosomes. Genome Res 12: 1316–1322.
Grützner F, Rens W, Tsend-Ayush E et al. (2004) In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Nature 432: 913–917.
Hughes JF, Skaletsky H, Pyntikova T et al. (2005) Conservation of Y-linked genes during human evolution revealed by comparative sequencing in chimpanzee. Nature 437: 100–103.
Kim UJ, Shizuya H, Deaven L, Chen XN, Korenberg JR, Simon MI (1995a) Selection of a sublibrary enriched for a chromosome from total human bacterial artificial chromosome library using DNA from flow sorted chromosomes as hybridization probes. Nucleic Acids Res 23: 1838–1839.
Kim UJ, Shizuya H, Chen XN et al. (1995b) Characterization of a human chromosome 22 enriched bacterial artificial chromosome sublibrary. Genet Anal 12: 73–79.
Kuroki Y, Toyoda A, Noguchi H et al. (2006) Comparative analysis of chimpanzee and human Y chromosomes unveils complex evolutionary pathway. Nat Genet 38(2): 158–167.
Pask A, Renfree MB, Graves JAM (2000) The human sex-reversing Atrx gene has a homologue on the marsupial Y chromosome, Atry: implications for the evolution of mammalian sex determination. Proc Natl Acad Sci USA 97: 13198–13202.
Rens W, O’Brien PC, Yang F, Graves JAM, Ferguson-Smith MA (1999) Karyotype relationships between four distantly related marsupials revealed by reciprocal chromosome painting. Chromosome Res 7: 461–474.
Sankovic N, Bawden W, Martyn J, Graves JAM, Zuelke K (2005) Construction of a marsupial BAC library from the model Australian marsupial, the tammar wallaby (Macropus Eugenii). Aust J Zool 53: 389–393.
Sharp P (1982) Sex chromosome pairing during male meiosis in marsupials. Chromosoma 86: 27–47.
Sinclair AH, Foster JW, Spencer JA et al. (1988) Sequences homologous to Zfy, a candidate human sex-determining gene, are autosomal in marsupials. Nature 336: 780–783.
Sinclair AH, Berta P, Palmer MS et al. (1990) A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346: 240–244.
Skaletsky H, Kuroda-Kawaguchi T, Minx PJ et al. (2003) The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423: 825–837.
Toder R, O’Neill RJ, Wienberg J, O’Brien PC, Voullaire L, Graves JAM (1997a) Comparative chromosome painting between two marsupials: origins of an Xx/Xy1y2 sex chromosome system. Mamm Genome 8: 418–422.
Toder R, Wienberg J, Voullaire L, O’Brien PC, Maccarone P, Graves JAM (1997b) Shared DNA sequences between the X and Y chromosomes in the tammar wallaby – evidence for independent additions to eutherian and marsupial sex chromosomes. Chromosoma 106: 94–98.
Toder R, Wakefield MJ, Graves JAM (2000) The minimal mammalian Y chromosome – the marsupial Y as a model system. Cytogenet Cell Genet 91: 285–292.
Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F (1996) Human Y chromosome azoospermia factors (Azf) mapped to different subregions in Yq11. Hum Mol Genet 5: 933–943.
Waters PD, Duffy B, Frost CJ, Delbridge ML, Graves JAM (2001) The human Y chromosome derives largely from a single autosomal region added to the sex chromosomes 80–130 million years ago. Cytogenet Cell Genet 92: 74–79.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sankovic, N., Delbridge, M.L., Grützner, F. et al. Construction of a highly enriched marsupial Y chromosome-specific BAC sub-library using isolated Y chromosomes. Chromosome Res 14, 657–664 (2006). https://doi.org/10.1007/s10577-006-1076-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10577-006-1076-z