The mouse Y chromosome: enrichment, sizing, and cloning by bivariate flow cytometry.
In this report, we demonstrate the utility of interleukin-2 (IL-2) stimulation of spleen cell cultures and bivariate flow cytometry in the analysis and purification of the C57BL/6J mouse Y Chromosome. We determined that the DNA content of the C57BL/6J Y Chromosome is approximately 94.7 Mb, making it similar in size to human Chromosome 16 and significantly larger than previous estimates. In addition, we describe the bulk isolation of mouse Y Chromosomes and demonstrate enrichment of the isolated material using a fluorescence in situ hybridization strategy. We detail the construction of two small insert Y Chromosome-specific libraries, ideal for sampling Y Chromosome sequences. From these libraries 1566 clones were analyzed. We provide a detailed characterization of 103 clones, generating nearly 50 kb of sequence. For 30 of these clones, we identify regions of homology to known Y chromosomal sequences, confirming the enrichment of the sorted DNA. From the remaining characterized clones, we describe the development of 15 male-specific PCR assays and 19 male-female PCR assays potentially originating from the pseudoautosomal region or other areas of X-Y or autosome-Y homology.
Bergstrom
D E
DE
Jackson Laboratory, Bar Harbor, Maine 04609, USA.
Grieco
D A
DA
Sonti
M M
MM
Fawcett
J J
JJ
Bell-Prince
C
C
Cram
L S
LS
Narayanswami
S
S
Simpson
E M
EM
D000818:Animals; D002885:Chromosomes, Human, Pair 16; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007376:Interleukin-2 / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013154:Spleen / Q000187:drug effects / Q000648:ultrastructure; D014998:Y Chromosome
D007376:Interleukin-2; D004247:DNA