A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Male sex determination is mediated sequentially by sex-determining region Y (SRY) and related SRY-box 9 (SOX9) transcription factors. To understand the gene regulatory hierarchy for SRY and SOX9, a series of chromatin immunoprecipitation and whole-genome promoter tiling microarray (ChIP-Chip) experiments were conducted with mouse gonadal cells at the time of sex determination. SRY and SOX9 bind to the promoters of many common targets involved in testis differentiation and regulate their expression in Sertoli cells. SRY binds to various ovarian differentiation genes and represses their activation through WNT/β-catenin signaling. Sertoli cell-Sertoli cell junction signaling, important for testis cord formation, is the top canonical pathway among the SRY and SOX9 targets. Hence, SRY determines Sertoli cell fate by repressing ovarian and activating testicular differentiation genes, promotes early Sertoli cells to form testis cord, and then passes on its functions to SOX9, which regulates common targets and activates its own gene regulatory program, beyond SRY actions, in sex determination.

In both humans and mice, two genes encode phosphoglycerate kinase, a key enzyme in the glycolytic pathway. The pgk-1 gene is expressed in all somatic cells, is located on the X chromosome, and contains 10 introns. The pgk-2 gene is expressed only in sperm cells, is located on an autosome, and has no introns. The nucleotide sequence of the pgk-2 gene suggests that it arose from pgk-1 more than 100 million years ago by RNA-mediated gene duplication. The pgk-2 gene may, then, be a transcribed retroposon. Thus, gene duplication by retroposition may have been used as a mechanism for evolutionary diversification.

Isolation and mapping of a mouse complementary DNA sequence (mouse Y-finger) encoding a multiple, potential zinc-binding, finger protein homologous to the candidate human testis-determining factor gene is reported. Four similar sequences were identified in Hind III-digested mouse genomic DNA. Two (7.2 and 2.0 kb) were mapped to the Y chromosome. Only the 2.0-kb fragment, however, was correlated with testis determination. Polymerase chain reaction analysis suggests both Y loci are transcribed in adult testes. A 3.6-kb fragment was mapped to the X chromosome between the T16H and T6R1 translocation breakpoints, and a fourth (6.0 kb) was mapped to chromosome 10. Hence, mYfin sequences have been duplicated several times in the mouse, although they are not duplicated in humans.