Genomic and expression analysis of multiple Sry loci from a single Rattus norvegicus Y chromosome

Abstract

Background: Sry is a gene known to be essential for testis determination but is also transcribed in adult male tissues. The laboratory rat, Rattus norvegicus, has multiple Y chromosome copies of Sry while most mammals have only a single copy. DNA sequence comparisons with other rodents with multiple Sry copies are inconsistent in divergence patterns and functionality of the multiple copies. To address hypotheses of divergence, gene conversion and functional constraints, we sequenced Sry loci from a single R. norvegicus Y chromosome from the Spontaneously Hypertensive Rat strain (SHR) and analyzed DNA sequences for homology among copies. Next, to determine whether all copies of Sry are expressed, we developed a modification of the fluorescent marked capillary electrophoresis method to generate three different sized amplification products to identify Sry copies. We applied this fragment analysis method to both genomic DNA and cDNA prepared from mRNA from testis and adrenal gland of adult male rats.

Which Sry Locus Is the Hypertensive Y Chromosome Locus?

“…14 All of the Sry loci sequenced from these strains have a full-length, potentially active coding region, based on conserved regions identified from previous sequencing results. 6 We had previously identified 6 Sry loci in the SHR/Akr strain, 6 and these results show 7 loci from the same strain. The Sry3 and Sry3A loci identified in the current data were cryptic, and only by using primers that amplified a larger region than previous results could these loci be differentiated, changing the number present from 6 to 7.…”

“…To avoid excessive sequencing of multiple clones of the same Sry copy identity, clones were screened by fragment analysis to determine copy identity. 6 At least 2 clones of each copy identity, from each PCR replicate, were selected for sequencing. Clone sequences were assigned a copy identity by comparison with SHR/Akr reference sequence.…”

“…The convention was adapted that, for a base difference to be scored, it must be seen from multiple clones from different amplification reactions. 6 Sry TOPO clones were screened by fragment analysis, and clones were selected for sequencing. Cycle sequencing reactions were set up in 0.2-mL 96-well PCR plates, in a total volume of 10 L, using the BigDye Terminator 3.1 Cycle Sequencing kit (Applied Biosystems).…”

“…We have sequenced the Sry gene complex from a single Y chromosome from the Akron SHR strain (SHR/Akr). 6 The analysis of these sequences identified 6 different Sry loci on the SHR Y chromosome (Sry1, Sry2, Sry3, Sry3BI, Sry3B, and Sry3C). These are paralogous copies consistent with a single phylogeny.…”

Switching on sex: transcriptional regulation of the testis-determining geneSry

“…Rats have 11 non-identical copies of Sry (Prokop et al, 2013), at least six of which are expressed (Turner et al, 2007); rabbits (Geraldes and Ferrand, 2006), pigs (Groenen et al, 2012) and dogs (Li et al, 2013) also have multiple Sry genes. It is not known which copy (or copies) carries the regulatory elements required for testis determination, confounding efforts to identify these sequences.…”