Turner Monte E, Martin Carey, Martins Almir S, Dunmire Jeffrey, Farkas Joel, Ely Daniel L, Milsted Amy
Department of Biology, The University of Akron, Akron, OH 44325-3908, USA.
BMC Genet. 2007 Apr 4;8:11. doi: 10.1186/1471-2156-8-11.
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.
Y chromosome fragments were amplified and sequenced using primers that included the entire Sry coding region and flanking sequences. The analysis of these sequences identified six Sry loci on the Y chromosome. These are paralogous copies consistent with a single phylogeny and the divergence between any two copies is less than 2%. All copies have a conserved reading frame and amino acid sequence consistent with function. Fragment analysis of genomic DNA showed close approximations of experimental with predicted values, validating the use of this method to identify proportions of each copy. Using the fragment analysis procedure with cDNA samples showed the Sry copies expressed were significantly different from the genomic distribution (testis p < 0.001, adrenal gland p < 0.001), and the testis and adrenal copy distribution in the transcripts were also significantly different from each other (p < 0.001). Total Sry transcript expression, analyzed by real-time PCR, showed significantly higher levels of Sry in testis than adrenal gland (p, 0.001).
The SHR Y chromosome contains at least 6 full length copies of the Sry gene. These copies have a conserved coding region and conserved amino acid sequence. The pattern of divergence is not consistent with gene conversion as the mechanism for this conservation. Expression studies show multiple copies expressed in the adult male testis and adrenal glands, with tissue specific differences in expression patterns. Both the DNA sequence analysis and RNA transcript expression analysis are consistent with more than one copy having function and selection preventing divergence although we have no functional evidence.
Sry基因对于睾丸决定至关重要，同时也在成年雄性组织中进行转录。实验大鼠褐家鼠（Rattus norvegicus）的Y染色体上有多个Sry拷贝，而大多数哺乳动物只有一个拷贝。与其他具有多个Sry拷贝的啮齿动物进行的DNA序列比较在多个拷贝的分歧模式和功能方面并不一致。为了验证分歧、基因转换和功能限制的假设，我们对自发性高血压大鼠品系（SHR）单个褐家鼠Y染色体上的Sry基因座进行了测序，并分析了各拷贝之间的DNA序列同源性。接下来，为了确定Sry的所有拷贝是否都有表达，我们改进了荧光标记毛细管电泳方法，以产生三种不同大小的扩增产物来鉴定Sry拷贝。我们将这种片段分析方法应用于成年雄性大鼠睾丸和肾上腺mRNA制备的基因组DNA和cDNA。
使用包含整个Sry编码区及其侧翼序列的引物扩增并测序Y染色体片段。对这些序列的分析在Y染色体上鉴定出6个Sry基因座。这些是与单一系统发育一致的旁系同源拷贝，任意两个拷贝之间的分歧小于2%。所有拷贝都有一个保守的阅读框和与功能一致的氨基酸序列。基因组DNA的片段分析显示实验值与预测值非常接近，验证了该方法用于鉴定各拷贝比例的有效性。对cDNA样本进行片段分析表明，表达的Sry拷贝与基因组分布有显著差异（睾丸p < 0.001，肾上腺p < 0.001），并且转录本中睾丸和肾上腺的拷贝分布彼此之间也有显著差异（p < 0.001）。通过实时PCR分析的Sry转录本总表达显示，睾丸中的Sry水平显著高于肾上腺（p < 0.001）。
SHR的Y染色体包含至少6个Sry基因的全长拷贝。这些拷贝具有保守的编码区和保守的氨基酸序列。分歧模式与作为这种保守机制的基因转换不一致。表达研究表明，多个拷贝在成年雄性大鼠的睾丸和肾上腺中表达，且表达模式存在组织特异性差异。DNA序列分析和RNA转录本表达分析均表明，虽然我们没有功能证据，但有多个拷贝具有功能且选择作用阻止了分歧。
