Expression of six novel Sry copies from the SHR/Akr rat strain. (Molecular Biology and Genetics 02:00 PM, Saturday, April 5, 2003 Brewer/Frost Science 141 Dr. Beth Berger Pritts-Presiding).
In mammals, testis determination is initiated by expression of the Sty gene. Sry, which is located on the non-recombining region of the Y chromosome, usually exists as a single copy in most mammalian species; however, in many rodent species including Rattus norvegicus more than one copy can exist. From the rat strain SHR/Akr, six copies of Sry have been identified through screening an SHR/Akr genomic library and PCR amplification of genomic DNA. Each PCR fragment was inserted into the cloning vector pC[R.sup.a]4-TOP[O.sup.a] (Invitrogen Corp., Carlsbad, CA). Sequence conservation between copies suggests that each is potentially active. To demonstrate the potential for gene activity, it must be determined if each Sry copy can be transcribed and translated. This investigation followed the hypothesis that multiple Sry copies isolated from male SHR/Akr will exhibit the potential for functionality through transcription and protein expression. Research began by subcloning the coding and 3' flanking regions into the expression vector pcDNA3.1(-) (Invitrogen Corp). Sequence analysis confirmed that each copy (n = 6) was successfully inserted. Next, in vivo (transfection into cultured vascular smooth muscle cells) and in vitro (PROTEIN[script.sup.a]-PRO Kit, Ambion, Inc., Austin, TX) transcription and translation techniques were used to express each copy. Analyses utilizing Northern blots and polyacrylamide gel electrophoresis revealed that all six Sry copies are potentially active. Results from this research demonstrate the potential for multiple Sry copy expression, and perhaps provide indications of the possible divergence of copy function.
ADAM C. UNDERWOOD ACU1@UAKRON.EDU, MONTE TURNER, DANIEL ELY, AND ANY MILSTED, DEPT OF BIOLOGY, UNIVERSITY OF AKRON, AKRON OH 44325-3908
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|Author:||Underwood, Adam C.; Turner, Monte; Ely, Daniel; Milsted, Amy|
|Publication:||The Ohio Journal of Science|
|Date:||Mar 1, 2003|
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