Robinson, Murray O. (1992) Gene regulation during spermatogenesis: transcriptional and translational control of phosphoglycerate kinase 2 in transgenic mice. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:08302011-152155117
The work presented in this thesis is an analysis of the transcriptional and translational control of the testis-specific gene for phosphoglycerate kinase (Pgk-2) in an effort to understand the mechanisms of gene regulation during spermatogenesis. To address transcriptional control (Chapter two), a genomic fragment containing the human gene for PGK-2 was expressed in transgenic mice, and then a 323 bp region of the promoter was shown to be necessary for tissue-specific, developmentally regulated expression. In Chapter three, a novel quantitation technique based on the polymerase chain reaction was developed and used to measure the accumulation of transgenic and endogenous Pgk-2 transcripts. The human PGK-2 transgene, the PGK-2/CAT transgene and the endogenous Pgk-2 gene all had similar levels and patterns of expression. Transcript levels of round spermatid-expressed protamine 2 were tenfold higher and showed delayed accumulation kinetics, implying that the peak expression of the Pgk-2-promoted genes occurred in pachytene spermatocytes. In Chapter four, the translational regulation of the Pgk-2 message was investigated, and the cis-acting sequences for proper translation of the PGK-2 message were shown to reside in the 5' untranslated region of the gene. Transgenes containing the PGK-2 promoter, the CAT coding sequence, and either the PGK-2 3' or SV40 3' sequences were both shown to behave like the translationally regulated endogenous Pgk-2 locus. Polysomal profiles of the PGK-2/CAT/SV40 3' construct demonstrated that the message shifts from a translationally inactive state to a translationally active state between post natal day 20 and day 33. This developmental shift occurs with the same timing as that of the endogenous Pgk-2 message. The Appendix investigates the relative levels of a number of transcripts in separated spermatogenic cells. Notably, the levels of Pgk-2 and the X-linked Pgk-1 were analyzed during spermatogenesis. Pgk-1 transcript levels diminish in the spermatogonial stages, whereas the Pgk-2 transcript first appears in early meiotic cells. This pattern of gene activity is consistent with the hypothesis that Pgk-2 expression substitutes for the inactivated Pgk-1 locus during the later stages of spermatogenesis.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||18 June 1991|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||John Wade|
|Deposited On:||30 Aug 2011 22:46|
|Last Modified:||26 Dec 2012 04:38|
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