Gene regulation during spermatogenesis: transcriptional and translational control of phosphoglycerate kinase 2 in transgenic mice

Author: Robinson, Murray O.

Year: 1992

Degree: Dissertation (Ph.D.)

Advisor: Simon, Melvin I.

Committee Member: Unknown, Unknown

Option: Biology

DOI: 10.7907/a6rw-je84

Abstract

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.

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