Date of Graduation

2007

Document Type

Thesis

Degree Type

MS

Committee Chair

Jianbo Yao

Abstract

Early embryonic development is a complex process that involves timely and quantitative control of gene transcription. Nuclear proteins, including transcription factors and chromatin remodeling proteins, are required for initiation of transcription in early embryos prior to embryonic genome activation. The nuclear transport of these proteins is mediated by transport factors such as importins. Through analysis of EST sequences from a bovine oocyte cDNA library and the database, we identified a novel transcript which shows sequence similarity to the importin α gene. The novel transcript (contig) is represented by multiple ESTs that are only present in the oocyte library and a 2-cell embryo library. The largest cDNA clone obtained from the oocyte library is 720 bp in length containing a poly (A) tail. Additional 5’ end sequence was obtained by RT-PCR amplification of cDNA from a fetal ovary using primers designed based on a bovine genomic sequence. The final assembled cDNA is 1680 bp (missing 5-UTR). Sequence analysis revealed that the cDNA has an open reading frame encoding a protein of 522 amino acids. The predicted protein shares 53%, 45%, 45%, 41%, 40% and 40% sequence identity with bovine importin α1, α3, α4, α5, α6 and α7 respectively. The protein contains a conserved importin α binding domain (IBB) at its N-terminus and four ARM (Armadillo/beta-catenin-like repeats) motifs in the central region. Analysis of tissue distribution of this novel transcript by reverse transcription polymerase chain reaction (RT-PCR) revealed that this gene is predominantly expressed in ovarian samples and

mature oocytes. Quantitative real time PCR analysis demonstrated that the expression of this novel importin α in GV stage oocytes is over 30 to 2000 times higher than all six known α importins. Analysis of temporal expression of the novel gene during early embryonic development showed that the transcript is abundantly present in GV and MII stage oocytes as well as in 2-cell, 4-cell, and 8-cell embryos, but barely detectable in morula and blastocyst. Western blot analysis using antibodies against this novel protein showed a similar expression pattern at the protein level. We therefore suggest that this novel importin α might be responsible for nuclear transport of certain key transcription factors and/or other essential nuclear proteins required for activation of the embryonic genome during early embryogenesis.

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