Progress 09/01/05 to 08/31/08
Outputs
OUTPUTS: The original objective of the project was to clone and characterize two novel oocyte expressed genes, JY-2 and JY-3. However, cloning of full-length cDNAs for both genes by library screening and RACE was not successful. Therefore, we turned our attention to another novel oocyte transcript which shows sequence similarity to importin α family genes. Full-length cDNA sequence for this gene was obtained by RT-PCR and 5'RACE. Sequence analysis revealed that the cDNA (1817 bp) has an open reading frame encoding a protein of 522 amino acids. A search of the Pfam protein database revealed that the protein contains a conserved importin β binding domain (IBB) and 7 armadillo (ARM) motifs, typical characteristics for importin α proteins. RT-PCR analysis revealed that the novel gene is predominantly expressed in ovarian tissues and mature oocytes. Quantitative real time PCR analysis demonstrated that the expression of this novel importin α gene in GV stage oocytes is over 30 to 2000 times higher than other known importin α genes. 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. A GST pull down assay revealed that the novel improtin α protein has a much stronger binding affinity with a nuclear protein, Nucleoplasmin 2, compared to other bovine importin α proteins. RNAi experiments demonstrated that knockdown of the expression of the novel importin α in bovine embryos results in a decrease of the proportion of embryos developing to the blastocyst stage. We 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. Recently, with the updating of the bovine genome database, we were able to identify a predicted bovine transcript that matches the partial JY-2 cDNA we isolated from the bovine oocyte library. Cloning of the full-length cDNA for this gene was therefore successfully completed by overlapping RT-PCR. The cDNA codes for a protein of 507 amino acids. Analysis of the predicted protein sequence revealed that the novel protein has conserved coiled-coil domains, a functional motif typically found in proteins involved in the regulation of gene expression such as transcription factors. Further functional characterization of the JY-2 gene is underway. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
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 largely unknown and the importin α/importin β-mediated import pathway may play a major role in this process. Identification of the novel importin α and an increased understanding of its functions in controlling early events of embryonic development may ultimately lead to the development of new strategies to improve the efficiency of nuclear transfer and reproduction in cattle.
Publications
- Tejomurtula, J. and Yao, J. 2007. Identification of a novel importin α predominantly expressed in bovine oocytes and early embryos. Biology of Reproduction. Special issue, Abstract 552.
- Tejomurtula, J. 2007. "Identification of a novel importin α predominantly expressed in bovine oocytes and early embryos". M.Sc. Thesis, West Virginia University.
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Progress 09/01/06 to 08/31/07
Outputs
Library screening for longer JY-2 and JY-3 clones has not been successful, most likely due to low levels of expression for both genes. However, we successfully identified another oocyte-expressed gene encoding a novel importin alpha. So far, we have obtained a cDNA of 1680 bp for this novel gene. It has an open reading frame encoding a protein of 522 amino acids and the predicted protein contains a conserved importin β binding domain (IBB) and 2 ARM motifs. The novel transcript is predominantly expressed in ovarian samples and isolated oocytes.
Impacts
The novel importin alpha may play an important role in nuclear transport of certain key transcription factors and/or other essential nuclear proteins required for activation of the embryonic genome during early embryogenesis.
Publications
- No publications reported this period
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Progress 09/01/05 to 08/31/06
Outputs
Genes expressed specifically in oocytes play important roles in the development of follicles and early embryos. The objective of this study is to clone full-length cDNA sequences for novel oocyte expressed genes JY-2 and JY-3 and to determine expression of both genes during oocyte maturation and early embryogenesis. Work has begun on screening a bovine fetal library to obtain cDNA closes for both JY-2 and JY-3genes.
Impacts
Reproductive efficiency is a major component in the economic success of domestic livestock operations. Genes expressed specifically in oocytes, in turn, strongly impact reproductive efficiency by affecting the development of follicles and early embryos. This study has the potential to generate significant economic benefit by increasing the efficiency of reproduction in cattle.
Publications
- No publications reported this period
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