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cis- and trans-acting sequences required for expression of simian virus 40 genes in mouse oocytes
L E Chalifour1, D O Wirak, U Hansen
1Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
To determine the requirements for gene expression in mammalian germ cells, circular double-stranded simian virus 40 (SV40) DNA molecules containing deletions in sequences controlling transcription and replication were injected into the nucleus of mouse oocytes. Expression of large (T-Ag) and small (t-Ag) tumor antigens ("early gene products") required at least three GGGCGG boxes, but did not require either the origin of viral DNA replication (ori) or a TATA box. Expression of capsid antigen VP1 ("late gene products") required at least three GGGCGG boxes, sequences between nucleotides 197 and 273 in the 72-bp repeat region, and transactivation by T-Ag. These results are consistent with the requirements for expression of the same genes in differentiated mammalian cells. Surprisingly, however, the 72-bp repeats ("enhancer elements") that are required for expression of T-Ag and t-Ag genes in differentiated cells were not required in mouse oocytes. Similarly, expression of both the early and late genes was unaffected in mouse oocytes by the absence of either DNA replication or an intact ori sequence, components required for maximum expression of late genes in differentiated cells. Thus, mammalian oocytes effectively utilize promoters that are fully active in mammalian differentiated cells only when associated with either enhancer elements or DNA replication. Furthermore, requirements for expression of SV40 genes in mouse oocytes are distinctly different from those reported for Xenopus oocytes. This suggests that caution should be exercised when extrapolating conclusions drawn from experiments with amphibian germ cells to mammalian germ cells.
Insights
Gene expression in mouse oocytes differs from differentiated cells. Mammalian germ cells utilize promoters without enhancer elements or DNA replication, unlike frog oocytes.
Area of Science:
- Molecular Biology
- Developmental Biology
- Virology
Background:
- Understanding gene expression in mammalian germ cells is crucial for reproductive biology.
- Simian virus 40 (SV40) provides a model system to study gene regulation.
- Previous studies established gene expression requirements in differentiated mammalian cells and Xenopus oocytes.
Purpose of the Study:
- To elucidate the specific requirements for SV40 gene expression in mouse oocytes.
- To compare germ cell gene expression with that of differentiated mammalian cells.
- To investigate the role of enhancer elements and DNA replication in oocyte gene expression.
Main Methods:
- Injection of modified SV40 DNA (with deletions) into mouse oocytes.
- Analysis of early (T-Ag, t-Ag) and late (VP1) gene product expression.
- Comparison of expression patterns with and without key regulatory sequences (e.g., 72-bp repeats, origin of replication).
Main Results:
- Expression of early and late SV40 genes in mouse oocytes requires specific promoter elements (GGGCGG boxes).
- Enhancer elements (72-bp repeats) and the origin of replication are not essential for SV40 gene expression in oocytes, contrary to differentiated cells.
- SV40 gene expression requirements in mouse oocytes differ significantly from those in Xenopus oocytes.
Conclusions:
- Mammalian oocytes can express genes using promoters that differ from those in somatic cells.
- Gene expression in mammalian germ cells does not necessitate enhancer elements or DNA replication, unlike differentiated cells.
- Findings highlight species-specific differences in germ cell gene regulation, cautioning against direct extrapolation from amphibian models.