Lamins are of fundamental biological importance. They provide a structural foundation for the nuclear envelope and may be involved in the regulation of DNA replication and gene expression. Although lamins are well characterized at the protein level, virtually nothing is known about the gene level. The long term goal of this project is to elucidate how lamin gene activity is controlled in sea urchin embryos during early development. There is strong evidence that the lamin proteins are differentially expressed during development and that the genes exist as a multigene family. As such, the lamin genes are excellent candidates for the studies on differential gene expression and maternal control mechanisms that we propose. The main objective of this proposal is to isolate and characterize lamin cDNA and genomic clones from Lytechinus variegatus DNA libraries. The construction of appropriate DNA probes will permit direct measurement of the expression of these genes during early sea urchin development. The proposed experiments will: (1) measure the steady state levels and synthetic rates of lamin protein and mRNA; (2) monitor the prevalence and heterogeneity of lamin RNA; (3) detect the presence of lamin mRNA precursors. These results will aid in sorting the maternal and embryonic contributions to the expression of the lamin genes and serve as ground work for future studies designed to define sequence requirements for efficient transcription and regulation. Four powerful bioassay systems that have recently been modified for use in sea urchin will facilitate these future studies. These are, (1) isolated nuclei- to monitor relative rates of transcription, (2) a homologous DNA dependent transcription extract- to delineate sequence requirements for transcription and regulation in vitro, (3) a microinjection system- to correlate the requirement of these sequences in vivo, (4) in situ hybridization- to localize lamin RNA throughout development.
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