The Genomic Organization and Expression of the Strongylocentrotus purpuratus Actin Gene Family

Citation

Lee, James Joseph (1986) The Genomic Organization and Expression of the Strongylocentrotus purpuratus Actin Gene Family. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/b4m8-v194. https://resolver.caltech.edu/CaltechTHESIS:04172019-114252554

Abstract

The actin gene family of the sea urchin Strongylocentrotus purpuratus was studied in detail, using subcloned probes specific to the 3'-terminal nontranslated actin gene sequences. By determining the often polymorphic restriction fragment band pattern displayed in genomic blots by each probe, the actin genes in this species could be classified. The evidence presented here shows that the S. purpuratus genome contains eight actin genes, and these can be assigned to four subtypes. Studies of the expression of the genes show that the actin genes of three of these subtypes code for cytoskeletal actins (Cy), while the fourth gives rise to a muscle-specific actin (M). There is a single CyI actin gene, three CyII genes (CyIIa, CyIIb, and CyIIc), three CyIII genes (CyIIIa, CyIIIb, and CyIIIc), and a single M actin gene. Pnmary sequence data shows that two of these genes, CyIIc and CyIIIc, have anomalous structures and are probably pseudogenes.

RNA gel blots, using the 3'-trailer probes, show that during embryogenesis, as well as in a number of adult tissues, the CyI, CyII, and M subtype genes are differentially expressed. Genes of the CyIII subtype are not active (i.e., transcripts do not accumulate) in any adult tissues. Moreover, molecular titration experiments, using Sp6 polymerase-generated RNA probes, measured the absolute prevalence of transcripts from five of the six active actin genes at different points in development. This analysis showed that actin transcripts begin to accumulate after seven hours postfertilization and are not prevalent in the maternal RNA pool. In situ hybridizations of sections through the embryo establish that the accumulation of actin transcripts occurs in a cell lineage specific manner. Together with the molecular titrations these analyses provide per-cell prevalence data for transcripts from each actin gene. In vitro nuclear run-off experiments show that actin transcripts accumulate as the result of the activation and continued transcription of each actin gene during embryogenesis. Additional in vivo kinetic labeling experiments were used to measure the rates of nuclear synthesis and decay. This data shows that once activated each actin gene is transcribed at a constant rate and these rates are the prominent kinetic parameters determining actin transcript prevalence levels.

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