Ph.D. - Biomedical Sciences (Genetics and Molecular Biology)

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    Studies of the TgHBox4 homeobox gene in Tripneustes gratilla
    ( 1994) Vansant, Gordon
    This study further characterized TgHBox4, an Abd-B class homeobox gene expressed as a 4.4kb and a 3.8kb mRNA in early and late embryogenesis, respectively, and as a 3.6kb mRNA in adult tissue of the sea urchin, Tripneustes gratilla. A full length cDNA clone was of 3623 base pairs encoding a 33,312kd protein was selected from an intestinal mRNA library. The cDNA clone was used to select a genomic clone containing a 5' exon of 830bp, which along with an original 3' exon of 2800bp accounted for the full cDNA sequence. Sequence analysis of the cDNA and the 5' genomic clone indicates that the A at the start site of the cDNA may be the transcription start site, with a TATA box 30 nucleotides upstream. The first in frame ATG codon is 210 nucleotides downstream of this transcription start site but the ORF encoding the homeobox protein extends 5' of the start site another 40 nucleotides including an upstream in frame ATG codon. The late embryonic message, which is slightly larger than the intestinal mRNA may be produced by use of an alternate upstream promoter. A TgHBox4 sequence was expressed in bacteria and the protein used as an immunogen. Immunofluorescent studies using the antibodies localized TgHBox4 expression during development. The TgHBox4 proteins appear generally early in embryogenesis and become slowly restricted to the ectoderm cells over the spicule forming primary mesenchyme cells by gastrula stage. As gastrulation proceeds TgHBox4 proteins accumulate in the invaginating gut and became progressively restricted to the region of the digestive tract by pluteus stage. The TgHBox4 bacterially expressed protein was also used in gel shift assays to study it's binding to an enhancer of the late H2B histone gene of S. purpuratus. The anti-TgHBox4 antibodies specifically blocked this binding as well as the binding of the endogenous nuclear factor to the enhancer sequence, indicating that the TgHBox4 gene may regulate late embryonic histone synthesis. It is speculated that the protein product of the larger TgHBox4 message produced in early embryogenesis is involved in the determination of cell patterns which guide mesenchyme cell localization. The protein product of the smaller message produced later in embryogenesis may function to enhance H2B histone gene and other genes expression in dividing cells restricted to the gut in late embryos. The late expression of TgHBox4 in the vegetal plate structures derived from the most posterior parts of the blastula may relate to the posterior expression of the Abd-B class genes in the Hox gene cluster.