Kazuko Takata

CAAT sites are required for the activation of the H. pulcherrimus Ars gene by Otx

Abstract A product of sea urchin homologues of the Drosophila orthodenticle gene, HpOtxL has been implicated as a transcription activator of the aboral ectoderm-specific arylsulfatase (Ars) gene during early development of the sea urchin embryo. Using an in vivo transactivation system, we present evidence that HpOtxL activates the target gene by interacting with co-factors. Otx binding sites alone have little effect on the activity of an Ars promoter, but when both Otx binding sites and CAAT sequences are present in the enhancer region of Ars, the fragment shows a high enhancer activity. A gel mobility shift assay reveals that a nuclear protein binds to the CAAT sequences present near the Otx binding sites in the enhancer region of Ars. The activation domain of HpOtxL resides in the C terminal region between amino acids 218 and 238. The N-terminal region is responsible for the enhancement of transactivation of the Ars promoter, although the region itself does not function as an activation domain. HpOtxE, which possesses an N-terminal region different from HpOtxL, does not activate the Ars promoter even in the presence of CAAT sequences. Together with previous findings, our results suggest that Otx regulates different genes by interacting with different co-factors in sea urchin development.

Utilization of a particle gun DNA introduction system for the analysis of cis-regulatory elements controlling the spatial expression pattern of the arylsulfatase gene (HpArs) in sea urchin embryos

Abstract. We applied a particle gun method to introduce DNA into fertilized sea urchin eggs for the analysis of cis-regulatory elements responsible for spatial gene expression during development. We introduced HpArs (sea urchin arylsulfatase gene) –GFP and HpArs–LacZ fusion constructs into the fertilized eggs and obtained high expression levels of the fusion genes. Using this assay system, we demonstrated that a fragment of HpArs (–3,484 to +4,636) is sufficient for aboral ectoderm-specific expression, and that the region in the first intron from +406 to +1,993 contains the control elements responsible for the repression of the HpArs promoter activity in secondary mesenchyme cells.

A new G-stretch-DNA-binding protein, Unichrom, displays cell-cycle-dependent expression in sea urchin embryos

We report the identification and characterization of Unichrom, a gene encoding a new G‐stretch‐DNA‐binding protein in the sea urchin embryo. The derived amino acid sequence of Unichrom contains plant homeodomain (PHD) finger and high mobility group (HMG) motifs as well as motifs required for cell‐cycle‐dependent degradation. The expression of a Unichrom‐green fluorescent protein (GFP) fusion protein in sea urchin embryonic cells indicates that Unichrom protein accumulates in nuclei during interphase and disperses into the cytoplasm at mitosis. Overexpression of dominant negative Unichrom, which contains the DNA binding domain lacking the motif for cell‐cycle‐dependent degradation, causes impairment of chromosome segregation. These results suggest that Unichrom binds to genome DNA at G‐stretch and that degradation of Unichrom is required for segregation of chromosomes.

Corrected Structure of the 5′ Flanking Region of Arylsulfatase Gene of the Sea Urchin, Hemicentrotus pulcherrimus

The nucleotide sequence of the 5′ flanking region of the arylsulfatase (Ars) gene of the sea urchin, Hemicentrotus pulcherrimus, is extensively corrected. The one previously reported from our laboratory (ref. 7, Develop, Growth & Differ., 34, 719–729, 1992) contains many sequencing errors and should be replaced with that presented here. Correction includes the addition of a 294 nucleotide (nt) sequence between −728 and −1,021. In the previous sequencing, this fragment was missed by overlooking one Hindlll site during subcloning.