Characterization and developmental regulation of a gene expressed specifically in the skeletogenic lineage of the sea urchin embryo

The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.

The important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.

Regulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.

Studies of the representation of interatomic distances in molecules and crystal as sum of atomic radii

The electron diffraction investigation of the following compounds has been carried out: sulfur, sulfur nitride, realgar, arsenic trisulfide, spiropentane, dimethyltrisulfide, cis and trans lewisite, methylal, and ethylene glycol.

The crystal structures of the following salts have been determined by x-ray diffraction: silver molybdateand hydrazinium dichloride.

Suggested revisions of the covalent radii for B, Si, P, Ge, As, Sn, Sb, and Pb have been made, and values for the covalent radii of Al, Ga, In, Ti, and Bi have been proposed.

The Schomaker-Stevenson revision of the additivity rule for single covalent bond distances has been used in conjunction with the revised radii. Agreement with experiment is in general better with the revised radii than with the former radii and additivity.

The principle of ionic bond character in addition to that present in a normal covalent bond has been applied to the observed structures of numerous molecules. It leads to a method of interpretation which is at least as consistent as the theory of multiple bond formation.

The revision of the additivity rule has been extended to double bonds. An encouraging beginning along these lines has been made, but additional experimental data are needed for clarification.

I. The crystal structure of a new dimer of triphenylfluorocyclobutadiene. II. A low temperature refinement of the cyanuric triazide structure

I. THE CRYSTAL STRUCTURE OF A NEW DIMER OF TRIPHENYLFLUOROCYCLOBUTADIENE

The crystal structure of thermal isomer of the “head-to-head” dimer of triphenylfluorocyclobutadiene was determined by the direct method. The Σ₂ relationship involving the low angle reflections with the largest E’s were found and solved for the signs by the symbolic method of Zachariasen. The structure was seen in the electron density map and the E-map, and was refined antisotropically by the method of least squares. The residual R was 0.065.

The structure is a gem-difluorohexaphenyldihydropentalene. All of the phenyl groups are planar as it is the cyclopentadiene ring of the dihydropentalene skeleton. Overcrowding at the position of the flourines causes some deviations from the normal bond angles in the cyclopentene ring.

The list of observed and calculated structure factors on pages 32-34 will not be legible on the microfilm. Photographic copies may be obtained from the California Institute of Technology.

II. A LOW TEMPERATURE REFINEMENT OF THE CYANURIC TRIAZIDE STRUCTURE

The structure of cyanuric triazide was refined anisotropically by the method of least squares. Three-dimensional intensity data, which has been collected photographically with MoK_α radiation at -110˚C, were used in the refinement. The residual R was reduced to 0.081.

The structure is completely planar, and there is no significant bond alternation in the cyanuric ring. The packing of the molecules causes the azide groups to deviate from linearity by 8 degrees.