Molecular cloning and developmental expression of two chick embryo gap junction proteins

The connexins are a family of related gap junction proteins which contain conserved transmembrane and extracellular domains but unique cytoplasmic regions. To identify connexins with potential roles in development, a chick embryo cDNA library was screened by hybridization at low stringency with a cDNA for rat connexin-43. cDNA clones for two previously undescribed connexins were isolated. Chick connexin-45 has a predicted molecular mass of 45,376 daltons; connexin-42 has a predicted molecular mass of 41,748 daltons. Both of these predicted connexin proteins share the homologous regions noted in other members of this family, and each has its own unique regions. Southern blots of chicken genomic DNA suggest that each connexin is encoded by a distinct single copy gene. RNA blots demonstrate that while chick connexin-43, -42, and -45 are each expressed in a number of chick organs, they each have a unique tissue distribution. Each connexin mRNA is present in heart. Blots of total RNA isolated from hearts of chick embryos of different ages demonstrate that the abundance of connexin-42 and -43 mRNAs varies no more than 2-fold between the embryo and the adult. However, connexin-45 mRNA shows a dramatic change, falling 10-fold from the 6-day embryonic heart to the adult. These multiple connexins are likely to have different physiological properties and may account for the multiple physiologically distinct gap junction channels which have been observed in cardiac myocytes. They may provide a mechanism for the formation of communication compartments in the developing myocardium.     

Molecular cloning and early expression of chick embryo SCO-spondin

SCO-spondin is a multidomain glycoprotein secreted by the subcommissural organ (SCO). It belongs to the thrombospondin type 1 repeat superfamily and has been identified in several vertebrate species. We report the cloning of the chick SCO-spondin ortholog and examine its temporal and spatial expression during early embryogenesis from Hamburger and Hamilton (HH) stage 12 to HH stage 21. Chick SCO-spondin cDNA contains a long open reading frame encoding a predicted protein of 5255 amino acids. Northern blot analysis has revealed SCO-spondin mRNA as a band of about 15 kb. Many conserved domains have been identified, including 27 thrombospondin type 1 repeats, 13 low-density lipoprotein receptor type A domains, one EMI domain (a cysteine-rich domain of extracellular proteins), three von Willebrand factor type D domains, and one cystine knot C-terminal domain. Whole-mount in situ hybridization enabled the first signal of mRNA expression to be detected at HH stage 17, exclusively in a thin area of the prosencephalon roof plate. During the following stages of development, SCO-spondin expression remained restricted to this region. The multidomain structure of SCO-spondin and its early expression suggest that it plays a role in developmental processes in the central nervous system.     

Recombinant expression of aryl hydrocarbon receptor for quantitative ligand-binding analysis

Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (B_max) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the K_d for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding.     

Isolation and biochemical characterization of nuclei from chick embryo liver

A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl₂ followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg²⁺ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl₂. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg²⁺ activated RNA polymerase, 4.3 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 10 min; and Mn²⁺-(NH₄)₂SO₄ activated RNA-polymerase, 136 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 45 min.     

Molecular characterization of the rostral-most somites in early somitic stages of the chick embryo

Segmentation consists on the progressive formation of repetitive embryonic structures, named somites, which are formed from the most rostral part of the presomitic mesoderm. Somites are subdivided into anterior and posterior compartments and several genes are differentially expressed in either compartment. This has provided evidence for the importance of establishing the anterior-posterior polarity within each somite, which is critical for the correct segmented pattern of the adult vertebrate body. Although all somites appear morphologically similar, fate map studies have shown that the first 4 somites do not give rise to segmented structures, in contrast to more posterior ones. Moreover, in several somitogenesis-related mutants the anterior somites are not affected while posterior somites present clear defects or do not form at all. Altogether these data suggest relevant differences between rostral and caudal somites. In order to check for molecular differences between anterior and posterior somites, we have performed a detailed expression pattern analysis of several Notch signalling related genes. For the first time, we show that the somitic expression pattern profile is not the same along the anterior-posterior axis and that the differences are not observed always at the same somite level.     

Beta-naphthoflavone induction of a cytochrome P-450 arachidonic acid epoxygenase in chick embryo liver distinct from the aryl hydrocarbon hydroxylase and from phenobarbital-induced arachidonate epoxygenase.

Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.     

Separation and characterization of 7 and 9 S forms of rat liver aryl hydrocarbon receptor

Two forms of rat liver aryl hydrocarbon receptor were separated by chromatography on DEAE-cellulose in the presence of molybdate. After labeling for 2 h at 0 degrees C, the receptor separated on the DEAE column into a flow-through peak (peak I) and a peak eluting at 80 mM KCl (peak II). It had been reported previously that exposure to high salt in the presence of molybdate caused the appearance of both 9 and 5-6 S receptor forms. After confirming this, I examined the relationship of the peak I and peak II receptors to these receptor forms. In high salt buffer containing molybdate, the peak I receptor sedimented in the 5-6 S region and the peak II receptor at 9 S. High salt buffer lacking molybdate converted both peak I and peak II receptors to forms sedimenting in the 5-6 S region. In low salt buffer containing molybdate, the peak I receptor sedimented at slightly more than 7 S and the peak II receptor at 9-10 S. Thus, the peak II receptor could be stabilized by molybdate as a 9 S form, and the peak I receptor was converted by high salt from a 7 to a 5-6 S form, despite the presence of molybdate. Most of the peak I receptor bound to a DNA-cellulose column and was eluted by high salt. The peak II receptor showed very little DNA binding.