Tandem duplication and divergence of a sea urchin protein belonging to the troponin C superfamily

The Spec1 and Spec2 proteins of the sea urchin Strongylocentrotus purpuratus are related to calmodulin, troponin C, and myosin light chains by sequence similarity in their four calcium binding domains. These domains, the EF-hands, are distinct helix-loop-helix structures of about 40 amino acids. The Spec1 and Spec2 genes are expressed specifically in aboral ectoderm cells of the developing embryo; however, the function of the Spec proteins in these cells is unknown. To find conserved regions of the proteins that might be important for structure and function, Spec homologues from Lytechinus pictus, a distantly related sea urchin, were sought. L. pictus embryos do not synthesize detectable amounts of the 14,000-17,000-Da Spec proteins as determined by two-dimensional gel electro-phoresis, but do synthesize three 34,000-Da proteins that cross-react with Spec1 antibodies and display a similar ontogenetic pattern of expression. cDNA clones were isolated by hybridization to a synthetic oligonucleotide corresponding to the EF-hand. One clone, LpS1, encodes an mRNA with developmental properties like those of the S. purpuratus Spec mRNAs. However, LpS1 contains an open reading frame for a protein of 34,000 Da rather than 17,000 Da, and antibodies raised against part of the LpS1 reading frame demonstrate that LpS1 encodes a 34,000-Da protein in L. pictus embryos. The sequence of LpS1 reveals the presence of eight EF-hand domains, which share structural homology with the Spec1 or Spec2 EF-hands; however, little else in the protein sequence is conserved. The results support the hypothesis that the LpS1 gene arose from a duplication of an ancestral Spec gene and that the overall structural features of the Spec family of proteins are more conserved than the amino acid sequences.     

Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm Bombyx mori

In order to establish a transient expression system for genes introduced into early embryos of the silkworm, Bombyx mori, we tested various promoters ligated with CAT reporter genes. The embryos into which we injected supercoiled plasmid DNA of pFb(-860/+10)CAT containing the Bombyx fibroin promoter region ligated to the CAT gene showed a reasonably high CAT activity beginning around 30 h after oviposition. This high activity was observed only when the plasmid was injected before termination of the early nuclear cleavage stage, which was about 8 h after oviposition, but not after this stage. This means that the expression of injected DNA is closely related to the presence of cleavage nuclei in early embryos. Promoters originating from insect genes, like the Bombyx sericin-1 gene, Drosophila hsp70 and Drosophila copia LTR, functioned as strong promoters in the embryos. On the contrary, promoters from mammalian virus genes, such as the SV40 early and Moloney murine leukemia virus LTR genes, functioned as weak promoters. Moreover, linearized DNAs showed no or weak activity of expression in embryos. From these results, we conclude that the silkworm embryo transient expression system is a useful tool for studying the mechanism of regulation of insect genes.     

Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.     

Differential stability of expression of similarly specified endogenous and exogenous genes in the sea urchin embryo.

The object of these experiments was to determine whether competitive titration in vivo of factors required for expression of the CyIIIa.CAT fusion gene would affect expression of the endogenous CyIIIa gene in the same embryos. Earlier work showed that expression of this fusion gene after injection into sea urchin eggs is stoichiometrically reduced when low molar excesses of DNA fragments containing only its regulatory domain are coinjected. In order to compare endogenous (i.e. CyIIIa) and exogenous (i.e. CyIIIa.CAT) expression simultaneously in embryos bearing excess competitor regulatory DNA, we developed, and here describe, a new procedure for generating transgenic sea urchin embryos in which all of the cells in many embryos, and most in others, bear the exogenous DNA. Such large reduction of mosaicism can be achieved by multiple injection of the exogenous DNA fragments into fertilized eggs. Using this method, we demonstrate that at a level of competitor DNA incorporation which reduces CyIIIa.CAT expression by 85%, endogenous CyIIIa mRNA levels are wholly unaffected. Nor is spatial expression of the endogenous CyIIIa gene disturbed. Since the CyIIIa.CAT genes are properly expressed under control of the CyIIIa regulatory sequences, they must participate in the same set of necessary DNA-protein interactions. However, we infer from the results that we report here that the regulatory complexes in the endogenous CyIIIa gene are greatly stabilized relative to those of the exogenous CyIIIa.CAT genes.     

Delivery systems for gene therapy

Introduction of foreign genes into mammalian cellsin vitro has been accomplished previously by a variety of methods. The few techniques that have been developed for transfection of mammalian cellsin vivo, are technically difficult or lack cell specificity.We have developed a soluble, targetable DNA carrier system consisting of an asialoglycoprotein covalently coupled to a polycation. The strategy was based on: 1) the presence of unique receptors on hepatocytes which internalize galactose-terminal (asialo-)glycoproteins; 2) polycations can bind DNA in a non-covalent, non-damaging interaction. Using chloramphenicol acetyltransferase (CAT) as a marker gene, specific delivery and expression of CAT was demonstratedin vitro using asialoglycoprotein receptor ( +) and (-) cell lines.Intravenous injection of conjugate-DNA complexes in rats resulted in detection of CAT DNA sequences in liver 10 min later by dot blots with a CAT cDNA probe. CAT enzyme activity 24 hrs later was found specifically in liver but no other tissues or control livers. Targeted hepatic CAT expression was transient, maximal at 24 hrs but declined to barely detectable levels by 96 hrs. Persistent foreign gene expression was achieved by injection of DNA complex followed by 67% partial hepatectomy. High levels of hepatic CAT activity were detected through 11 weeks post-hepatectomy.The data indicate that a targetable gene delivery system can permitin vivo expression of an exogenous gene after simple intravenous injection. The foreign gene expression can be enhanced and made to persist by induction of hepatocyte replication.     

Smad‐induced alterations of matrix metabolism by a myristoyl tetra peptide

Regulation of extracellular matrix (ECM) components is essential for tissue homeostasis and function. We screened a small peptide that induces ECM protein synthesis for its usefulness in protecting keratinocytes. In this report, we demonstrate that myristoyl tetrapeptide Ala‐Ala‐Pro‐Val (mAAPV) stimulates the expression of ECM proteins and inhibits the expression of metalloproteinases (MMPs) that degrade ECM proteins in Hs68 human fibroblast cells. In order to elucidate the underlying molecular mechanisms for the effects of mAAVP, we investigated the changes in gene expression in the presence of mAAPV using a cDNA microarray. Treatment with mAAPV resulted in decreased expression of MMP‐related genes such as MMP1, MMP3, TIMP1 and TIMP3 and increased expression of collagen genes, including COL1A1, COL1A2, COL3A1, COL5A1 and COL6A3. The pattern of gene expression regulated by mAAPV was very similar to that of gene expression induced by transforming growth factor (TGF)‐β, indicating that the TGF‐β signaling pathway is crucial for simultaneous activation of several ECM‐related genes by mAAPV. We examined whether the activation of SMAD, a downstream protein of TGF‐β receptor, is involved in the signal transduction pathway induced by mAAPV. The results demonstrate that mAAVP directly activates SMAD2 and induces SMAD3 to bind to DNA. In conclusion, our results demonstrate that mAAPV both enhances the expression of collagen and inhibits its degradation via production of protease inhibitors that prevent enzymatic breakdown of the ECM. The results suggest that mAAPV would be a useful ECM‐protecting agent. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of cDNA clones for differentially expressed genes in embryos of dormant and nondormant Avena fatua L. caryopses

The molecular regulation of seed dormancy was investigated using differential display to visualize and isolate cDNAs representing differentially expressed genes during early imbibition of dormant and nondormant Avena fatua L. embryos. Of about 3000 cDNA bands examined, 5 cDNAs hybridized with mRNAs exhibiting dormancy-associated expression patterns during the first 48 h of imbibition, while many more nondormancy-associated cDNAs were observed. Dormancy-associated clone AFD1 hybridized with a 1.5 kb mRNA barely detectable in dry dormant and nondormant embryos that became more abundant in dormant embryos after 24 h of imbibition. Clone AFD2 hybridized with two mRNAs, a 1.3 kb message constitutively expressed in dormant and nondormant embryos and a 0.9 kb message present at higher levels in dormant embryos after 3 h of imbibition. Nondormancy-associated clones AFN1, AFN2 and AFN3 hybridized with 1.5 kb, 1.7 kb and 1.1 kb mRNAs, respectively, that were more abundant in nondormant embryos during imbibition. Expression patterns of some mRNAs in dormant embryos induced to germinate by GA₃ treatment were different than water controls, but were not identical to those observed in nondormant embryos. DNA sequence analysis revealed 76% sequence identity between clone AFN3 and a Citrus sinensis glutathione peroxidase-like cDNA, while significant sequence similarities with known genes were not found for other clones. Southern hybridization analyses showed that all clones represent low (1 to 4) copy number genes.