RESUMO
Autocatalysis has been recognized to be involved in the emergence of life and intrinsic to biomolecular replication. Recently, an efficient template autocatalysis driven by solvent-free crystallization has been reported. Herein, we unveil the role of intermolecular hydrogen bonds formed by amides in crystallization-driven template autocatalysis (CDTA), which involves the autocatalytic activity, template selectivity, and thermal responsiveness. We found that the thermal-induced cis-trans isomerization of amides possibly affects the H-bonding-mediated template ability of products for autocatalytic transformation. As a result, CDTA can be reversibly inhibited and activated by tuning the reaction temperatures. Our work sheds light on the significance of noncovalent H-bonding interactions in artificial self-replicators.
RESUMO
The total RNA was extracted from porcine ovary. Porcine Follistatin cDNA was cloned by RT-PCR. Complete porcine follistatin cDNA coding sequences are presented including 1038 bp of open reading frame. The purified porcine follistatin cDNA was inserted into pGEX-4T-3 vector to construct the prokaryotic fusion protein expression vector. The recombinant expression plasmid was transformed into BL21 (DE3) and expression was induced by IPTG. Protein products were detected by SDS-PAGE and confirmed by Western blotting analysis, which showed that the yield of the Follistatin cDNA was a 63kD protein expression vector. Follistatin protein was expressed in the form of glutathione-S-transferase (GST) fusion protein in E. coli.
Assuntos
Escherichia coli/genética , Folistatina/genética , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Folistatina/química , Dados de Sequência Molecular , Filogenia , SuínosRESUMO
Despite recent successes in cloning various mammals and amphibians, the low efficiency of animals production and abnormal symptoms in many cloned animals are crucial problems in cloning technology. To overcome these problems, scientists focus on mechanisms of cloning. A possible cause of the low success frequency of cloning is the insufficient dedifferentiation and the inadequate reprogramming of the high differentiated adult somatic nucleus in enucleated oocytes, which caused by incomplete methylation and premature de novo remethylation of donor DNA. In cloned embryos the methylation level is higher than normal embryos, and this may cause aberrant expression of several important genes, especially imprinting genes. Study on these mechanisms is very important to improve the rate of successful cloned animals.
Assuntos
Clonagem de Organismos , Epigênese Genética/fisiologia , Animais , Metilação de DNA/genética , Metilação de DNA/fisiologia , Epigênese Genética/genética , Impressão Genômica/genética , Impressão Genômica/fisiologia , HumanosRESUMO
By the method of single preimplantation embryos differential display polymerase chain reaction (SPEDDRT-PCR), 25 reprogramming cDNA fragments were obtained from single 2-cell, 8-cell embryos and blastula. After cloning and sequencing, five of them were identified by reverse-Northern and characterized with stage-specific expression during reconstructed embryo development. This results will help to isolate full length reprogramming genes and study their function during embryonic development.
Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Animais , Blastocisto/fisiologia , Northern Blotting , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Reação em Cadeia da Polimerase , Gravidez , CoelhosRESUMO
Lack of or abnormal expression of developmentally important genes is believed to hamper early development of the nuclear transfer (NT) embryo. To identify stage-specific genes in rabbit NT embryo development, mRNA differential display was used to compare the mRNA content of rabbit NT embryos at different developmental stages, from Metaphase II oocytes to 8-16-cell stage embryos. Thirty-four zygotic transcripts, which abruptly appeared at the 8-16-cell stage in rabbit NT embryos, were isolated; 11 of these were potential novel genes with no matches in the current databases. Of the remaining 23, 12 were matched with established sequence tags with functions uncharacterized and the other 11 were homologous to those in the European Molecular Biology Laboratory (EMBL) and GenBank databases. The differential expression of eight of the 34 amplicons were confirmed by reverse Northern blotting, and four positive clones were validated. Previous studies and present data indicated that these three genes were probably related to preimplantation rabbit embryo development.
