The RNA-directed DNA Methylation (RdDM) is one type of epigenetic modification which was firstly discovered in plant. RdDM can directly cause DNA modifications of the genome through RNA-DNA interactions. In plant, both of RdDM and mRNA degradation induced by siRNA can silence sequence specific genes through RNA. They play very significant roles in chromosome rearrangement, defence of virus invasion, regulation of gene expression and many processes of plant development. However, the mechanisms of RdDM are still unclear. In this paper the basic characteristics of RdDM were briefly summarized and advances in studies on mechanisms of RdDM were reviewed. These include the kinds of DNA methyltransferases and their functional mechanisms in RdDM, the relationships between DNA methylation and chromatin modification, and important proteins involved in the RdDM process. In plants, RdDM may occur at both the transcriptional and post-transcriptionnal levels, both of which induce gene silencing. Methylation of the target gene promoter correlates with transcriptional gene silencing (TGS) whereas methylation of the coding sequence is associated with post-transcriptional gene silencing (PTGS). RdDM and RNAi all depend on the similar siRNA and enzymes, such as DCL3, RdR2, SDE4 and AGO4. There are at least three kinds of DNA methyltransferases, DRM1/2, MET1 and CMT3, in pants. They can interact with and modifies all cytidines within the DNA regions homologous to RNA sequence. Furthermore, methylation of lysine 9 in Histone H3 can affect the methylation of cytidines.
DNA Methylation ; DNA Modification Methylases ; metabolism ; Gene Silencing ; Plants ; genetics ; metabolism ; Protein Processing, Post-Translational ; RNA ; metabolism

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.
Animals ; Cell Line ; Chloride Channels ; physiology ; Cricetinae ; Cystic Fibrosis Transmembrane Conductance Regulator ; biosynthesis ; genetics ; Dependovirus ; genetics ; Genetic Vectors ; Humans ; Membrane Potentials ; genetics ; Protein Processing, Post-Translational ; Protein Splicing

Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.
Animals ; DnaB Helicases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Inteins ; genetics ; Natriuretic Peptide, Brain ; biosynthesis ; genetics ; Protein Engineering ; methods ; Protein Splicing ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

The recent identification of the transactive response DNA binding protein with a molecular weight of 43 kDa (TDP-43) as the major pathological protein, in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U), provides the new insight into understanding disease processes. The pathogenesis of both diseases is unclear, although they are related by having some overlap of symptoms and now by the shared histopathology of TDP-43 deposition. The number of degenerative diseases associated with TDP-43 has increased, leading to the new designation "TDP-43 proteinopathy". TDP-43 is a highly conserved protein ubiquitously expressed in many tissues including the central nervous system where it is present in neuronal and glial nuclei and to a lesser extent in the cytoplasm. Currently, TDP-43 has been implicated in regulating gene transcription and alternative splicing, in addition to maintaining mRNA stability. However, we still need to investigate the effects of posttranslational modifications of TDP-43, including phosphorylation, ubiquitination, and cleavage, on its regulation of various cellular processes. We review recently published studies of TDP-43 and its relationship to human disease with a special focus on ALS and FTLD-U. We conclude that the TDP-43 proteinopathies represent a novel class of neurodegenerative disorders and both ALS and FTLD-U are closely related conditions linked to similar mechanism of neurodegeneration.
Alternative Splicing ; Amyotrophic Lateral Sclerosis ; Central Nervous System ; Cytoplasm ; DNA ; DNA-Binding Proteins ; Frontotemporal Dementia ; Frontotemporal Lobar Degeneration ; Humans ; Molecular Weight ; Neurodegenerative Diseases ; Neurons ; Phosphorylation ; Protein Processing, Post-Translational ; RNA Stability ; TDP-43 Proteinopathies ; Ubiquitin ; Ubiquitination

A library with potential to produce six amino acids cyclic peptides was prepared using pET-28a as the starting plasmid. pVmut was used to amplify the Int(C)-dnaB-N-Int(N) fragment that was inserted into pET28a to give pEV. On pEV, DnaB split intein was expressed under the strong T7 promoter. Analyses of Escherichia coli transformed with pEV showed that DnaB split intein was produced in large quantity and the fusion protein self-spliced efficiently to produce cyclized DnaB-N. A synthesized 115 bp fragment mixture encoding 5 random amino acids was inserted into pEV to generate pEV-IS. The ligation mixture was transformed into E. coli. A library of 10(3) clones was obtained, 20 randomly picked clones were sequenced. All of them contain different sequences. Nine clones were chosen for further analysis. Split-intein-ISs were expressed in large quantity, and 90% of them self-spliced under 16 degrees C in 20 hours. After induction at 30 degrees C for 3 hours, the expressed DnaB split intein was purified using His-column, and then a molecular weight of target cyclic peptide was detected by MALDI-TOF-MS.
Amino Acids ; chemistry ; Base Sequence ; DnaB Helicases ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Inteins ; genetics ; Molecular Sequence Data ; Peptide Library ; Peptides, Cyclic ; chemistry ; Protein Splicing ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Synechocystis ; chemistry

OBJECTIVETo investigate the influence of fluorosis on nicotinic acetylcholine receptors (nAChRs) in protein and gene levels in SH-SY5Y cells and the mechanism of the receptor modification.

METHODSSH-SY5Y cells, a human neuroblastoma cell line, were incubated with different concentrations of fluoride or with antioxidant for 48 hours. The functions of cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and protein oxidation detected by carbonyl content; the alpha3 and alpha7 nAChR subunits in protein level were measured by Western blotting and in mRNA level by RT-polymerase chain reaction (RT-PCR).

RESULTSIn high-dose group as compared to the control, the decreased MTT (49%), increased protein oxidation (72%), and lower expression of alpha3 (51%) and alpha7 (47%) nAChR subunit proteins were obviously observed in SH-SY5Y cells. There were no changes in expression of nAChR subunit mRNAs between the cells treated with fluoride and those un-treated in controls. Prior treatment with antioxidant resulted in preventing the decrease of nAChR protein in cells exposed to the high doses of fluoride.

CONCLUSIONFluorosis should result in damage of cells and the declined expression of nAChRs in protein levels, but no influences on gene expression of the receptors in human neuroblastoma neurons. The decreased nAChR proteins might be involved in the mechanism of oxidative stress induced by fluorosis.

Cell Line, Tumor ; Fluoride Poisoning ; metabolism ; Fluorides ; toxicity ; Humans ; Neuroblastoma ; metabolism ; pathology ; Protein Processing, Post-Translational ; drug effects ; Proteins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Nicotinic ; biosynthesis ; genetics

Filamentous fungi can secrete large amounts of proteins, glycosylate proteins and grow faster than plant, insect or mammalian cells. With the advances in fungal molecular genetics, strain improvement, and especially fungal genomics, filamentous fungi are developed as microbial cell factories for the production of heterologous proteins. This review focuses on recent developments of filamentous fungi as production hosts, such as protein quality control mechanisms, the secretion pathways, protein modification, strain stability, and most importantly the application of functional genomics in protein expression. At the same time, the strategies for improving heterologous protein production were also discussed in details.
Biotechnology ; methods ; trends ; Cloning, Molecular ; Fermentation ; genetics ; physiology ; Fungi ; genetics ; metabolism ; Genetic Engineering ; Protein Processing, Post-Translational ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVEScreening and characterizing high affinity completely humanized single-chain antibody (ScFv) against PreS1 of hepatitis B virus.

METHODSA combinatorial library of phage-displayed human ScFv, genes of which were derived from peripheral blood lymphocytes immunized by PreS1 of Hepatitis B Virus in vitro, was constructed. The library contained 7 10(8) clones.

RESULTSAfter 3 rounds panning, a high affinity (K=10(7) to 10(8) mol/L) ScFv specific to PreS1 was obtained. The V(H) belonged to human V(H4) family, and V(1) to V(4) by sequence analysis.

CONCLUSIONThis study suggests that the method of antigen stimulation in vitro is an expeditious way for the source of human immune antibody. And the ScFv may provide a more satisfactory therapy.

Antibodies, Monoclonal ; biosynthesis ; Antibody Affinity ; Hepatitis B Antibodies ; biosynthesis ; Hepatitis B Surface Antigens ; immunology ; Humans ; Peptide Library ; Protein Precursors ; immunology

Humans ; Oxidative Stress ; genetics ; Proteasome Endopeptidase Complex ; metabolism ; Protein Modification, Translational ; genetics ; Ubiquitination ; genetics

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.
Animals ; Cells, Cultured ; Chlorides ; metabolism ; Codon ; genetics ; Cricetinae ; Cystic Fibrosis ; therapy ; Cystic Fibrosis Transmembrane Conductance Regulator ; genetics ; metabolism ; DNA, Complementary ; genetics ; Dependovirus ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Inteins ; physiology ; Kidney ; cytology ; Protein Processing, Post-Translational ; Recombinant Proteins ; genetics ; metabolism ; Trans-Splicing ; Transfection