Ultrashort peptides have higher stability, tissue penetrability, biocompatibility, and less immunogenicity, and are widely applied in biology and medicine. GHK (glycyl-l-histidyl-l-lysine) and GQPR (glycyl-l-glutamyl-l-prolyl-l-arginine) can stimulate collagen renewal and inhibit collagen degradation. GHK and GQPR have been used in cosmetic anti-wrinkle skincare and make-up products. The most common approach for ultrashort peptide production is the solid-phase synthesis, which is eco-unfriendly due to heavy usage of organic chemical reagents during the manufacturing process. Here we report a new approach to the production of ultrashort peptides. Recombinant expression of ultrashort peptides is usually unfeasible because of the short amino acid sequences. A vector pET28a-Trxm harboring the thioredoxin gene was first constructed for subsequent fusion expression. The tandem repeats of GHK and GQPR genes were used as the templates for rolling circle amplification (RCA). The RCA reaction was tuned to incorporate noncanonical nucleotides 5-methylcytosine to obtain long DNA fragments. Gene sequences with various lengths were generated through double digestion of Acc65 Ⅰ and Apa Ⅰ. The resulting digestion products were gel recovered by size (from 500 bp to 1 500 bp) and cloned into pET28a-Trxm to obtain the recombinant vector pET28a-Trxm-(TRSP)_n. The pET28a-Trxm-(TRSP)_n was introduced into E. coli BL21(DE3) to generate a library of Trxm-(TRSP)_n sequences with a controlled distribution of lengths. Through double digestion and sequencing, positive clones with tandem repeats n=1, 2, 3, 4, 6, 7, 8, 9 were obtained. Protein expression results showed protein bands with corresponding molecular weight, and the protein expression level decreased as the tandem repeats increased. The expression level of Trxm-(TRSP)₁ achieved 50% of the total protein, while the expression level of Trxm-(TRSP)₂ was 30% of the total protein. The crude extracts from cell pellets were further treated with enterokinase cleavage, and the supernatants containing (TRSP)₁ were collected after ultrafiltration and then subjected to trypsin cleavage. HPLC analysis indicated that the ultrashort peptides GHK and GQPR were successfully obtained through two-step cleavage. This study may facilitate the commercial production of ultrashort peptides.
Escherichia coli/metabolism* ; Peptides/chemistry* ; Amino Acid Sequence ; Gene Library ; Tandem Repeat Sequences

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.
Coculture Techniques ; DNA ; Endogenous Retroviruses ; Gene Expression ; Genome ; HeLa Cells ; Humans ; Kidney ; MicroRNAs ; Parents ; Proviruses ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; RNA-Directed DNA Polymerase ; Selective Breeding ; Terminal Repeat Sequences ; Transfection

OBJECTIVES: Chronic otitis media (COM) is followed by irreversible tissue damage and destruction of the middle ear structures, with the possibility of complications under the maintenance of inflammation. Inflammatory mediators such as cytokines play a crucial role in the initial stage of inflammation. The aim of this study was to evaluate the association of the polymorphisms in two innate immunity/inflammation cascade genes from interleukin-1 (IL-1) gene cluster with COM with regard to cholesteatoma. METHODS: In the cross-sectional case-control study, DNA samples were collected from 189 patients with COM and 119 controls from a population of Serbia. The +3953 C/T (rs1143634), TaqI polymorphism in interleukin-1 beta (IL-1β) gene and 86 bp variable number tandem repeat (VNTR, rs2234663) polymorphism in the IL-1 receptor antagonist (IL-1RA) gene were analyzed by polymerase chain reaction. RESULTS: The IL-1β TaqI polymorphism was not significantly different in patients compared with the control group. The significant difference between patients and controls was observed for both, genotype and allele frequencies of IL-1RA VNTR polymorphism (chi-square P < 0.01). We found that carriers of IL-1RA allele 2 (odds ratio, 0.47; 95% confidence interval, 0.29 to 0.76; P=0.004) have a favorable association with COM, using multivariate logistic analysis that included both polymorphisms, age and sex. The IL-1RA allele frequency distribution was significantly different with regard to cholesteatoma. CONCLUSION: The carriers of allele 2 of VNTR IL-1RA polymorphism had a decreased odds ratio for COM, which is in agreement with findings in other inflammatory disease and its previous association with higher IL-1RA levels. Possible down-regulation of IL-1 mediated proinflammatory signaling pathways via IL-1RA in COM as well as results of our study should be further investigated and replicated.
Alleles* ; Case-Control Studies ; Cholesteatoma ; Cytokines ; DNA ; Down-Regulation ; Ear, Middle ; Gene Frequency ; Genotype ; Humans ; Inflammation ; Interleukin 1 Receptor Antagonist Protein* ; Interleukin-1 ; Interleukin-1beta ; Multigene Family ; Odds Ratio ; Otitis Media* ; Otitis* ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Serbia ; Tandem Repeat Sequences

AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.
Animals ; Baculoviridae ; DNA, Single-Stranded ; Dependovirus ; Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Sf9 Cells ; Terminal Repeat Sequences ; Transfection

DNA microarray and next-generation sequencing provide data that can be used for the genetic analysis of multiple quantitative traits such as gene expression levels, transcription factor binding profiles, and epigenetic signatures. In particular, chromatin opening is tightly coupled with gene transcription. To understand how these two processes are genetically regulated and associated with each other, we examined the changes of chromatin accessibility and gene expression in response to genetic variation by means of quantitative trait loci mapping. Regulatory patterns commonly observed in yeast and human across different technical platforms and experimental designs suggest a higher genetic complexity of transcription regulation in contrast to a more robust genetic architecture of chromatin regulation.
Chromatin* ; Epigenesis, Genetic ; Epigenomics ; Gene Expression ; Genetic Variation ; Humans ; Oligonucleotide Array Sequence Analysis ; Quantitative Trait Loci ; Regulatory Sequences, Nucleic Acid ; Research Design ; Transcription Factors ; Yeasts

To obtain the genome sequence of human bocavirus 2 (HBoV2), different regions of HBoV2 genome were amplified through PCR in fecal specimens which had been identified as single-positive for HBoV2 in 2010. A genome sequence of HBoV2 (HBoV2-NC, 5444 bp) was obtained after sequence assembly. The phylogenetic analysis showed that HBoV2-NC had the closest evolutionary relationship with HBoV2 Lanzhou strain. The predication of inverted terminal repeats of HBoV2-NC by DINAMelt showed that inverted terminal repeats were contained in HBoV2-NC 5' terminal, which had the typical stem-loop structure in other parvoviruses. Finally, some flanking sequences of HBoV2-NC were amplified by linker-PCR.
Base Sequence ; Gene Amplification ; Genome, Viral ; Human bocavirus ; chemistry ; classification ; genetics ; Humans ; Molecular Sequence Data ; Nucleic Acid Conformation ; Parvoviridae Infections ; virology ; Phylogeny ; RNA, Viral ; chemistry ; genetics ; Terminal Repeat Sequences

Approximately 45% of the human genome is comprised of transposable elements (TEs). Results from the Human Genome Project have emphasized the biological importance of TEs. Many studies have revealed that TEs are not simply "junk" DNA, but rather, they play various roles in processes, including genome evolution, gene expression regulation, genetic instability, and cancer disposition. The effects of TE insertion in the genome varies from negligible to disease conditions. For the past two decades, many studies have shown that TEs are the causative factors of various genetic disorders and cancer. TEs are a subject of interest worldwide, not only in terms of their clinical aspects but also in basic research, such as evolutionary tracking. Although active TEs contribute to genetic instability and disease states, non-long terminal repeat transposons are well studied, and their roles in these processes have been confirmed. In this review, we will give an overview of the importance of TEs in studying genome evolution and genetic instability, and we suggest that further in-depth studies on the mechanisms related to these phenomena will be useful for both evolutionary tracking and clinical diagnostics.
DNA ; DNA Transposable Elements* ; Gene Expression ; Gene Expression Regulation ; Genome* ; Genome, Human ; Human Genome Project ; Humans ; Terminal Repeat Sequences

Genome-wide association studies have proven the highly polygenic architecture of complex diseases or traits; therefore, single-locus-based methods are usually unable to detect all involved loci, especially when individual loci exert small effects. Moreover, the majority of associated single-nucleotide polymorphisms resides in non-coding regions, making it difficult to understand their phenotypic contribution. In this work, we studied epistatic interactions associated with three common diseases using Korea Association Resource (KARE) data: type 2 diabetes mellitus (DM), hypertension (HT), and coronary artery disease (CAD). We showed that epistatic single-nucleotide polymorphisms (SNPs) were enriched in enhancers, as well as in DNase I footprints (the Encyclopedia of DNA Elements [ENCODE] Project Consortium 2012), which suggested that the disruption of the regulatory regions where transcription factors bind may be involved in the disease mechanism. Accordingly, to identify the genes affected by the SNPs, we employed whole-genome multiple-cell-type enhancer data which discovered using DNase I profiles and Cap Analysis Gene Expression (CAGE). Assigned genes were significantly enriched in known disease associated gene sets, which were explored based on the literature, suggesting that this approach is useful for detecting relevant affected genes. In our knowledge-based epistatic network, the three diseases share many associated genes and are also closely related with each other through many epistatic interactions. These findings elucidate the genetic basis of the close relationship between DM, HT, and CAD.
Coronary Artery Disease ; Deoxyribonuclease I ; Diabetes Mellitus ; Diabetes Mellitus, Type 2 ; DNA ; Gene Expression ; Genome-Wide Association Study ; Hypertension ; Korea ; Polymorphism, Single Nucleotide ; Regulatory Sequences, Nucleic Acid ; Transcription Factors

OBJECTIVETo evaluate the anti-viral effects of a plasmid expressing an inverted-repeat RNA targeting dengue virus type-2 (DENV-2) pre-membrane (prM) gene.

METHODSuckling mice were inoculated with live DENV-2 in the brain. The total RNA was extracted from the brain tissue of the infected mice, and the prM gene fragments were amplified by RT-PCR and then subcloned into XhoI/EcoR I of the pcDNA3.1(+) plasmid in antisense orientation to construct the plasmid pcDNA-asprM. DENV-2 prM sequences were also subcloned into pMD18-T-vector in sense orientation to construct the plasmid pMD18-T- prM. pcDNA-irRNA was constructed by inserting in sense orientation the prM fragment isolated from pMD18-T-prM into the NheI/Kpn I of pcDNA-asprM. The plasmid pcDNA-irRNA was transfected into BHK-21 cells and the anti-viral effects were analyzed by semi-quantitative PCR and real-time PCR.

RESULTSTransfection with the plasmid pcDNA-irRNA caused a reduction of NS3 mRNA expression level by 28% in BHK-21 cells following a 96-h challenge with DENV-2 as compared to the cells without plasmid transfection (positive control). The viral copies in pcDNA-irRNA-transfected cells was 1.44-fold lower than those in the positive control cells following a 72-h virus challenge, and the mRNA expression levels of NS1 were also significantly lower in the transfected cells at 96 h after viral challenge (P<0.05) as shown by real-time quantitative PCR.

CONCLUSIONThe inverted-repeat RNA for DENV-2 prM gene silencing can suppress DENV-2 replication in BHK-21 cells, which provides a basis for developing dengue virus gene vaccine.

Animals ; Base Sequence ; Cells, Cultured ; Cricetinae ; Dengue Virus ; physiology ; Gene Silencing ; Mice ; Mice, Inbred Strains ; RNA, Viral ; genetics ; Terminal Repeat Sequences ; Viral Envelope Proteins ; genetics ; Virus Replication ; genetics

Transposable elements (TEs), particularly, long terminal repeat retrotransposons (LTR-RTs), are the most abundant DNA components in all plant species that have been investigated, and are largely responsible for plant genome size variation. Although plant genomes have experienced periodic proliferation and/or recent burst of LTR-retrotransposons, the majority of LTR-RTs are inactivated by DNA methylation and small RNA-mediated silencing mechanisms, and/or were deleted/truncated by unequal homologous recombination and illegitimate recombination, as suppression mechanisms that counteract genome expansion caused by LTR-RT amplification. LTR-RT DNA is generally enriched in pericentromeric regions of the host genomes, which appears to be the outcomes of preferential insertions of LTR-RTs in these regions and low effectiveness of selection that purges LTR-RT DNA from these regions relative to chromosomal arms. Potential functions of various TEs in their host genomes remain blurry; nevertheless, LTR-RTs have been recognized to play important roles in maintaining chromatin structures and centromere functions and regulation of gene expressions in their host genomes.
Evolution, Molecular ; Gene Silencing ; Genome, Plant ; genetics ; Plants ; genetics ; Retroelements ; genetics ; Terminal Repeat Sequences ; genetics