RESUMO
The generation of true random and pseudorandom control sequences is an important problem of computational biology. Available random sequence generators differ in underlying probabilistic models that often remain undisclosed to users. Random sequences produced by differing probabilistic models substantially differ in their outputs commonly used as baselines for evaluations of the motif frequencies. Moreover, modern bioinformatics studies often require generation of matching control transcriptome with emulated partitions into ORFs, 5'- and 3'-UTRs as well as the proportion of non-coding RNAs within model transcriptome rather than relatively simple continuous control sequences. Here we describe novel random sequence generating tool RANDTRAN that accounts for the length distribution of 5' and 3' non-translated regions in given transcriptome and the partition-specific di- and trinucleotide compositions in translated and non-translated regions. RANDRAN presents matching control transcriptomes in ready-to-use UCSC genome browser-compatible input files. These features may be useful for generating of control sequence sets for common types of computational analysis of various sequence motifs within various sets of RNA. RANDTRAN is available for free download at http://www.genereseairch.ru/images/Randtran.rar.
Assuntos
Biologia Computacional , Células Eucarióticas , RNA Mensageiro , Software , Transcriptoma , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Algoritmos , Fases de Leitura Aberta , Distribuição AleatóriaRESUMO
It was determined the ratio of viral DNA and DNA from Vero cells using the polymerase chain reaction in real time in Vero cell lysate, infected with L2 strain of the herpes simplex virus type 1. Copy number of the virus reached a maximum after 24 hours of incubation of infection. Total DNA was isolated and sequenced using NGS technology by Ion Torrent device. Nucleotide sequences of the thymidine kinase gene (UL23) and DNA polymerase (UL30) were determined for a population of HSV-1 strain L2. Comparison of the primary structure of these genes with the corresponding nucleotide sequences of known strains of HSV-1 KOS and 17 was conducted. Differences in the structure of genes UL23 and UL30 between strain L2 and reference strains KOS and 17 are not important, because changes are found in non-conservative regions.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Herpesvirus Humano 1/genética , Timidina Quinase/genética , Proteínas Virais/genética , Animais , Chlorocebus aethiops , Reação em Cadeia da Polimerase , Análise de Sequência de DNA/métodos , Células Vero/virologiaRESUMO
Gene-directed therapy with small interfer-ring RNA (siRNA) has a tremedous potential and in the future will undoubtly occupy one of the leading positions among other therapeutic methods. The lack of efficient and targeted delivery vectors delays the successful implementation of this method in clinic. To develop such systems, one needs a comprehansive insight into the processes of interactions between siRNAs, its delivery systems and an organism. This review covers properties of therapeutic siRNAs and non-viral systems for their delivery.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Terapia Genética , Interferência de RNA , RNA Interferente Pequeno/química , Aptâmeros de Nucleotídeos/química , Hemorreologia , Humanos , Lipossomos/química , Nanopartículas/química , Tamanho da Partícula , RNA Interferente Pequeno/genética , Eletricidade EstáticaRESUMO
To date, RNA interference remains the most powerful and promising tool for gene-targeted therapy. Several problems still have to be solved for its successful use in clinics. One of the main issues is the siRNA's efficient delivery. This review covers various types of nonviral siRNA delivery systems.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Terapia Genética , Interferência de RNA , RNA Interferente Pequeno/química , Aptâmeros de Nucleotídeos/química , Materiais Biocompatíveis/química , Humanos , Lipídeos/química , Lipopeptídeos/química , Lipossomos/química , Nanopartículas/química , Polímeros/química , RNA Interferente Pequeno/genética , Eletricidade EstáticaRESUMO
Cloned laboratory mutants of herpes simplex virus type I resistant to acycloguanosine H-phosphonate have been investigated. For all clones were shown that mutations resulted to increasing of sensitivity to acting of sidofovir. Thymidine kinase of mutant viruses partially preserves the ability to phosphorilate thymidine, but loses the ability to phosphorilate BVDU.
Assuntos
Aciclovir/análogos & derivados , DNA Polimerase Dirigida por DNA/metabolismo , Farmacorresistência Viral/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/enzimologia , Organofosfonatos/farmacologia , Timidina Quinase/química , Aciclovir/farmacologia , Animais , DNA Polimerase Dirigida por DNA/genética , Humanos , Mutação , Timidina Quinase/genética , Células VeroRESUMO
The primary structures of DNA-polymerase (ul30) and thymidine kinase (ul23) genes from several herpes simplex virus type 1 (HSV-1) clinical isolates which differed in sensitivity for a number of antiherpetic drugs were determined and compared with those for two laboratory HSV-1 strains one of which was ACV-sensitive (L2), while the another was resistant (L2) to ACV. The phylogenetic analysis of the sequences showed that conserved regions of ul30 gene of HSV-1 clinical isolates and L2 strain were homologous with the exception of point mutations and degenerated substitutions. Several new mutations in the HSV-1 DNA-polymerase and thymidine kinase functional domains were established and identified as the substitutions associated with the strain-resistance to ACV and other drugs.
Assuntos
Aciclovir/farmacologia , Antivirais/farmacologia , DNA Polimerase Dirigida por DNA/genética , Farmacorresistência Viral/genética , Exodesoxirribonucleases/genética , Herpes Simples/genética , Herpesvirus Humano 1/genética , Mutação Puntual , Timidina Quinase/genética , Proteínas Virais/genética , Linhagem Celular , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Herpes Simples/tratamento farmacológico , Herpes Simples/enzimologia , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/isolamento & purificação , Humanos , Timidina Quinase/metabolismo , Proteínas Virais/metabolismoRESUMO
A real-time PCR procedure is proposed for assaying E. coli residual DNA in the pharmaceutical substance of human recombinant insulin. For the quantitative analysis of the DNA content, an amplification of fragments of the bla gene plasmid DNA and E. coli genomic DNA of the 16S RNA gene were used. The contents of plasmid and genomic DNA were detected both in intermediates at various stages of the insulin purification process and in the finished product.
Assuntos
DNA Bacteriano/análise , Insulina/química , Escherichia coli/genética , Humanos , Reação em Cadeia da Polimerase , Engenharia de Proteínas , Sensibilidade e Especificidade , beta-Lactamases/genéticaRESUMO
Up-to now three variants of antisense technologies are known, namely antisense oligonucleotides, RNA interference, and ribozymes. In spite of different mechanisms of action, all of them are united by the common principle: an antisense preparation works after binding with RNA-target by forming a duplex. Today all three variants are intensively used in vivo. Present posture of affairs in use of antisense technologies for treating various diseases is considered in the review.
Assuntos
Terapia Genética/métodos , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Interferência de RNA , Animais , Humanos , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/metabolismo , RNA/genética , RNA/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Catalítico/uso terapêuticoRESUMO
Antisense regulation of gene expression is a widespread but poorly understood mechanism of gene expression regulation. The potential role of antisense transcripts in tumorigenesis is the most intriguing for the functional research. Here we experimentally characterize an antisense mRNA asLZK overlapping human MAP3K13/LZK gene that is involved in mitogenesis related JNK/SAPK signal transduction pathway. According to the functional annotation of the human genome, asLZK transcript (LOC647276) is expressed at the relatively high level and overrepresented in tumor samples. To our surprise, experimental study of human asLZK revealed that this sequence is not expressed, but represents a silent pseudogene of ribosomal protein L4 encoding gene RPL4. This pseudogene resulted from relatively recent retroposition of RPL4 mRNA into the first intron of MAP3K13 gene and does not participate in the regulation of MAP3K13 expression. This study stresses that, after initial in silico mapping efforts, experimental verification of the expression landscape is warranted.
Assuntos
Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase Quinases/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , RNA Antissenso/biossíntese , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Íntrons/genética , MAP Quinase Quinase Quinases/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Pseudogenes/genética , RNA Antissenso/genética , Retroelementos/genética , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genéticaRESUMO
IgA1-specific proteinases (Igase) are acknowledged as a pivotal pathogenicity factor in meningococcus (Neisseria meningitidis) and in some related bacteria. These enzymes belong to trypsin-like clan of serine proteases. They exhibit high substrate selectivity being able to discriminate between IgA1 and IgA2. On the other hand, these enzymes are able to distinguish the human IgA1 from IgA1 of non-primate species of mammals. In addition to conventional IgA1-processing enzymes, alternative enzymes were recently reported to occur in meningococci. However, the substrate specificity of the conventional Igase, its role in pathogenesis, and ability to complement functionality remains obscure. Within the framework of the present project we studied the structure of the Igase genes and their products in two highly virulent N. meningitidis serogroup A strains M9 and A208. In particular, we succeeded to find both conventional and alternative Igase genes in each genome: nucleotide sequences of these genes were deposited in the NCBI Gene Bank under the access number AY770504, AY558158, AY558159. The DNA sequence of the conventional Igase was almost entirely conserved in the two strains, whereas the recently discovered alternative Igase (formerly known as meningococcal adhesine, type 1) exhibited occurrence of a variable region spanning about 900 bp in the 5'-terminal part of the gene. Conventional genes from both strains were expressed in E. coli rendering inclusion bodies. The recombinant products were used for immunization of rabbits and exhibited reaction with both recombinant and native antigen from the N. meningitidis cultural medium.
Assuntos
Genes Bacterianos , Neisseria meningitidis Sorogrupo A/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Imunização , Dados de Sequência Molecular , Neisseria meningitidis Sorogrupo A/enzimologia , Polimorfismo Genético , Coelhos , Análise de Sequência de DNA , Serina Endopeptidases/imunologiaRESUMO
A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10 microg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.
Assuntos
DNA/análise , Insulina/química , Reação em Cadeia da Polimerase/métodos , beta-Lactamases/genética , DNA/genética , Humanos , Proteínas Recombinantes/química , Sensibilidade e EspecificidadeRESUMO
2-Methylthioadenosine 5'-mono- and diphosphates were prepared by chemical synthesis, and enzymatically phosphorylated to 5'-[beta-32P]-diphosphate and 5'-[gamma-32P]-triphosphate, respectively. They were isolated by HPLC and had activity exceeding 1000 Ci/mmol and radiochemical purity higher than 95%.
