RESUMO
RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.
Assuntos
Regiões Promotoras Genéticas , RNA Polimerase III/genética , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência de Metionina/genética , Sequência de Bases , Northern Blotting , Linhagem Celular , Expressão Gênica , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , Sondas RNA , RNA Catalítico/genética , RNA Mensageiro/química , Retroviridae/genéticaRESUMO
An immune-tolerizing protocol was employed to generate monoclonal antibodies to a variant protein isoform of bovine growth hormone arising from alternative pre-mRNA processing. Variant bovine growth hormone used for immunization was obtained by expression in bacteria and electroelution of the protein from preparative sodium dodecyl sulfate-polyacrylamide gels. Balb/c mice were first immunized with wild-type bovine growth hormone in the presence of the cytotoxic drug cyclophosphamide, thereby tolerizing the mouse to common epitopes shared among the two proteins. Subsequently, the mice were immunized with variant bovine growth hormone to produce antibodies specific to variant epitopes. Comparisons of fusions resulting from standard and tolerizing immunization protocols resulted in a significantly enhanced production of variant bovine growth hormone-specific antibodies as a result of the immunotolerizing protocol. The specificity of the antibodies to the variant growth hormone was substantiated by differential enzyme-linked immunosorbent assay and Western blot. Nearly all hybridomas positive for variant growth hormone were negative for wild-type growth hormone. Finally, the antibodies were used to demonstrate intracytoplasmic staining of COS I cells transiently transfected with a variant growth hormone-producing plasmid. Given the power of the polymerase chain reaction to conveniently clone alternatively processed mRNA species, followed by expression in bacteria to provide antigen, the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms.
Assuntos
Anticorpos Monoclonais/biossíntese , Hormônio do Crescimento/imunologia , Tolerância Imunológica , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Bovinos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Variação Genética , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Haplorrinos , Imunização , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos , Processamento de Proteína Pós-Traducional , Precursores de RNA/genética , RNA Mensageiro/genética , Baço/citologia , Baço/imunologia , TransfecçãoRESUMO
Chinese hamster ovary cells were pretreated with Neplanocin A, a potent inhibitor of RNA methylation. Analysis of polyadenylated RNA from treated cells by high-pressure liquid chromatography revealed marked decreases of 2'-O-methylation within mRNA cap structures and of internal N6-methyladenosine residues. In these Neplanocin A-treated cells, influenza viral mRNA accumulation was virtually abolished. Cellular RNA from Neplanocin A-treated cells was substantially less efficient than RNA from control cells in priming cell-free influenza transcription reactions. These results suggest that the observed inhibition of influenza virus replication is due at least in part to impaired recognition of undermethylated cellular mRNA cap structures by the influenza polymerase complex.
Assuntos
Antivirais/farmacologia , Orthomyxoviridae/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Metilação , Hibridização de Ácido Nucleico , Orthomyxoviridae/genética , Capuzes de RNA/efeitos dos fármacos , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/efeitos dos fármacos , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/biossínteseRESUMO
Influenza virus mRNA is posttranscriptionally methylated at internal adenosine residues to form N6-methyladenosine (m6A). It has been previously shown that there is an average of three m6A residues per influenza virus mRNA (R. M. Krug, M. A. Morgan, and A. J. Shatkin, J. Virol. 20:45-53, 1976). To determine the distribution of m6A in the different influenza virus mRNAs, we purified six of the mRNAs by hybrid selection, digested them with nuclease, and determined their methylation patterns by high-pressure liquid chromatography. The amount of m6A in the different mRNAs varied from one in matrix to eight in hemagglutinin.
