RESUMO
We present for the first time experimental evidence that in vitro synthesized RNA (cRNA) is trans-spliced after microinjection into the nuclei of mammalian tissue culture cells. The template used for cRNA synthesis was the early SV40 BstXl/BamHI DNA fragment. This DNA fragment encodes exclusively for the second T-antigen exon and contains the intact small t-antigen intron. To generate the corresponding mRNA (T1-mRNA) by trans-splicing, the cells utilize a 5' cryptic splice site located within the second T-antigen exon of one cRNA molecule which is spliced to the small t-antigen 3' splice site of another cRNA molecule. Formation of the T1-mRNA by trans-splicing was confirmed by RT-PCR analysis and DNA sequencing. Efficient trans-splicing required that competitive small t-antigen cis-splicing be inhibited by deletion of the small t-antigen 5' splice site. The T1-mRNA was not generated when the cryptic 5' splice site was mutated.
Assuntos
Splicing de RNA , RNA Complementar/metabolismo , RNA Viral/metabolismo , Vírus 40 dos Símios/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Northern Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Primers do DNA , Eletroforese em Gel de Ágar , Éxons/genética , Imunofluorescência , Genes Virais , Microinjeções , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , RNA Complementar/síntese química , RatosRESUMO
The purpose of this investigation was to study the effect on cap structure for trans-splicing in mammalian cells. The early SV40 Bst/Bam pre-mRNA (cRNA) was synthesized in vitro in both capped (cap-Bst/Bam-cRNA) and non-capped (Bst/Bam-cRNA) versions and microinjected into the nuclei of TC7 cells. Trans-splicing was monitored by immunofluorescence staining (T1-antigen) and by RT-PCR analysis. Cap-Bst/Bam-cRNA was trans-spliced with high efficiency, but not the Bst/Bam-cRNA molecules. Northern blot analysis revealed that both the capped and uncapped cRNA molecules had similar stability in the microinjected cells. The coinjected m7G(5')ppp(5')G cap analog did not inhibit the trans-splicing reaction in vivo and did not prevent nuclear export of the mRNA.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Capuzes de RNA , Precursores de RNA/metabolismo , Splicing de RNA , RNA Viral/metabolismo , Vírus 40 dos Símios/genética , Animais , Antígenos Transformantes de Poliomavirus/análise , Northern Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Primers do DNA , Eletroforese em Gel de Ágar , Imunofluorescência , Microinjeções , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Precursores de RNA/síntese química , Precursores de RNA/genética , RNA Complementar/genética , RNA Complementar/metabolismo , RNA Viral/genética , RatosRESUMO
The early SV40 BstXI-BamHI (Bst/Bam) DNA fragment encodes exclusively for the second exon of the large T-antigen and contains the intact small t-antigen intron. Rat cells transformed by the p14T, a construct that carries the Bst/Bam DNA fragment as a tail-to-head tandem duplication, synthesize a truncated T-antigen (T1-antigen) without having a direct equivalent at the DNA level. Formation of the T1-mRNA occurs by means of two distinct mechanisms: alternative-tandem-cis-splicing and trans-splicing. To generate the T1-mRNA the cells utilize a cryptic 5' splice site, located within the second exon of the large T-antigen and the regular small t-antigen 3' splice site. Since these splice sites are in an inverted order two Bst/Bam transcripts are required to generate one T1-mRNA molecule. For alternative-tandem-cis-splicing the cells utilize a 4.4 kb pre-mRNA that contains the sequence of the entire Bst/Bam tandem repeat. The proximal Bst/Bam segment provides the 5' donor splice site and the distal segment the 3' acceptor site. This requires that the pre-mRNA not be cleaved after the RNA polymerase II has passed the polyadenylation signal of the proximal Bst/Bam DNA segment. Synthesis of the 4.4 kb pre-mRNA was demonstrable by RT-PCR but not by Northern blot analysis. For trans-splicing, the cells utilize two separate pre-mRNA molecules. One transcript provides the cryptic 5' splice donor site and the other the 3' splice acceptor site. To demonstrate this a three base pair deletion was introduced into the proximal Bst/Bam segment of the p14T DNA (p14Tdelta-3) as a marker, destroying the recognition site for Pf/MI restriction enzyme. This deletion allowed the differentiation between the proximal and distal Bst/Bam segment. RT-PCR analysis and DNA sequencing confirmed that the p14Tdelta-3 transformed cells generate the T1-mRNA by intra- and inter-molecular RNA splicing.
Assuntos
Processamento Alternativo/genética , Antígenos Transformantes de Poliomavirus/genética , Splicing de RNA/genética , Vírus 40 dos Símios/imunologia , Animais , Antígenos Transformantes de Poliomavirus/biossíntese , Sequência de Bases , Linhagem Celular Transformada , Éxons/genética , Dados de Sequência Molecular , Precursores de RNA/biossíntese , Precursores de RNA/genética , RNA Mensageiro/biossíntese , Ratos , Sequências Repetitivas de Ácido Nucleico/genética , Deleção de Sequência/genéticaRESUMO
We present evidence that mammalian cells have the ability to generate functional mRNA molecules by trans-splicing. Rat cells, transformed by an early SV40 DNA fragment (Bst/Bam) synthesize a truncated T antigen (T1 antigen), although the cells do not have a direct sequence homology for the T1 antigen at the DNA level. The Bst/Bam DNA fragment encodes exclusively for the second SV40 T antigen exon (aa 83-708) and contains the entire small t antigen intron. To synthesize the corresponding mRNA (T1 mRNA), the cells utilize a cryptic 5' splice site within the second exon (codons for aa 131/132) as donor site and the upstream small t antigen 3' splice site as the acceptor site. Since these sites are in an inverted order on the pre-mRNA, two Bst/Bam transcripts are required to generate one T1 mRNA molecule. HeLa cell nuclear extracts also performed the trans-splicing reaction in vitro.
Assuntos
Mamíferos/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Antígenos Virais de Tumores/biossíntese , Antígenos Virais de Tumores/química , Antígenos Virais de Tumores/genética , Sequência de Bases , Células Cultivadas , Éxons , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Íntrons , Modelos Genéticos , Dados de Sequência Molecular , RNA/biossíntese , RNA/química , RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/química , RNA Viral/química , RNA Viral/genética , RatosRESUMO
Bacterially-expressed fusion proteins containing the DNA-(region C) or hormone-binding (region E) domains of the chicken progesterone receptor (cPR) fused to the C terminus of Escherichia coli beta-galactosidase were analysed for the specificity of interaction with natural and synthetic hormone-responsive elements (HREs) and progestins, respectively. The purified fusion protein containing the progestin-binding domain bound progesterone with an apparent Kd of 1.0-1.5 nM and was specifically photocross-linked with the synthetic progestin R5020 in crude bacterial lysates. Labelling of intact bacterial cells with [3H]R5020 revealed that the majority, if not all, of the bacterially produced hormone-binding domain was active. No differences in the binding to a synthetic palindromic glucocorticoid/progestin-responsive element (GRE/PRE) were found when the bacterially produced cPR DNA-binding domain was compared in methylation interference assays with the full-length chicken progesterone receptor form A expressed in eukaryotic cells. The study of dissociation kinetics, however, revealed differences in the half-life of the complexes formed between the palindromic GRE/PRE and either the receptor form A or the fusion protein containing the cPR DNA-binding domain. DNase I protection experiments demonstrated that the bacterially produced region C of the cPR generated specific 'footprints' on the mouse mammary tumour virus long terminal repeat (MMTV-LTR) which were nearly identical to those previously reported for the rat glucocorticoid receptor.
Assuntos
Receptores de Progesterona/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Sequência de Bases , Galinhas/genética , DNA/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Vírus do Tumor Mamário do Camundongo/genética , Dados de Sequência Molecular , Progesterona/metabolismo , Promegestona/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequências Repetitivas de Ácido NucleicoRESUMO
Intraspecific somatic cell hybrids between T-lymphoma cells and lymphocytes are highly tumorigenic whereas fusion of T-lymphoma cells with normal fibroblasts leads to reduced or even completely suppressed tumorigenicity of the hybrid cells. A particular cytogenetic phenomenon defines these two classes of hybrids. DNA replication analysis via bromodeoxyuridine pulse labelling reveals an aberrant banding pattern in the c-myc chromosomal domain in tumour cells and highly tumorigenic hybrids. In hybrids with suppressed tumorigenicity the tumor parent derived chromosomes have reverted to normal DNA replication banding. Aberrant DNA replication in tumour cells and highly tumorigenic hybrids coincides with enhanced c-myc expression. In hybrids with suppressed tumorigenicity and with normal DNA replication banding c-myc expression is also reduced. Thus, a correlation between aberrant DNA replication and enhanced expression of a gene located in the same chromosomal domain is observed. Reversion of aberrant DNA replication and reduction of c-myc expression to normal in hybrid cells may be due to a site-specific trans effect which overrides the control brought about in cis by retroviral insertion near the c-myc gene.
Assuntos
Replicação do DNA , Linfoma/genética , Oncogenes , Animais , Regulação da Expressão Gênica , Células Híbridas , Camundongos , Provírus/genética , Linfócitos TRESUMO
Trisomy 15 is described as a common finding in all T-cell leukemias induced by a single dose of methylnitrosourea (MNU) in BDF1 mice and in the leukemias induced by 7 doses of benzo(a)pyrene. Additional trisomies were found in about half of the leukemias. The organ distribution suggests that the leukemic cells with trisomy 15 originate in the thymus. Trisomy 15 was detected in the thymus as early as 6 weeks after the application of MNU, i.e. during the latency period.
