RESUMEN
Survivin is a member of the inhibitor apoptosis family that is overexpressed in many malignancies. It has five known alternative splice forms, some of which differ in their antiapoptotic properties and expression levels in human cancers. Here we describe a novel donor splice site (DSS), 2B+32 DSS, which is used in conjunction with survivin alternative exon 2B, resulting in the inclusion of 32 additional nucleotides from intron 2 at the 3' end of this exon. Sequence analysis showed that both the classical exon 2B DSS and 2B+32 are provided by an Alu sequence, which is inserted in intron 2 downstream of a functional acceptor splice site, leading to the exonization of part of the repetitive element. Minor transcripts including the 2B+32 alternative exon, or retaining the whole intronic region comprised between exons 2B and 3, were detected in several human cell lines and in some human tissues. Survivin 2B+32 containing variants acquire a premature stop codon (PTC) and may therefore be degraded by the nonsense mediated decay pathway. The implication of these novel isoforms, as well as other PTC+ survivin variants, in the overall regulation of survivin expression is discussed. Sequence analysis of intron 2 which contains the Alu Y element was performed on different primate species in order to trace its insertion and exonization during primate evolution.
Asunto(s)
Empalme Alternativo , Elementos Alu , Exones , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Primates/genética , Isoformas de Proteínas/genética , Animales , Secuencia de Bases , Línea Celular , Evolución Molecular , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Homología de Secuencia de Ácido Nucleico , SurvivinRESUMEN
In situ hybridization (ISH) for JC virus (JCV) is generally applied for the diagnosis of progressive multifocal leukoencephalopathy (PML). To explore the usefulness of immunohistochemistry (IHC) for JCV early proteins, 14 paraffin-embedded postmortem brain specimens with histologic features compatible with PML were tested for the presence of JCV by means of DNA-DNA ISH with a biotinylated probe corresponding to the entire JCV genome, for JCV early proteins IHC with both PAb 2003 and anti-SV40 large T antigen monoclonal antibodies, and polymerase chain reaction (PCR) amplification of JCV virion protein 3 (VP3) and transcriptional control region (TCR) sequences. ISH was positive in 13 cases and IHC in all 14 cases, the number of IHC-positive cells generally being far in excess of ISH-positive cells. Of the 2 monoclonal antibodies used, PAb 2003 proved to be more sensitive than anti-SV40 large T antigen. Occasional neuronal nuclei were positive for JCV early proteins in 5 cases. As for PCR, VP3 was amplified in all 14 cases and TCR in 9 cases. Consequently, PAb 2003 IHC for JCV early proteins seems to be a powerful tool for viral demonstration in PML and may well become the diagnostic recourse of choice in this setting.