Exon

About: Exon is a research topic. Over the lifetime, 38308 publications have been published within this topic receiving 1745408 citations. The topic is also known as: exons.

Normal Human Tissues, in Addition to Some Tumors, Express Multiple Different CD44 Isoforms

Abstract: At least 20 different isoforms of the human CD44 lymphocyte-homing receptor/hyaluronan receptor have been described to date that arise from the differential splicing of up to 10 alternative exons (termed v1-v10) encoding the membrane-proximal extracellular domain. Although numerous analyses at the mRNA level have indicated tissue-specific expression of CD44 variants, few analyses have been performed at the protein level because of limited availability of suitable monoclonal antibodies. Recently, however, exon-specific monoclonal antibodies have been generated using bacterial fusion proteins, and these have been reported to detect high levels of vCD44 containing the v6 exon on human tumors. Together with earlier evidence linking this particular exon with tumor metastasis in the rat, these latter experiments have led to the interpretation that v6 splice variants play a causative role in tumor dissemination. In this paper we describe the use of a new and comprehensive panel of CD44 exon-specific monoclonal antibodies generated against a recombinant CD44(v3-10)-immunoglobulin chimera to study vCD44 expression in a large number of normal and neoplastic tissues. We show that the expression of vCD44 varies greatly among different human tumors and that some express either very low levels of vCD44 or no CD44 at all. Furthermore, we demonstrate that expression is not limited to isoforms containing the v6 exon but includes variants carrying v3, v4/5, and v8/9. Additionally, normal epithelial tissues are shown to express considerable levels of these same vCD44 isoforms. Such results argue against a ubiquitous role for vCD44 isoforms in promoting tumor growth and metastasis.

Functional significance of different human CYPlAl genotypes

Abstract: At least two different polymorphisms in the human CYP1A1 gene have been associated with an increased risk for tobacco-related lung cancer; however, the functional significance of these polymorphisms has not been determined. We measured CYP1A1 genotypes, gene expression levels and enzymatic activity levels in mitogen-stimulated lymphocytes to determine whether genetic polymorphisms in CYP1A1 alter transcriptional and/or post-transcriptional regulation of the gene. Genotypes were determined at two sites previously associated with lung cancer: a point mutation in exon 7 near the catalytic region of the enzyme and an Msp1 RFLP in the 3' non-coding region of the gene. Variant genotypes at the Msp1 site had no effect on CYP1A1 gene induction, however, variant genotypes at the exon 7 site were significantly associated with increased CYP1A1 gene inducibility. We also observed a significant interaction between the exon 7 polymorphism and smoking on mRNA levels. There was a 3-fold elevation in CYP1A1 enzymatic activity in exon 7 variant genotypes. When Msp1 and exon 7 genotypes were combined, there was an increased CYP1A1 inducibility and enzymatic activity in subjects with the exon 7 polymorphism, and in subjects with both polymorphisms.

Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein.

Abstract: Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.

Saturation editing of genomic regions by multiplex homology-directed repair

Abstract: Saturation mutagenesis--coupled to an appropriate biological assay--represents a fundamental means of achieving a high-resolution understanding of regulatory and protein-coding nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or "safe-harbour" integration fail to capture the native context of the endogenous chromosomal locus. This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/Cas9 RNA-guided cleavage with multiplex homology-directed repair using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a six-base-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences of large numbers of mutations with saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.