Alternative splicing of mGlu6 gene generates a truncated glutamate receptor in rat retina.

A novel splice variant of metabotropic glutamate receptor type 6 (mGlu6 receptor) was identified by reverse transcription-polymerase chain reaction amplification and sequence analysis of rat retina cDNA. The new rat receptor isoform (mGlu6b receptor) is characterized by an additional exon of 88 nucleotides containing an inframe stop codon, thus predicting the expression of a truncated protein of 508 amino acids. In situ hybridization reveals mGlu6b receptor mRNA to be predominantly expressed in the outer part of the inner nuclear layer of rat retina, containing ON-bipolar cells. The mGlu6b protein would comprise the extracellular domain of the receptor containing the ligand-binding site, but would lack the transmembrane and intracellular portions, thus possibly acting as a retinal soluble receptor for glutamate.

Identification of novel alternatively-spliced mRNA isoforms of metabotropic glutamate receptor 6 gene in rat and human retina.

Novel splice variants of metabotropic glutamate receptor type 6 (mGlu6 receptor) were identified by reverse transcription-polymerase chain reaction (RT-PCR) amplification and sequence analysis of rat and human retina cDNAs. The new rat mGlu6 receptor mRNA isoform is characterized by an additional exon of 88 nucleotides containing an in frame stop codon, thus predicting the expression of a truncated protein of 508 amino acids. The human retina was found to express two different mGlu6 receptor mRNA variants: one lacking 97 nucleotides from exon 6, the other including five nucleotides of intron 5. These mRNAs would encode truncated receptors of 425 and 405 amino acids, respectively. Both in rats and in humans, the truncated mGlu6 receptor proteins would comprise the extracellular domain but lack the transmembrane and intracellular portion of the receptor, thus possibly acting as retinal soluble receptors for glutamate. Though generated by different patterns of alternative splicing, the inter-species conservation of truncated mGlu receptor molecules strongly suggest their relevance in the regulatory network of glutamatergic neurotransmission.

Three homozygous PTC mutations in the collagen type VII gene of patients affected by recessive dystrophic epidermolysis bullosa: analysis of transcript levels in dermal fibroblasts.

The Hallopeau-Siemens variant of recessive dystrophic epidermolysis bullosa (HS-RDEB) is a severe inherited skin disease characterized by the absence of collagen type VII (COLVII) and anchoring fibrils (AF), caused by mutations in collagen type VII gene (COL7A1). Mutations leading to the formation of premature termination codons (PTCs) of translation are the characteristic genetic lesions in HS-RDEB patients; many PTC mutations have been found to be associated with a marked reduction or complete absence of COLVII mRNA. In this article, we report homozygosity for three different mutations in the COL7A1 of HS-RDEB patients. One mutation, the R2685X, falling in exon 109, is a novel mutation, whereas the other two, the 425A-->G falling in exon 3 and the 497insA in exon 4, have been previously identified in compound heterozygosity with different mutations in other unrelated RDEB patients. Haplotype analysis in three Italian families carrying the 497insA mutation suggested a common origin of this mutation and indicated that this is an ancestral Italian mutation. All these mutations generate PTCs and are associated with the absence of COLVII expression, as detected by immunofluorescence analysis of the patient's skin. Evaluation of the levels of the mutated COLVII mRNAs in cultured skin fibroblasts of the patients and of their parents showed that all the mutated transcripts were expressed at consistent levels. Therefore, our results indicate that a marked mRNA reduction is not a constant feature associated with PTC mutations in COL7A1.

The type III-9 repeat of human fibronectin is encoded by a single exon which is not alternatively spliced.

Type III homologies of human fibronectin are generally encoded by two exons, with the exception of the ED-A and ED-B repeats which are encoded by a single exon undergoing alternative splicing. We report that also the type III-9 homology is encoded by a single exon. Further more, RT-PCR analysis, performed on mRNA purified from fetal and adult tissues and from normal and tumor-derived cell types, showed that the III-9 region is not undergoing alternative splicing in all samples tested.

One-lane chemical sequencing of 3'-fluorescent-labeled DNA.

A method for DNA sequencing that combines limited chemical degradation of 3'-fluorescent-labeled DNA with densitometric analysis using an automatic sequencer is presented. The DNA sequence is determined in a single electrophoretic lane by monitoring the intensities of bands representing products of cleavage at the four bases obtained by solvolysis in hot aqueous piperidine (10%) followed by treatment with hot formamide. An application of the method for the detection of point mutations is reported.