Characterization of the Gene and Messages for Vasoactive Intestinal Polypeptide in Rat and Mouse: a Thesis

Abstract

The organization and transcription of the gene for vasoactive intestinal polypeptide (VIP) in rats and mice was investigated using northern- and Southern-blot hybridizations, selective genomic cloning, Sl-nuclease protection assays, oligonucleotide-directed RNase H digestions, and genomic cloning by standard methods. The center of the rat VIP gene and the entire mouse gene were cloned and sequenced. Selective genomic cloning was used to isolate a strongly-hybridizing fragment of the rat VIP gene identified in Southern-blot hybridizations with an existing human VIP cDNA. This fragment contains separate exons encoding VIP and a closely related neuropeptide, peptide histidine-isoleucine (PHI-27). This organization is the same in the mouse gene, which bears a total of seven exons and a close similarity to the human gene for VIP. Although the arrangement of exons suggested that VIP transcripts could be subjected to differential splicing to alter the coding capacity of the final messages, no evidence was found for this possibility. Two bands were seen in northern-blot hybridizations, but exon-specific probes and Sl-protection experiments provided evidence that they differed not in their coding regions but in the extent of their 3'-untranslated ends. RNase H digestions targeted to specific portions of transcripts from the VIP gene were used to resolve the principal band, to demonstrate that it represented a single species of message with sequence from both the VIP and PHI exons. In the course of the characterization of the murine VIP gene, a new method was developed for generating subclones for DNA sequencing in M13 bacteriophage. The central feature of the partial deletion subcloning method is its employment of frequent-cutting restriction endonucleases to detach different extents of the insert from a construction. The viral construct is first linearized at a unique site between the insert and the site for hybridization of the M13 sequencing primer. The linearized construct is then subjected to partial digestion with different frequent-cutting restriction enzymes. Partially digested products are repaired and religated. Products with deletions from the insert now have the sequencing priming site religated to a portion of the insert that formerly had been distant. Most of the products with deletions in the viral genome are not viable and do not survive the procedure. Subclones are sorted from the pool of transfected products by sizing of single-stranded viral DNA by agarose gel electrophoresis. Selected subclones are subjected to a simple test for the presence of the sequencing priming site. With this method and its associated tests, a variety of restriction enzymes can be used to generate a spectrum of deletion subclones for sequencing. In a simple trial of this method with an unknown 3.3 kilobasepair cDNA, a set of subclones was generated to allow sequencing to span the cDNA in one direction.