GSBS Dissertations and Theses

Title

Characterization of the Two Non-Allelic Preproinsulin Genes in Mice: a Thesis

Approval Date

8-1-1987

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Department of Molecular Genetics & Microbiology

Subjects

Proinsulin; Mice; Academic Dissertations

Abstract

The two non-allelic preproinsulin genes of the mouse have been cloned and their nucleotide sequences determined. The mouse preproinsulin I gene, like its rat counterpart, has only one intron. Homology between the two mouse genes extends in the 5' direction to about position -500. Homology 3' of the coding sequence terminates shortly after the polyadenylation signal with a dA rich region found in gene I.

The coding sequences of the two genes have been compared. The deduced amino acid sequences of the mature hormones are identical to the published protein sequences and to the corresponding sequences of rat insulins I and II. The prepeptides of mouse insulin I and II differ at six positions. However, they maintain hydrophobic cores that are required for transport of the nacent peptide across microsomal membranes. The B-peptide of mouse insulin I differs from insulin II at two positions: at position B9 a proline has replaced a serine, and at position B29 a lysine has replaced a methionine, compared to the sequence of insulin II. The A-peptides of the two hormones are identical. The C-peptide of mouse proinsulin I has a deletion eliminating amino acids C17 (Gly) and C18 (Ala) compared to the sequence of proinsulin II. The presence of this deletion in mature RNA was confirmed through an S1 nuclease assay.

The transcriptional start sites for the preproinsulin genes were determined with S1 and Mung Bean nuclease assays, and with a primer extension assay. The data indicate that transcription of the mouse preproinsulin genes starts 6 bp 5' of the site reported for the rat II gene.

Single-stranded DNA probes were used to determine the structure of the 3' ends of the preproinsulin mRNAs. Hybridization conditions were used which only allowed each probe to detect its cognate mRNA. Digestion of the resulting DNA-RNA duplex molecules with S1 nuclease followed by gel electrophoresis demonstrated that transcription of mouse preproinsulin I mRNA terminates 18 bases after the polyadenylation signal. Transcription of preproinsulin II mRNA terminates 43 bases after the polyadenylation signal, thus extending 25 bases past the last point of homology between the two genes.

The 3' end-specific probes were used in experiments designed to determine the ratio of preproinsulin I and II mRNA in pancreatic extracts of normal, fasted and fasted and refed mice. In all cases the amount of preproinsulin I mRNA exceeded preproinsulin II by about 2.3:1. These results were extended to include an analysis of preproinsulin mRNA from freshly isolated islets and islets incubated for 48 hours in the presence of 2.8 mM or 16.7 mM glucose. With both high and low glucose concentrations, the amount of preproinsulin I mRNA exceeded preproinsulin II by about 2.3:1. The mouse islets were also incubated with 3H-leucine and the ratio of insulin I and II determined after fractionation by HPLC. Unlike the mRNA results, the level of insulin II, within the islets and secreted into the media, exceeded insulin I by about 2:1 under all conditions.

The available preproinsulin prepeptide amino acid sequences have been compared. The sequence of mouse I prepeptide differs from most other insulin prepeptides at amino acid position 4. At that position a tryptophan residue that is conserved in most insulin prepeptides has been replaced by a leucine in the mouse I prepeptide. This change causes a shift in the hydropathy profile in that region of the mouse insulin I prepeptide making it more hydrophobic. Every other insulin prepeptide is relatively hydrophilic at that position. This difference is postulated to interfere with signal recognition particle mediated regulation of translation and/or transport of nacent mouse preproinsulin I to microsomal membranes, and nay account for the discordant mRNA-peptide ratios.

The structure of the insulin genes in a number of myomorph rodents has been examined. The data indicate that only members of the sub-family Murinae have two insulin genes.

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