| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1988: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1987: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
I have previously characterized the NON mice as an animal model of human non-obese NIDDM, and suggested that the glucose intolerance in the animal was mainly due to the abnormal function of pancreatic B cells.
In the present research project, I examined the hypothesis that the glucose intolerance in the NON mice is caused by the reduced expression before the translation on account of structural changes in the insulin gene of the NON mice through the determination of its nucleotide sequence.
1. Construction of the genomic library of the NON mice : High-molecular-weight genomic DNA was isolated from the mouse liver, and partially digested with Lamda Dash/BamHI "arms", assembled into phages, and 1.1x10^6 pfu of genomic library was constructed.
2. Isolation and subcloning of the insulin genes from the genomic library of the NON mice : 6x10^5 pfu of phages were screened by plaque hybridization using rat preproinsulin I cDNA as a probe. 5 positive clones consisted of the two independent clones,
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iambdaNins I and iambdaNins II. They were digested with EcoRI and BamHI, and subcloned into Bluescript KS (+) plasmid.
3. Determination of nucleotide sequence of the insulin genes : After the preparation of deletion mutant from the two subclones, pNins I and pNins II, DNA sequencing was accomplished by the dideoxy chain termination method using synthetic primers to T7 promoter and [alpha-^<35>S] dATP.
It was confirmed that 1.4kbp pNins I and 2.5kbp pNins II contained full length of the NON mouse preproinsulin I and II gene respectively. The nucleotide sequence of the NON mouse preproinsulin I gene including its 5' transcriptional regulatory region was completely identical with that of BALB/c mouse gene. In the structure of the NON mouse preproinsulin II gene, Its 5' transcriptional regulatory region (about 350bp upstream from the transcription initiation site), exons, and introns are completely identical with those of BALB/c mouse gene. But in contract with the preproinsulin I gene, the NON mouse preproinsulin II gene had an additional 113bp sequence that the BALB/c mouse didn't have at the further 5' upstream site, 7 point mutations near the BamHI subcloning site, and 3 point mutations in the 3' flanking region.
In the present project, I was able to determine the nucleotide sequence of the NON mouse preproinsulin genes, and got an interesting finding of an additional 113bp sequence in the NON mouse preproinsulin II gene with regard to whether it would affect the expression of the gene as a cis-acting transcriptional regulatory factor. Less
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