Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters

Item Type:	Article
Title:	Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters
Creators Name:	Graw, J. and Loester, J. and Puk, O. and Muenster, D. and Haubst, N. and Soewarto, D. and Fuchs, H. and Meyer, B. and Nuernberg, P. and Pretsch, W. and Selby, P. and Favor, J. and Wolf, E. and de Angelis, M.H.
Abstract:	PURPOSE. To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs. METHODS. Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients. RESULTS. The new mutations-ADD4802, Aey11, and Aey18- were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C→T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G→A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G→A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6 Aey11 and Pax6Aey18 mutants but not in Pax6 ADD4802. This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aey11 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6ADD4802 allele stimulated both promoters. CONCLUSIONS. Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6Aey11 and Pax6 Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.
Keywords:	Alleles, Amino Acid Sequence, Base Sequence, Ethylnitrosourea, Eye Proteins, Homeodomain Proteins, Inbred C3H Mice, Indirect Fluorescent Antibody Technique, In Situ Hybridization, Messenger RNA, Microphthalmos, Molecular Sequence Data, Mutagenesis, Mutation, Nerve Tissue Proteins, Paired Box Transcription Factors, Phenotype, Promoter Regions, Repressor Proteins, Reporter Genes, Reverse Transcriptase Polymerase Chain Reaction, Animals, Mice
Source:	Investigative Ophthalmology & Visual Science
ISSN:	0146-0404
Publisher:	Assoc Research Vision Ophtamology Inc
Volume:	46
Number:	12
Page Range:	4671-4683
Date:	1 December 2005
Official Publication:	https://doi.org/10.1167/iovs.04-1407
PubMed:	View item in PubMed

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