title: Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters.
abstract: OBJECTIVE
  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 Pax6Aey11 and Pax6Aey18 mutants but not in Pax6ADD4802. 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 Pax6Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.
authors.affiliation: Institute of Developmental Genetics, GSF-National Research Center for Environment and Health, Neuherberg, Germany. graw@gsf.de
authors.forename: Jochen
authors.initials: J
authors.lastname: Graw
authors.forename: Jana
authors.initials: J
authors.lastname: Löster
authors.forename: Oliver
authors.initials: O
authors.lastname: Puk
authors.forename: Doris
authors.initials: D
authors.lastname: Münster
authors.forename: Nicole
authors.initials: N
authors.lastname: Haubst
authors.forename: Dian
authors.initials: D
authors.lastname: Soewarto
authors.forename: Helmut
authors.initials: H
authors.lastname: Fuchs
authors.forename: Birgit
authors.initials: B
authors.lastname: Meyer
authors.forename: Peter
authors.initials: P
authors.lastname: Nürnberg
authors.forename: Walter
authors.initials: W
authors.lastname: Pretsch
authors.forename: Paul
authors.initials: P
authors.lastname: Selby
authors.forename: Jack
authors.initials: J
authors.lastname: Favor
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
authors.forename: Martin
authors.initials: M
authors.lastname: Hrabé de Angelis
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005038:Ethylnitrosourea / Q000633:toxicity; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics*; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008850:Microphthalmos / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097607:Homeobox Protein SIX3
chemical_list: D005038:Ethylnitrosourea; D005136:Eye Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; D012097:Repressor Proteins; D000097607:Homeobox Protein SIX3; C000605732:Pax6 protein, mouse