Gnb1 spans at least 50 kb of genomic DNA and consists of 12 exons and 11 introns. The coding sequence starts in exon 3.
20 Our FISH results showed that in the
Rd4/+ mouse the breakpoint is located in intron 2 of
Gnb1. Therefore, its 5′-untranslated region (UTR) is interrupted and separated from the rest of the gene. Because of this interruption/separation, we hypothesized that expression of the
Rd4 Gnb1 allele would be completely shut down. To test this, the developmental expression patterns of
Gnb1 and other retina-specific mRNAs were measured by Northern blot analysis in normal and
Rd4/+ retinas. There were no evident differences in expression levels of
Gnat1 and
Rho mRNAs between
Rd4/+ and C57BL/6J retinas at postnatal day (P)8 and P11
(Fig. 6) . However, from P16, mRNAs for these two genes drastically decreased in
Rd4/+ retinas, correlating with the photoreceptor loss that characterizes the
Rd4/+ retina at this postnatal age.
11 In contrast, expression of
Gnb1 mRNA in
Rd4/+ relative to C57BL/6J control retinas was reduced by approximately 50% at P8 and P11
(Fig. 6) , suggesting haploinsufficiency due to the absence of expression of the
Rd4 Gnb1 allele, but normal expression from the normal
Gnb1 allele. These results were confirmed by real-time quantitative RT-PCR. The results show that the expression level of
Gnb1 mRNA in
Rd4/+ retina is 58% of wild-type at P8, and 36% at P11 (
Fig. 7 , top). For the
Gnat1 and
Rho transcripts, there is no significant difference in expression at P8 (
Fig. 7 , middle and bottom, respectively). Because the
Rho primer pair used for real-time quantitative RT-PCR detects two transcripts (2.9 and 3.5 kb) of five, the relative quantity of
Rho gene measured in adult retinas in these experiments is only 20- to 25-fold that obtained at P8. To examine for the possible presence of transcripts fused with the inverted
Gnb1 promoter-exon 1 and 2 sequence, we used a cDNA probe comprised of exons 1 to 7 for Northern blot analysis. No other transcripts were observed from
Rd4/+ retina after a 5-day exposure to x-ray film (data not shown).