a subject that still needs to be studied much more, particularly because the relative paucity of cones in the mammalian retina has made their study difficult and has left behind the understanding of the molecular nature of our most used photoreceptors. In fact, very few cone genes have been characterized in detail and the functional significance of the majority of these genes remains unknown.

One of our goals has been precisely to isolate and characterize novel genes expressed in cones. We have already studied a few of these genes (i.e., Viczian et al. 1995; Ahkmedov et al. 1997; Ahkmedov et al. 1998; Reid et al. 1999; Akhmedov et al. 2002) and are currently involved in the characterization of ZBED4, a gene isolated by microarraying the output of the second round of Representational Difference Analysis using the mRNA from adult cd dog retina (that has lost a large number of its cones due to an autosomal recessive cone degeneration) and normal adult dog mRNA.

10.2 Methods and Results

The human ZBED4 gene contains two exons. The first exon (169 base pairs) is a part of the 5 UTR; the second exon contains the rest of the 5 UTR, the complete open reading frame and the 3 UTR. After cloning and sequencing the complete ZBED4 transcript, we confirmed that the generated sequence encodes a putative DNA-binding protein with several DNA binding domains and a dimerization domain (Fig. 10.1). In addition, human ZBED4 contains two LXXLL motifs, which are found in proteins that function as co-activators of nuclear hormone receptors. The presence of these motifs suggests a co-activator function for ZBED4. Overall, human, dog, mouse and rat ZBED4 proteins share 81–82% homology while chicken only exhibits 62% homology with the human counterpart.

10.2.1 ZBED4 mRNA is Expressed in Human Retina

Northern blots using human retinal RNA and a human 3 UTR ZBED4 probe revealed two transcripts, 5.0 and 7.0 kb long. Both of these transcripts encode the same ZBED4 protein. The blot was also hybridized with a 5 UTR/coding region probe. In this case, three transcripts were detected (5.0, 7.0 and 4.0 kb). All these mRNAs have been recently reported in the databases for ZBED4.

10.2.2 ZBED4 mRNA is Expressed in Mouse and Human Cones

Studies using real time PCR on mouse cone cells sorted by activated flow cytometry revealed the expression of ZBED4 in cone photoreceptors. Dissociated mouse retinal cells were incubated with FITC-conjugated peanut agglutinin (PNA). PNA binds preferentially to galactosyl (ß-1, 3) N-acetylgalactosamine, which is present only in

Fig. 10.1 (a) Diagrammatic representation of the genomic organization of human ZBED4, its corresponding mRNA and the conserved domains of the encoded protein; these domains are numbered: the zinc BED fingers (1–4) are shown as light gray boxes and the dimerization domain (5) as a dark gray box

the matrix surrounding cone photoreceptors, but not in that surrounding rods. FITClabeled cone cells were then sorted by FACS (Fig. 10.2a) and their total RNA as well as that of the dissociated retinal cells was obtained. Figure 10.2b shows the results of the quantitative PCR experiments using these RNA samples and appropriate primers for ZBED4, cone PDEα , and rod PDEα mRNAs. As we anticipated, the level of rod PDEα in the cone enriched cell fraction was barely detectable since PDEα is a rodspecific enzyme, whereas that of PDEα mRNA was about 29-fold more abundant than PDEα in the cone enriched cell fraction. Accordingly, ZBED4 mRNA was also enriched by about 16-fold compared to PDEα mRNA.