Progress 10/01/05 to 09/30/06
Outputs
Aim #1: Develop cichlid crosses between species with different adult expression patterns and quantify opsin expression. Cichlid fishes have seven cone opsin genes which are spectrally distinct. This includes SWS1, SWS2b, SWS2a, RH2b, RH2a1, RH2a2 and LWS. Typical cichlid adults express only 3 or 4 of these though species differ in the combination they use. Our previous Hatch project identified two species which differed in opsin expression and made a cross between them. This cross, Copadichromis eucinostomus x Dimidiochromis compressiceps, showed that opsin expression is controlled by only a few genes. We have now developed two more crosses which differ in opsin expression, Metriaclima zebra x Metriaclima zebra gold and Aulonocara baenschi x Tramitichromis intermedius. We have sampled retina for over 60 F2 individuals from the Metriaclima cross. The other cross is still being bred to generate F2. Both crosses will prove useful for mapping studies. Aim #2: Course scale
mapping to identify how many genetic factors are controlling opsin expression. Using the F2 cross between C. eucinostomus and D. compressiceps, we have utilized markers which are very close or within each of the opsin genes to test for linkage of cis regulatory regions surrounding the opsin genes with opsin gene expression. We have now used markers that are within intron 3 of the SWS1 gene, one 300 bp upstream of the RH2b-Rh2a1-RH2a2 cluster and one 20 kb from the SWS2-LWS cluster. These do not show linkage with cone opsin expression. This suggests that opsin expression differences are not the result of cis acting factors. To look for trans acting factors, we have performed a genome scan to try to identify genomic regions which contribute to the control of opsin expression. We have now identified one region which is on tilapia LG 13. Four markers show linkage to SWS1 opsin expression. We plan to add more markers to more tightly locate the region.
Impacts
We have identified one factor which may be important in opsin expression and so help in development of gene therapies for retinal disease.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
1. Identifying opsin regulatory regions. We have sequenced the BAC clones containing the cichlid opsin genes and confirmed that cichlid fishes have seven cone opsin genes. This was done in collaboration with DOEs Joint Genome Institute. By expressing these genes in vitro and reconstituting the corresponding visual pigments, we can relate gene sequence with visual pigment function. Each of the seven genes produces a spectrally distinct visual pigment (SWS1-360nm, SWS2b-423nm, SWS2a-456nm, RH2b-472nm, RH2ab-518nm, RH2aa-528nm and LWS-561nm). These opsins are differentially expressed through development in tilapia with each gene being turned on at some developmental stage (Spady et al submitted). The opsins fall into clusters in the genome. The SWS2a-SWS2b-LWS gene cluster is on LG 5. The RH2b- RH2ab- RH2aa cluster is also on LG5 but is approximately 20 cM from the SWS2-LWS cluster. The SWS1 gene is on LG 17. Regulatory regions important for control of opsin gene
expression have been examined. In this genomic footprinting, we compared the cichlid BAC sequences with other fishes (zebrafish, medaka, tetraodon, fugu) for whom large scale genome sequences have been done. This has identified genomic regions 2-3 kb upstream of each of the opsins which are candidate regulatory regions. 2. Mapping of cis and trans acting factors in crosses between species differing in opsin expression. We have identified two Lake Malawi cichlids that differ greatly in cone opsin gene expression. D. compressiceps expresses SWS2a, RH2a and LWS genes while C. eucinostomus expresses SWS1, RH2b and RH2a genes in the adults. In order to map the genomic location of factors controlling the SWS1-SWS2a and RH2b-LWS expression differences, we have made an F2 cross between these two species. Real time RT-PCR was used to quantify opsin expression from both eyes of each individual. The distribution of SWS1 expression amongst the individuals is broad with several individuals having
close to the parental gene expression values. Using the Castle Wright estimator of the number of genes controlling SWS1 gene expression yields an estimate of 1.9 genes controlling the switch in gene expression. This is a lower bound and relies on the simplifying assumptions of additivity and lack of environmental variance. Since a small number of genes likely control this difference, we tested whether markers associated with any of the opsin clusters was a controlling factor. Individuals were genotyped for three markers: one in the SWS1 gene, one 20 kb from the SWS2-LWS cluster and one 3 kb upstream of the RH2 cluster. The individuals were grouped by genotype and analyzed by ANOVA to look at the difference in SWS1 expression between the different groups. For the SWS1, SWS2-LWS and RH2 markers, there was no linkage between genotype and phenotype (p=0.34, p=0.78, and p=0.35 respectively). Because of the small number of individuals analyzed, we can not rule out the presence of regulatory
regions cis to the opsin genes. However, this demonstrates that trans acting factors elsewhere in the genome must contribute to the shift in expression between the species.
Impacts
We have determined that trans acting factors are critical to the control of opsin gene expression. We are now working to identify these factors using a whole genome scan with microsatellite markers.
Publications
- Carleton KL, Spady TC, and Cote RH. 2005. Rod and cone opsin families differ in spectral tuning domains but not signal transducing domains as judged by saturated evolutionary trace analysis. J Molecular Evolution 61: 75-89.
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Progress 10/01/03 to 09/30/04
Outputs
1. Identifying opsin regulatory regions: Cichlids have five classes of cone opsin genes: SWS1, SWS2a, SWS2b, RH2 and LWS. We have identified contigs of BAC clones containing these opsin genes in the cichlid fish tilapia. The genes fall in three contigs: one containing the SWS1 gene, another containing the RH2 gene, and a third containing SWS2a, SWS2b and LWS. One clone for each contig has been shotgun sequenced (10x coverage) by the Joint Genome Institute (JGI). We are in the process of finishing these clone sequences but already have considerable regulatory sequence for analyses. The long range sequence provided by these opsin BAC clones will be useful for several reasons. First, they can be used in phylogenetic footprinting in comparisons with other fishes such as zebrafish, fugu and medaka. This will identify conserved regulatory regions that are important to opsin expression in all fishes. Second, these sequences will enable us to resequence regulatory regions in
closely related cichlid fishes differing in cone opsin expression. These studies will identify regulatory elements that are important to cone opsin expression. 2. Mapping of cis and trans acting factors in crosses between species differing in opsin expression: We have identified two Lake Malawi cichlids that differ greatly in cone opsin gene expression. D. compressiceps expresses SWS2a, RH2 and LWS genes while C. eucinostomus expresses SWS1, RH2 and LWS genes in the adults. These species therefore differ only in SWS1-SWS2a expression. In order to map the genomic location of factors controlling this expression difference, we have made an F2 cross between these two species and generated 33 individuals which were sampled at day 15. Real time RT-PCR was used to quantify opsin expression from both eyes of each individual. The distribution of SWS1 expression amongst the individuals is broad with several individuals having close to the parental gene expression values. Using the Castle Wright
estimator of the number of genes controlling SWS1 gene expression yields an estimate of 1.9 genes controlling the switch from SWS1 to SWS2a in gene expression. This is a lower bound and relies on the simplifying assumptions of additivity and lack of environmental variance. Since a small number of genes likely control this difference, we tested whether either of the cone opsin genes (SWS1 or SWS2a) was a controlling factor. Genomic DNA was made from each individual and used to genotype those individuals for a marker in the SWS1 gene, and another marker close to SWS2a. The individuals were grouped by genotype and analyzed by ANOVA to look at the difference in SWS1 expression between the different groups. For both the SWS1 and SWS2a markers, there was no linkage between genotype and phenotype (p=0.34 and p=0.78 respectively). Because of the small number of individuals analyzed, we can not rule out the presence of regulatory regions cis to the opsin genes. However, it seems clear that
there are trans acting factors elsewhere in the genome which contribute to the shift in expression from SWS1 to SWS2a.
Impacts
Opsins are the first proteins in the signaling transduction cascade which convert light into a neural output of the retinal photoreceptors. Because of their critical function, misexpression of opsins or mutation at sites which are key to opsin function and signal transduction lead to photoreceptor cell death and often blindness. In this work, we hope to identify the mechanisms by which opsin gene expression is controlled as well as the sites which are critical for opsin function. This would enable molecular testing to identify the cause of disease, but could also lead to cures through gene rescue therapy.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
The project has focused on two areas of research during its first year. The first is the genetic control of opsin gene expression. The second is the molecular basis for the difference between rod and cone phototransduction. To examine the cis regulatory elements of the rod and cone opsin genes, we are utilizing the cichlid fish tilapia. Using real-time RT-PCR, we have shown that this species differentially expresses five cone opsin genes through the first six months of development. To identify the regulatory elements for these opsin genes, we have screened a tilapia BAC library to identify clones that contain each of the opsin genes. This library has been recently fingerprinted and contains 3500 contigs. We then used this information to identify the corresponding contigs. For each contig, all clones were PCR screened for the opsin genes and sized by pulsed field gel electrophoresis. BAC end sequences were also obtained from these clones and primers designed to confirm
the physical arrangements of these clones. We hope at the end of this process to identify individual BAC clones which contain the maximum sequence surrounding the opsin genes for shotgun sequencing. This information will be used in comparisons with other model fishes (fugu and zebrafish) for which we have isolated regulatory sequence. In examining the molecular basis for the difference between rod and cone phototransduction, we have utilized evolutionary trace analysis. By comparing an evolutionarily diverse group of sequences for both the rod and cone proteins, we hope to identify the molecular mechanisms by which the rod and cone electrophysiological responses to light differ. In our first study, we have examined 182 rod and cone opsin sequences available in Genbank (Carleton and Cote, submitted). In this work, we have identified sites which distinguish each of the five opsin classes (one for rods and four for cones). Several of these sites are in the transmembrane regions, directed
into the retinal binding pocket, and involve chances in amino acid polarity or charge. These sites are likely important for tuning the opsin classes for sensitivity over the range of visible wavelengths. In comparing the cytoplasmic domains where opsins interact with other phototransduction genes (transducin, G protein kinase and arrestin), we determined these regions to be highly conserved with strong identify between the rod and cone opsins. This suggests that opsins have been selected for spectral sensitivity, but that there is no difference in the interactions of opsins with the rest of the phototransduction cascade. These results support recent experimental evidence which makes similar findings. In addition, we have compared the conserved sites in the opsins with those sites known to cause disease. Over 80% of the disease sites are identical in all opsins or in the opsin class known to cause the disease. This suggests that ET analysis is useful for identifying functionally
important sites which when mutated cause disease.
Impacts
Opsins are the first proteins in the signaling transduction cascade which convert light into a neural output of the retinal photoreceptors. Because of their critical function, misexpression of opsins or mutation at sites which are key to opsin function and signal transduction lead to photoreceptor cell death and often blindness. In this work, we hope to identify the mechanisms by which opsin gene expression is controlled as well as the sites which are critical for opsin function. This would enable molecular testing to identify the cause of disease, but could also lead to cures through gene rescue therapy.
Publications
- No publications reported this period
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