Introduction

332 R.W. Beuerman, L.K. Goh and V.A. Barathi

A Brief Introduction to Comparative Genomics

It may not be immediately clear as to what can be accomplished by finding genes in animals associated with experimental myopia. In this “omics” era, the number of genes in a species has been bantered about as something associated with the more advanced the organism, which we normally consider to be humans and their closest relatives, the apes and other nonhuman primates. Indeed, the number of human genes had been variously projected to be from 20,000 to over 100,000. Now it is fairly clear that mammals have about 30,000 protein encoding genes,2 which brings parity to the initial consideration of the genome of apparently diverse species such as the mouse and human. To make full use the mouse genome to uncover homologies, an important goal has been to delineate and develop catalogs of the protein encoding genes of several species. Recently, more interest has developed in the chicken genome due to its prominence in agriculture as well as biological sciences.3 However, it can be said that the chicken genome is smaller than either the mouse or human, but there are 39 chromosome pairs, with 20 in the mouse and 23 in humans.4,5 Functional gene encoding of proteins is, however, important for the value of animal models, as the somatic mutation, unless aided by a specific knock-out, will be missing. With some understanding of what protein similarities can be expected, the animal model becomes more or less useful. By comparison with other species, such as mouse and chicken where the emphasis is on similarities between the species with the human genome, work on the non-human primate genome, and in particular the chimpanzee, has concentrated on differences. The genome is about the same size, but other great apes have an additional pair of chromosomes, 48 compared to 46 for humans, but other monkeys have a variable number of chromosomes. Protein homologs were found to differ by on average only two amino acids. A recent study found that the genome-wide nucleotide divergence between chimpanzee and human was only 1.23%.6

Comparative Expression

The foregoing points to the fact that gene numbers are not the source of the phenotypes between mammals but rather differences in their expression at the protein level. The complexities multiply at the protein level; gene protein interactions can control genes, and one to several proteins

333 Gene Analysis in Experimental Myopia

can be produced by adjunct mechanisms such as alternative splicing or post-translational processing. Clearly, these mechanisms that control the eventual final expression are species specific. However, genes act in networks, as the gene products activate other genes, which often remain poorly defined even after a gene is defined, and in experimental models of myopia, the work most often concentrates on particular proteins associated with some specific aspect of the biological process. Therefore, despite the knowledge of all potential gene homologies and numbers, it is an additional task to examine and to correlate the transcriptome between species. Due to the utilitarian value of the mouse, much of the effort has been to examine transcription between these two species. When a panel of 79 human and 61 mouse tissues were subjected to custom expression arrays that included over 44,000 unique human and 36,000 mouse transcripts, the authors found that 52% and 59% of human and mouse transcripts were found in at least one tissue.7 The comparison of the human and mouse genome is not yet finished and a recent innovative approach began with a gene discovery method starting with a statistical analysis of sequence alignment for gene prediction.8

However, with the vast amount of public databases, it has been necessary to examine these for consistency of the representation of genes, transcripts, and proteins between human and mouse. A large-scale network, the collaborative consensus coding sequence (CCDS) project has identified 20,159 human and 17,707 mouse consensus coding regions from approximately the same number of genes.9 This new CCDS database found that at least 77% of mouse genes have a homologous human gene and should be a major resource for those working on myopia and who intend to make available for human gene searches the results from mouse studies.

While there are apparently about 2.5–3 billion base pairs distributed among about 30,000 genes in mammals, there are significant phenotype differences between the mammalian species in general and those used for myopia studies. However, there are some differences in the number and homologies of genes, which may also contribute to the inter-species differences along with complexities due to factors such as changes in duplicated genes, alternate splicing, and post-translational modifications, which are not universal across mammals. Therefore, an important origin of these differences must be examined in the makeup of the genes, and the ultimate form their expression-protein products. A triplet base sequence provides the amino acid code or codon that is universal, but for most

334 R.W. Beuerman, L.K. Goh and V.A. Barathi

amino acids there is more than one codon. Moreover, mitochondrial coding is different.

The strategy then becomes directed toward making information from experimental myopia studies that contribute specific genes of functional interest, which could be passed along to those working with human samples to provide data for more focused candidate gene studies.

Genes in Retina and Sclera in Animal Models of Myopia

The finding of Zhou and Williams (1999) that eye weight is a heritable trait in various species of mice was independent of body weight or brain size, but the correlation to the retinal area was high.10 Within the 50 strains of mice that were examined, both overall weight and lens weight, as well as the number of retinal ganglion cells showed strain specific variations. As the mouse eye continues to grow past sexual maturity, a mouse opportunities to examine and modify growth with correlative molecular and cellular data could be presented. A somewhat similar study in three strains of chickens found that eye size did not vary, but induced changes in axial length due to posterior chamber elongation varied between strains.11 However, the strain differences appeared to be overcome by within from species differences.

ZENK (EGR-1)

Early gene expression studies in association with experimental myopia were all centered on the chick model as that has been prominent and primarily focused on using mRNA and Northern blots of retina for analysis. However, these papers did uncover interesting candidates such as sonic hedgehog, among a few possible genes that were examined.12,13 Sonic hedgehog continues to be of interest in developmental anomalies of eye and brain. Although this was present in the retina in a few cases, bone morphgenetic protein (BMP4) was more associated with myopia.14 Work on association of ZENK in glucagon containing amacrine cells, the avian homolog of EGR-1 — an early immediate gene and transcription factor whose protein is a member of the C2H2-type zinc-finger proteins — was discovered.15,16 Acting as a nuclear protein, Egr-1 is involved in the activation of genes required for differentiation and mitogenesis. Schaeffel and