The mapping, molecular characterization, cellular localization and functional studies of a novel gene with predominant muscle and brain expression

Author

John Gerard Geisler

Date of Award

12-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Michael L. Mucenski

Abstract

One of the most frequently asked questions from non-scientists is, "Why do you study mice?". If you respond by saying that you are actually trying to study human diseases using mice as a model organism, they look even more puzzled. Monkeys always seem the only natural, model organism of choice for humans. Probably one of the most fascinating lessons for me in science, besides learning that a nucleus from any cell (even intestinal) is capable of being reprogrammed into becoming an embryo (Gurdon, 1962), is the lesson on evolutionary conservation of genes. At a global level, the conservation can be seen in the large blocks of chromosomes rearranged among the different species' genome, but within the block conservation of gene synteny (linked on a thread) often remains intact (Carver and Stubbs, 1997; Copeland et al., 1993; Stubbs et al., 1996).

So why mice? For starters, a substitute for humans is necessary. Mutagenesis experiments are not allowed on humans for moral and ethical reasons, and even if possible, very few experiments could be accomplished with a 9 month gestation. For these reasons, historically mice became the substitute due to their similarity in anatomical structure and short gestation time. Mice are mammals. They have the same organs as humans: bones, kidneys, livers, spleens, brains, testes, ovaries, uterus, etc.. Best of all, they breed very well. Gestation of mice is approximately 20 days, with litters averaging 8-12 pups and the female is immediately receptive to the male after giving birth. This being the case, it becomes immediately apparent that numerous experiments could take place within a given year. Studies performed starting in the 1950s used mice models to study radiation risk after exposures to radiation from atomic bomb tests. By exposing mice at varying distances to low doses of radiation continuously or high doses in short intervals, it was possible to extrapolate to humans, predictions about the effects on the individual and their progeny, resulting from such an exposure (Russell et al., 1958).

On the molecular level, orthologous genes (a term referring to genes that represent direct evolutionary counterparts in two species), which are found within the conserved chromosomal blocks are strikingly similar in mice and humans. For instance, mouse proximal chromosome 7 contains large blocks of chromosomal DNA which correspond to human chromosome 19 (Stubbs et al., 1996). Although there are numerous blocks of homology that demonstrate this conservation, there are also numerous examples of evolutionary rearrangement as well (Carver and Stubbs, 1997). It is interesting to note, that mice and humans are predicted to share the same approximate total number of genes, each containing approximately 80,000 within a haploid genome (Antequera and Bird, 1993).

The study of mouse models would not makes sense, however, if the genes' functions were not conserved as well. The expectations of conservation of function between mice and humans have met with a spectrum of outcomes. A classic example of the benefits of using mice as a model for human disease can be demonstrated with a dominant human trait called the piebald trait. The mutation is autosomal dominant and is displayed as a white or hypopigmented patch of skin in the midforehead, abdomen and extremities of afflicted people. There is a mutation in mice called "dominant white spotting" (W), which is also associated with head spot and belly spot in heterozygous mice. The gene which causes this phenotype had been determined to be c-kit (Fleischman et al., 1991). When the human c­kit gene probe was used on piebald patients' DNA, it was determined that these patients were heterozygous for a mutation at c-kit (Fleischman et al., 1991). Mice homozygous for the mutation lack pigmented cells, and are also anemic and infertile. One case in humans was reported in which both parents were heterozygous for the piebald trait, and had a homozygous child for the mutation. The baby lacked pigmented cells and was severely retarded. From the phenotype of a human, it was possible to gain access to the gene of interest due to a similarity in phenotype displayed in mice. In other cases, the function is a much less conserved. An example of this can be seen with the gene that causes Lesch­Nyhan syndrome in humans, hypoxanthine-guanine phosphoribosyl-transferase (HPRT). HPRT encodes an enzyme in the purine salvage pathway. Disruption of this gene in humans results in self-injurious behavior (SIB) as a result of purine build-up. The orthologous gene, has been disrupted in the mouse, but no behavioral abnormalities were found and the mice developed quite normally (Doetschman et al., 1988). It was latter found that mice carry a gene called adenine phosphoribosyltransferase (APRT), which can substitute for the function of Hprt. When both genes are silenced, the purine salvage pathway is finally disrupted and the mice excrete adenine (Wu and Melton, 1993). In this case, the doubly-deficient mice seem to mimic the biochemical abnormalities of Lesch­Nyhan syndrome in humans, however they do not display any of the behavioral abnormalities such as SIB (Engle et al., 1996). These mice do have phenotypes that can be used as models for studying purine salvage pathways, but there are cases in which no substitute gene can be found and a model for the disease is presently unknown.

A similar situation can be seen with the gene that causes Myotonic dystrophy (DM), Dystrophia myotonica protein kinase (DMPK). DM is an autosomal dominant disorder associated with symptoms of muscular dystrophy (degenerative myopathies), myotonia (tonic spasms), hypogonadism, cataracts and frontal balding (McKusick, 1994). Onset of the disease symptoms is usually delayed until middle life. Unlike other muscular dystrophies, DM affects the distal extremities first, then moves more proximal. A smaller population of patients show congenital symptoms and in these cases the child is usually mentally retarded. The disorder is caused by an expansion of a trinucleotide repeat (CTG) located in the 3-prime untranslated region (3' UTR) of DMPK, which lowers the levels of DMPK protein expression. The murine orthologue (Dmpk), located on proximal chromosome 7, has been knocked out, and the normal gene has been overexpressed in mice (Jansen et al., 1996). The results showed that in all phases of mouse development (development includes aging), the null mutation or overexpression of the normal Dmpk gene had very mild effects. Although a spectrum of functional equivalencies between human and mouse genes can be found, the mouse still remains an excellent genetic model organism to study human genes due to their ease of manipulation and maintenance in the laboratory, similarities as a mammal to humans, and their short life spans. Eventually the primate and great ape genomes will be sequenced and compared to that of humans. In this case, the very small fraction of the genes that are different to that of humans will be studied. Our understanding of these genes may provide the key to understanding speech and higher order cognition.