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Dissertation/Thesis Abstract

Lineage-Specific Gene Families and Evolutionary Innovation
by Bedell, Victoria M., Ph.D., College of Medicine - Mayo Clinic, 2012, 193; 3553003
Abstract (Summary)

The mechanisms underlying organism evolution and innovation has long been studied. Currently there are three theories detailing how innovation is encoded in the genome. First, the gene duplication theory states that creation of new genomic material, ranging from a single exon to the entire genome, via duplication and subsequent mutational processes encodes for diversity. Second, the gene regulation theory states that mutation of a gene's regulatory sequences changes the temporal and spacial expression pattern of that gene and that, in turn, creates diversity. Finally, the de novo gene synthesis theory states that new genes are needed for creating new structures.

Substantial progress has been made in understanding how conserved gene families, through both duplication and changes in regulatory sequences, encode for evolutionary innovation. However, very little is known about the function of de novo genes and their role in evolution. De novo genes fall within two categories. First, if de novo genes are found only within one particular species and share no homology with any other protein or domain, they are called orphan genes. Second, if orphan genes become fixed within the lineage they are called lineage-specific or taxonomically-restricted gene families. Here, we focused on lineage-specific gene families and investigated their significance in chordate and vertebrate evolution.

We defined a set of vertebrate-specific gene families using two established protein homology databases, HomoloGene and TreeFam. We found that 20% of all known gene families are vertebrate-specific. Once we created a list of families, we identified biological processes with a significant overrepresentation of vertebrate-specific genes. We found that vertebrate-specific genes are enriched in the sensory, immune and neural systems, all of which are sites of vertebrate innovation. This suggests that vertebrate-specific genes are important in vertebrate diversity and innovation.

Using the zebrafish (Danio rerio) as a model for vertebrate evolution, we identified a chordate-specific gene family, ponzr, and determined the role of ponzr1 in zebrafish kidney evolution. We found that ponzr1 is required for pronephric glomerular development, which is a vertebrate innovation. Furthermore, when ponzr1 is knocked down, the kidney retains some functionality. However, the filtering appears further posterior along the pronephric ducts. This suggests that, without ponzr1, a functional kidney is still patterned. However, the zebrafish patterns a simplified kidney demonstrating tubular secretion, similar to those seen in aglomerular fish.

Finally, to make the zebrafish a more tractable model for vertebrate evolution and development and to create ponzr1 a knockout, we developed a technology for site-specific genome engineering in the zebrafish. Using a second generation transcription activator-like effector nuclease (TALEN) scaffold, GoldyTALEN, we saw increased efficiency in TALEN-created mutations. In 3 out of the 5 TALENs tested, we saw biallelic conversion in zebrafish larvae. The mutagenesis efficiency was sufficient to identify an F0 phenotype in larvae, similar to the phenotype in knockdown experiments. With this increased TALEN efficiency, we asked whether we could introduce exogenous sequences via homology directed repair. Using single stranded DNA as a template along with the ponzr1 GoldyTALEN, we were able to engineer an EcoRV and modified loxP site into the second exon of ponzr1. Furthermore, we were able to see germline transmission of the EcoRV sequence, creating the first successful HDR addition of an exogenous sequence in the zebrafish.

In conclusion, this thesis demonstrates that lineage-specific gene families are important for evolutionary innovation. Using the zebrafish, we show that ponzr1 is necessary for pronephric glomerular innovation. Finally, we demonstrate that, using TALENs, we can engineer the zebrafish genome, making it a tractable model to study vertebrate innovation.

Indexing (document details)
Advisor: Ekker, Stephen C.
Commitee: Harris, Peter C., Isaya, Grazia, Maher, Louis J., Mukhopadhyay, Debabrata
School: College of Medicine - Mayo Clinic
Department: Biochemistry and Molecular Biology
School Location: United States -- Minnesota
Source: DAI-B 74/06(E), Dissertation Abstracts International
Subjects: Evolution and Development, Bioinformatics
Keywords: Evolution, Kidney development, Transcription activator-like effector nuclease, Vertebrate specific genes
Publication Number: 3553003
ISBN: 978-1-267-92187-1
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