Richard L. Borowsky - US grants

With National Science Foundation support, Dr. Todd Disotell and his collaborators at New York University will purchase an ABI PRISM 377 system which consists of an electrophoresis and detection unit, a Power Macintosh computer and software for DNA sequencing analysis or PCR fragment sizing and quantification. The system will allow high throughput through the use of multicolored fluorescence technology, allowing multiplex electrophoreses by co-loading the products of multiple PCR or sequencing reactions in the same lane. To increase the efficiency of the group of utilizing scientists, a thermocycler (Perkin-Elmer Geneamp PCR System 9700) along with a microcentrifuge (Eppendorf 5400 Series) and microconcentrator (Savant DNA SpeedVac) will be acquired and housed with the ABI genetic analyzer. A Power Macintosh computer will allow data collected on the ABI genetic analyzer to be analyzed off-line, allowing the computer controlling the ABI machine to be dedicated to data collection. This instrumentation will greatly improve the efficiency of six research projects, pursued by a number of investigators. These include the study of 1. the genetic and developmental mechanisms of morphogenesis and the evolution of form in rhabditid nematodes; 2. genetics of organogenesis in the gonad of C. elegans; 3.primate molecular evolution and population genetics; 4. evolutionary genetics of cave adaptations in fish; unidentified proteins in fission yeast that interact with Ras G-proteins and microtubules; and 6. recognition of DNA structure by HMG proteins. The machine will also be used at both graduate and undergraduate levels and thus serve a valuable educational as well as research function.

0216103/0217178
Browsky
Tabin

The long term goal of this research is to understand the nature of the developmental genetic differences that make all individuals of a species unique, and distinguish the various species from one another. Towards this end, differences between two types of fishes that are closely related, but genetically distinct, will be studied. The Mexican tetra, Astyanax mexicanus, has some populations that live in rivers and others that live in caves. Cave forms differ from their surface relatives by having greatly reduced eyes and pigmentation. The caves in which they live provide little food and they have adapted to this by developing a slower metabolism. Other senses are overdeveloped to compensate for the lack of vision: e.g., smell, taste and the ability to detect vibration and current. Not only can they survive eternal darkness, they thrive in caves.

The genome of this species will be mapped at a fine scale using microsatellite DNA markers, in hybrids between the cave and surface forms. The map and further analysis of the hybrids will permit the detection and characterization of genes (QTL) that are responsible for the differences between cave and surface forms. The results will give estimates of the "genetic architecture" of trait differences: the number of genes involved and magnitudes of their individual effects, and their positions in the genome relative to one another. Positional information of some of the major effect QTL will be used to identify and clone the genes; this will facilitate their functional analysis. The results will shed light on the genetic pathways involved in development and maintenance of complex traits, like the vertebrate eye and other aspects of the sensory system, control of pigmentation, behavior and metabolism. The results will also inform on the nature of genetic differences distinguishing populations and species, and on the constraints on evolutionary change resulting from trait correlations.

[unreadable] DESCRIPTION (provided by applicant): The long-term objectives of this project are to identify genes important for the development and maintenance of the human eye and visual system, to understand their normal actions and interactions, and how these may fail, leading to abnormalities of the visual system. These will be studied in the cave fish model system (Astyanax mexicanus) in which genes causing eye and visual disorders can be mapped and identified. The project has three specific aims: first, we will examine the abnormal eyes of cave/surface hybrids histologically and do QTL mapping in order to better characterize the abnormal phenotypes and understand their genetic bases. This will allow us to recognize similarities to known human eye disorders and increase our knowledge of their causes. Second, we will map genes known to affect eye development or to be expressed in the eye. Some of these genes will prove to be QTL candidates and our analyses will increase our understanding of the ways in which they function in controlling eye development or maintenance. The third aim is to do histological and genetic analyses on the restoration of eye structure and function that occurs in hybrids between two different cave populations. An understanding of the ways in which normal phenotype can be restored by the interaction of elements from two flawed genomes will increase our understanding of the genetic pathways involved in human eye development and disease. [unreadable] [unreadable] [unreadable]

Cave fishes differ from surface fishes in numerous ways because of their evolutionary adaptations to life in the dark. Most noticeably, they have smaller eyes and less pigmentation, but they also have better senses of smell and taste, and are more efficient metabolically. All of these differences are under genetic control; the gene copies in the cave populations affecting important developmental systems differ from those in the surface populations. The Mexican Tetra is unique in that the species has both cave and surface forms. Because they can hybridize, they afford the opportunity to investigate the differences through genetic analysis. In this collaborative research project, the genes responsible for these differences will be identified by genetic mapping and functional analyses of selected candidate genes. The results will lead to a better understanding of the genetic pathways that control the development and maintenance of: (1) the visual system; (2) pigmentation; (3) metabolic efficiency; and (4) various other physiological and anatomical systems. In terms of Broader Impacts, the work on cave fishes has been well covered by the media, and the extraordinary ability of this system to engage people's interest and attention will have broad positive impacts on public education in biology, genetics and evolution. The research will also impact the training of undergraduate and graduate students and postdoctoral fellows, particularly in novel concepts of evolution and development (evodevo). In terms of societal impacts, the results may also contribute to a general understanding of the causes of eye degeneration and metabolic disorders and could lead to a better insight into how environmental changes (i.e., the shift from surface to cave environments) can modulate the direction of evolutionary change.

It has been accepted that the characteristics of sperm are determined by the diploid genotype of the male producing them. However, recent and unexpected findings indicate that the phenotype of a sperm cell may also be influenced by its own genotype. This innovative project examines the degree to which the haploid genotypes of sperm cells affect their individual characteristics and the relative importance of sperm competition versus selection post-fertilization in causing biases in transmission of alleles from one generation to the next. It will provide basic information to illuminate a previously unsuspected but potentially important avenue for natural selection in evolution. The expected results have the potential to open up an important new line of research in sperm biology. They are expected to have broad implications in developmental biology, evolutionary biology, and for improvements in reporductive technologies in humans and domestic animals.

This research investigates the epistatic interactions among genes observable in hybrids between two incipient species of freshwater fish (Astyanax spp.). Previous work has shown that sperm from hybrid males pass along biased subsets of alleles and allelic combinations derived from unlinked genes. This observation suggested the hypothesis that the phenotypes of individual sperm (in this case fertilization efficiency) are determined by their individual genotypes, i.e., the probability that a package gets delivered depends upon what it contains. Consistent with this hypothesis are preliminary studies that show that sperm subpopulations - defined by differences in swimming ability or by their response to a chemical challenge - differ significantly in allelic content. This research tests whether the observed transmission biases are indeed caused by sperm competition or whether there is a contribution from zygotic selection, post fertilization. Single sperm will be genotyped from embryos formed using intracytoplasmic sperm injection. This will allow different sub-populations of sperm (e.g. slow versus fast swimmers) to be compared for allelic content. Additionally, the offspring of hybrid X non-hybrid individuals will be genotyped, allowing for the detection of transmission biases. The hypothesis predicts that biases will be restricted to families derived from hybrid males but not from hybrid females.