Lawrence Harshman - US grants

This is a proposal for multi-year funding in support of an integrated and expanded instructional program for community college faculty. The proposal will introduce molecular biology and molecular diagnostics as continuing education. The proposed program will continue to offer the week-long summer institute in molecular biology and add to that a new summer institute in molecular diagnostics in which the principles of molecular biology find applications in several areas of biotechnology. In addition, it will expand follow-up program services and other participant contacts with university faculty and industry researchers. Internships for community college faculty will continue as a part of the program.

human mortality; human population genetics; alleles; longevity; gene frequency; human very old age (85+); clinical research; human subject; genetic mapping; human genetic material tag;

Although a tremendous amount of information is available on physiological, biochemical, and molecular aspects of endocrinology, very little is known about the evolution of endocrine regulation. Genetically-based variations in endocrine traits provide the raw material upon which natural selection acts during the initial stages of adaptive evolution. Thus, a key step in understanding the evolution of hormonal control is the characterization of genetically-based variation in endocrine regulators within species. The goal of the present multi-disciplinary study is to identify the variable molecular and physiological factors that cause genetically-based variation in the enzyme juvenile hormone esterase (JHE) in a cricket species. JHE degrades and regulates the key insect hormone, juvenile hormone, and has been the subject of intensive study in the Principal Investigator's laboratory during the past 15 years. This proposal has three primary research objectives. First, using both in vivo and in vitro approaches, the following hypothesis will be tested: genetic variation in the concentration of or tissue sensitivity to neurohormones causes genetically based variation in JHE activity and tissue distribution. Experiments will largely involve assessing the effect of extracts or implants of neural tissue derived from high- or low- JHE activity genetic stocks on JHE activity and related endocrine traits in the same vs alternate lines. Second, the role of variation in JHE messenger RNA abundance as the cause of genetically-based variation in JHE activity will be tested. JHE mRNA abundance will be compared between high and low activity lines using Northern blots employing a probe derived from a recently obtained, nearly full-length JHE cDNA. Third, the high and low JHE activity lines will be crossed and backcrossed to determine the degree of co-segregation among (1) JHE activity and associated endocrine traits, (2) molecular correlates of these traits (JHE mRNA level), and (3) potential regulators of these traits. This research will simultaneously provide important new information for several different areas of biology. This will be the first study to identify the molecular and physiological causes of genetically-based variation in an endocrine regulator in natural populations. This project will result in the first detailed synthesis of endocrine physiology, molecular biology, and quantitative genetics. Finally, these studies will constitute the first investigations of genetic variation in neuroendocrine regulation in natural populations and will set the stage for subsequent identification of these regulators and the genes that encode them.

Enzymatic and molecular bases of trade-offs in lipid metabolism that underlie a life-history trade-off
Anthony J. Zera
University of Nebraska


The ultimate goal of this project is to identify genetically-based modifications in intermediary metabolism that underlie genetically-based variation in life history traits. Experiments will be undertaken in the wing-polymorphic cricket, Gryllus firmus, which exists as a flight-capable morph that delays egg production and a flightless morph with substantially enhanced egg production. Previous studies demonstrated that egg production in the flight-capable morph is reduced because a large amount of triglyceride flight fuel must accumulate. Previous studies also have documented that the increased accumulation of triglyceride results from increased fatty-acid biosynthesis and increased diversion of fatty acids into the production of triglyceride as opposed to phospholipid. Proposed research will build on this extensive data base and has two goals: The first is to identify the enzymological characteristics and gene-regulatory mechanisms in the flight-capable morph responsible for the increased biosynthesis of fatty acid. The second goal is to identify functional modifications of enzymes at the triglyceride/ phospholipid biosynthetic branch point that result in the preferential diversion (i.e. trade-off) of fatty acid into triglyceride flight fuel in the flight-capable morph. Four key lipid-biosynthetic enzymes will be purified to homogeneity, antibodies will be raised against them, and then used to measure enzyme concentration. This will test the hypothesis that elevated enzyme activity of lipid-biosynthetic enzymes is due to elevated enzyme concentration. Purified enzymes will be characterized kinetically to test the alternate hypothesis that morph-differences in enzyme activities result from changes in catalytic efficiency, due to factors such as differential phosphorylation. Message abundance of genes encoding the four lipogenic enzymes will be compared to determine the extent to which morph-differences in enzyme activity/concentration result from differences in gene expression. Finally, five enzymes controlling flux through the triglyceride/phospholipid biosynthetic branch point will be compared between flight-capable and flightless morphs with respect to a variety of functional characteristics such as specific activity, differential phosphorylation, and binding to membranes. The goal will be to identify differences in the enzymes that are responsible for the morph-specific trade-off in the diversion of fatty acid into triglyceride (flight fuel) vs. phospholipid (key egg component) in flight-capable vs. flightless morphs.

This study will contribute significantly to our understanding of the functional causes of life history evolution, a poorly understood topic in evolutionary biology. It will comprise the most detailed analysis of alterations in intermediary metabolism that give rise to life history variation and trade-offs that occur in natural populations. This will be the first study to identify specific enzymatic alterations at a branch point (triglyceride/phospholipid) in intermediary metabolism that underlies a life history trade-off. This project will contribute to the development of a deep integration of life history evolution and evolutionary biochemistry, which was initiated in the previous NSF-funded study. This project will also contribute to the development of human resources by providing integrative training for a postdoctoral associate in evolutionary biology, enzymology, and molecular biology, and will also provide numerous significant undergraduate research opportunities.

This project is designed to investigate genes expressed in sperm storage organs (SSOs) in female Drosophila melanogaster. SSOs potentially play an important role in a range of evolutionary phenomena including co-evolution and speciation. Given the prominent role of D. melanogaster as a model for evolutionary and genetic studies, it is surprising that so little is known about the evolution of genes characteristically expressed in SSOs and that little is known about the function of these genes. The investigators will examine the function of a set of these genes defined by molecular evolution data, molecular population genetic data and information suggesting that certain genes play a direct role in sperm storage.

The sequences generated from this project will be deposited in the public database, NCBI/GenBank, in timely manner. A project website will be developed to provide information about the progress of the research and to provide the research community access to all sequence data. The grant will contribute to educational outcomes in various ways. First, both graduate and undergraduate students will be involved in the research. Second, the co-PI (Moriyama) will incorporate statistical methods used in the project and the results into her course. Third, the PI (Harshman) will expand the population genetic component of his honors human genetics course and its graduate student counterpart.