Gavin Budhram, M.D.
Education: Bachelor of Science in biology (May 2000)
Doctor of Medicine specializing in emergency medicine from the University of Kansas
McNair Project: Pearl and White as Homologous Genes in Tribolium and Drosophila (1998)
Mentor: Richard Beeman, Ph.D.
Most of the economically significant pest insect species are either beetles or moths. The fruit fly, Drosophila, has served as the sole genetic model for pest insects by default, since other insect models have been unavailable. Because of the great diversity of insect adaptations, and genetic pathways, other pest insect species are needed as genetic models to supplement and enhance information gained from study of Drosophila. The widely distributed pest species, Tribolium castaneum ("Tribolium" means red flour beetle), is an extremely facile and tractable genetic model, and represents the most speciose of all animal Orders. Tribolium is twice as recombinogenic as Drosophila, and has a suitably small genome size of 200Mb. We have recently succeeded in cloning the white cDNA from Tribolium -- the first time this gene has been cloned in an insect outside the Drosophilid Order, Diptera.
There are several genes in the Drosophila genome that control the eye color of the individual. One of these genes, white, has the power to correct a common mutation in eye pigment. It is a recessive allele. Therefore an individual who is either homozygous wildtype or heterozygous will display the normal black eye pigment, while the homozygous recessive individual will display the mutant white eyes. It is believed that a homologous region exists in the Tribolium genome, called "pearl," after the mutation it causes in Tribolium. The objective of the research has been to study Tribolium with the pearl mutation and prove that their mutation can be traced back to an area on the Tribolium genome that is homologous to Drosophila. That is, pearl and white are homologous genes in Tribolium and Drosophila. The research began by mating a wildtype mother (homozygous dominant, with black eyes) with a mutant father (homozygous recessive, with white eyes.) The results of such a cross should produce offspring that all display the black phenotype, but carry the recessive pearl allele. That is, the offspring should all be heterozygous. Next, a daughter from this family was backcrossed to the father. This second filial generation would therefore produce progeny that are half white-eyed (homozygous recessive) and half black-eyed (homozygous dominant). In other words, they were either "pure white" or "pure black." One hundred white progeny and one hundred black progeny then underwent DNA extraction and PCR, amplifying the pearl genes in their genomic libraries. The primers Wh-ll and Wh-12 RC were chosen because they reside on opposite sides of the pearl gene sequence, and PCR using them would amplify the complete pearl sequence. The next phase was Single Strand Confirmation Polymorphism (SSCP). The purpose of this was to denature each insect's copy of the pearl gene (wildtype or mutant) and check to see which allele was inherited by which insect. As was expected, the black-eyed progeny displayed a middle band that the white-eyed progeny lacked. This indicates that the white-eyed progeny inherited two copies of the recessive pearl allele whereas the black-eyed progeny inherited only one.
The fact that no recombination occurred during this experiment is strong evidence that pearl is closely linked to the white gene in Drosophila. The success rate of the experiment was so high that out of the 179 beetles prepped, all showed evidence of correct pearl segregation. That is, the fact that all the black-eyed progeny displayed a homozygous recessive genotype for pearl and all the white-eyed progeny displayed a heterozygous genotype indicates that the mutation can be traced to an area of the Tribolium genome that is homologous to the white gene in Drosophila.