Student Research Project
Homeobox genes in the medicinal leech

Student
Robert Jawetz
Major: Biochemistry
Future Plans: Medical school

Professor
Eduardo Macagno, Professor, Department of Biological Sciences, Columbia University, New York

The homeobox genes code for a group of highly conserved transcription factors that have been found in organisms as diverse as Drosophila, C. elegans, mice, and human beings. Different combinations of homeobox proteins bind to the DNA of embryonic neurons, thereby activating certain genes that probably direct neuronal growth and develoment. My research in Dr. Macagno's lab has focused on elucidating the order in which these genes are found on the chromosomes of the medicinal leech, Hirudo medicinalis. The leech is an excellent choice of experimental organism because its simple nervous system and large neurons are ideally suited for studying the effects of the homeobox genes on the embryonic nervous system. The order of the homeobox genes is essential, as the genes at the 3´ end of the chromosome are expressed toward the anterior portion of the embryo, while genes further upstream are expressed further down the anterior-posterior axis. Determining the order will contribute to the understanding of embryonic neuronal development.

In order to map the leech homeobox (Lox) complex, I used restriction endonucleases to cut the leech genome into thousands of small pieces. Then I ligated the fragments into cosmids (vectors used to carry 35-45 kbase fragments of foreign DNA into bacteria) and transformed E. coli with the cosmids I had created. Eventually I was able to isolate and purify cosmid DNA from three colonies that contained either a Lox 2, Lox 4, or Lox 15 gene. I digested the cosmid DNA with EcoRI and probed it with DNA from the other purified cosmids. For example, the Lox 2 cosmid was probed with the Lox 4 cosmid in one experiment and with the Lox 15 cosmid in another. If the genes were adjacent, they would theoretically, but not necessarily, produce cosmids that had overlapping regions.

Thus far, I have not located any overlapping sequences. This would suggest that, unlike vertebrate homeobox genes, the Lox genes are separated by long sequences in the chromosome. However, this does not necessarily mean that the genes are far away from each other. It is quite possible that the three colonies I isolated contained cosmids missing the overlapping sequences due to the vagaries of restriction enzyme function, but that other clones might contain overlapping sequences.

There are two possible paths that should be investigated before concluding that these Lox genes are distant from one another. The first is to use the cosmids to produce in situ probes for screening prophase nuclei from leech cells. The probes will bind to the regions of the chromosome where these Lox genes are located, and their relative positions may be determined. The second approach is to actually rescreen the genomic library for more colonies and then once again look for overlap. By applying these two methods, the map of the Lox genes may one day be completed.

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