Program of the 2001 International Worm Meeting - Sternberg Lab ...

599
599. Using PCR in an undergraduate
lab course to detect deletions in the
unc-93 gene
James L. Lissemore, Laura L.
Lackner, George D. Fedoriw
Biology Department, John Carroll University,
University Heights, OH 44118
PCR is a powerful and common molecular
biology technique with many applications. We
have developed an exercise for an
undergraduate molecular biology lab course
using PCR to detect deletions in the unc-93 gene
of C. elegans. unc-93 encodes a putative
transmembrane protein muscle protein of
unknown function.
Wildtype and three different unc-93 deletion
mutant strains (carrying alleles lr12, lr28, and
lr81) were grown, harvested, and frozen at -20 C
by the instructor. Students isolate genomic DNA
from each strain using a genomic DNA isolation
kit from Gentra Systems, Inc. (Minneapolis,
MN). We have used this kit for several years
and it seems to be almost foolproof in the hands
of undergraduates. Following proteinase K and
RNase A treatment, proteins in the worm extract
are precipitated by high salt and the genomic
DNA is recovered by isopropanol precipitation.
PCR reactions using two sets of primers (A and
B) from two different regions of the unc-93
gene are carried out on the genomic DNA from
wildtype and mutant strains and the results
analyzed by agarose gel electrophoresis. Primer
pair A yields a 789 bp fragment, while primer
pair B yields a 728 bp fragment. The A primers
detect a 173 bp deletion in lr28 and a 78 bp
deletion in lr81 while the B primers detect a 517
bp deletion in lr12. The use of wildtype DNA
and primers for amplifying two different regions
of the unc-93 gene provide internal controls.
Five student groups carried out this exercise in a
molecular biology lab course during the Spring
2001 semester and their results will be
presented.
We gratefully acknowledge the assistance of Dr.
Beth DeStasio, Biology Dept., Lawrence
University, Appleton, WI, who provided the
unc-93 strains and primers used in this exercise.
600. Caenorhabditis elegans in the
Undergraduate Physiology
Curriculum
James Priest, Jerrold Sulcove,
Taylor Allen
Biology, Oberlin College, Oberlin, OH
44074-1082
600
Given the broad range of mutant strains with
deficits in behaviors relevant to an
undergraduate course in physiology, we sought
to develop two discovery-based experiments
with C. elegans that would both amplify
concepts being learned in class and permit
students to participate in experimental design.
The first experiment challenges students to
characterize quantitatively the deficits in
muscular function arising from selected
missense mutations of unc-54 (myosin heavy
chain B). Cyclic interaction of myosin with
actin is the basis of tension development in
muscle cells, as well as eukaryotic cells
generally, and the mechanical events are
coupled to the hydrolysis of MgATP by myosin.
The experiment exploits the availability of
unc-54 mutations (e.g., s74 and s95 alleles) that
have salient functional deficits without
disruption of muscle structure. Following
discussion of viscosity and Reynolds number,
students develop swimming assays in which
tail-beat frequency is examined as a function of
bulk solution viscosity. The concepts that the
students consider when designing their
experiments include the following: osmolarity
and its measurement; bulk solution viscosity and
its measurement; muscular fatigue and
endurance; and force-velocity relations and their
interpretation in the context of muscular
efficiency. Because of the linkage between
mutations of human myosin isoforms and a
range of muscle and non-muscle diseases,
including lethal heart disease and hereditary
deafness, students have found the experiment
engaging. Moreover, the accessibility of the
crystalline structure of the myosin head
(containing the catalytic domain and the
actin-binding regions) permits students to
correlate their results with the emerging
structure-function relationship of myosin. The
experiment can also be extended to the analysis
of other mutations affecting muscular function
(e.g., unc-27/Troponin-I-2; please see abstract