Gene Cloning

Gene Cloning in the Functional Analysis of Proteins 293
Q10.5. Why was it necessary in designing the primers to ensure that they
would put the GFP in the same reading frame as the ORF?
A second primer was designed that was homologous to the region downstream
from the his3 gene in the plasmid, followed by a stretch of DNA
homologous to the sequence downstream from the ORF in the genome.
PCR reactions were then done for each ORF, and each PCR product was
individually transformed into a haploid yeast strain that was auxotrophic
for histidine. The transformants were plated out on minimal medium lacking
histidine. Colonies that arose on these plates are candidates for ones
where a homologous recombination event has taken place between the
PCR product and the ORF, since this will insert the his3 gene onto the chromosome,
enabling the growth of the transformed strain in the absence of
histidine. PCR on individual transformants was then used to check that the
PCR product had indeed integrated at the correct locus. A total of 6029
transformants were obtained. The others presumably represent cases
where the presence of the GFP is deleterious to the normal function of the
protein, and where the protein is essential for cell growth.
All transformants were screened for fluorescence, and approximately
two thirds showed an increase. The remainder probably represent fusions
with genes which were not expressed, or were expressed at very low levels,
under the conditions of the experiment. Those colonies that showed fluorescence
were then studied individually using fluorescence microscopy,
and categorized into different groups according to where the fluorescence
signal was seen to localize (nucleus, nuclear periphery, actin cytoskeleton,
mitochondrion, plasma membrane, cytoplasm, etc.). Careful analysis
enabled 22 different cellular locations to be defined. It was found that 44%
of the proteins studied were found in locations other than the two major
ones (nucleus and cytoplasm), and in many cases these were assignments
that had not been made before.
10.4 Mapping of Membrane Proteins
One of the most interesting and challenging areas of cell biology is the
study of membrane proteins. These proteins are of great importance to the
cell, acting as pumps and channels that maintain internal homeostasis,
and detectors that enable the cell to respond to conditions and materials
outside the cell even when they do not penetrate the membrane. They are
of considerable interest to structural biologists since they have to interact
with two very different environments (the internal hydrophobic region of
the membrane and the hydrophilic aqueous milieu on both the inside and
outside of the cell). They are also difficult to study, particularly from a
structural point of view, because they can only maintain their structure