Biol 3054
1.
Worksheet 7
1
You have identified a cDNA that is 2Kbp long. You want to see know long the gene is so you carry out
Southern analysis. You cut genomic DNA with EcoRI and ran it on an agarose gel. The DNA fragments were
then blotted onto a nylon membrane and probed. You used the 2Kbp cDNA as the probe to detect those
genomic fragments which contain the gene. The results of the Southern blot hybridization are shown below.
Read the figure legend to understand the data.
a. Explain what a cDNA is and what it represents
b.
c.
What is the approximate length of the complete gene. ________________
Explain why the apparent size of the gene is larger than the cDNA.
d.
This cDNA is not present in persons with a particular disease. The sizes of the bands detected by the cDNA
probe are different in diseased persons. Describe the difference between the size of the gene in healthy
vs diseased persons and how this difference might result in the loss of expression of this gene (and
only this gene).
1
Figure 1. Southern blot of
genomic DNA cut with
EcoRI and probed with a
2Kbp cDNA probe. DNA
from a healthy individual
was loaded in lane 1, DNA
from a person with the
disease was loaded in lane 2.
Molecular size standards are
in lane 3. Sizes are given in
Kbp.
2
3
10
8
6
4
3
2
1.55
1.4
1
.75
.5
.4
.3
.2
.1
.05
Biol 3054
2.
Worksheet 7
2
The structure of a gene is given below. The length of each exon is given in the boxes and the length of the
introns is given below the hash marks (exons & introns not to scale). This gene encodes a mRNA that is 2871 nt
long. The polyA tail of this mRNA is 275 nt long. This mRNA encodes a protein of 673 amino acids.
280
/
//
1214
471
/
//
774
522
/
//
987
343
/
//
2177
619
/
//
157
402
/
//
204
521
a.
How many nucleotides are in the ORF of this mRNA? _____________
b.
How many nucleotides total are in the untranslated regions of this mRNA?________
c.
Indicate a plausible position for the start codon and stop codon for this gene on the diagram. Provide
the exact number of the base where the 1 st nucleotide of the start codon is positioned and the 1 st
nucleotide of the stop codon is positioned. Don’t CHEAT or COPY or COLLABORATE or I’ll give
you a BIG FAT ZERO!
3.
If molecules like cAMP, Ca2+, & DAG are 2nd messengers, what are the 1st messengers?
4.
You are trying to clone a receptor tyrosine kinase from the genome of an organism whose genome has 3 x 10 9
bp. The average size of a gene is 50Kbp and you screened a genomic library made in a cosmid vector. How
many cosmid plaques would you need to screen to guarantee that you would find the receptor gene? (show all
your math for any credit)
5.
Describe in concise detail how the protein kinases A and C would be simultaneously activated within a cell.
Begin your description with the arrival of ligands at the cell.
Biol 3054
6.
3
In Drosophila melanogaster, a signaling pathway is responsible for the differentiation of the photoreceptor
cells of the eye. Each unit of the compound eye has 8 cells. A mutation was discovered in which the 7 th cell did
not form. This mutation (and the gene) was named sevenless (sev). Flies lacking cell 7 can’t see UV light. After
this discovery a 2nd mutation that had the same phenotype was discovered, although the mutation was in a
different gene. Because of the similarity, the 2nd mutation was named bride of sevenless (boss). It was
postulated that one of these genes encoded a receptor and the other gene encoded a ligand. An elegant
experiment was performed that showed in sev eyes, the mutant cells could not be corrected by transplanting in
wildtype cells. However, in boss eyes, the mutant phenotype could be corrected by transplanting in wildtype
cells. In technical terms, sev behaved in a cell autonomous manner, while boss behaved in a cellnonautonomous manner. With this information and a lot of hard thinking, tell me, which gene encodes the
ligand and which gene encodes the receptor?
Ligand = _______________
7.
Worksheet 7
Receptor = ________________
Diagram the ras signal transduction pathway. Included everything!