Molecular Biology, Spring 2006

Problem Set #8 (Lectures 15 & 16)

Lodish et al. Molecular Cell Biology “Review the Concepts” chapter end problems:
3-3 – Ubiquitin is a 76 amino acid protein that serves as a molecular tag for proteins destined for
degradation. Ubiquitination of a protein involves an enzyme-catalyzed transfer of a single
ubiquitin molecule to the lysine side chain of a target protein. This ubiquitination step is repeated
many times, resulting in a long chain of ubiquitin molecules. The resulting polyubiquitin chain is
recognized by the proteasome, which is a large, cylindrical, multisubunit complex that
proteolytically cleaves ubiquitin-tagged proteins into short peptides and free ubiquitin molecules.

9-3 – Bacteria that synthesize restriction enzymes also synthesize a DNA modifying enzyme to
protect its own DNA. The modifying enzyme is a methylase, which methylates the host DNA.
Methylated DNA is no longer a substrate for the encoded restriction enzyme. Restriction enzyme
sites commonly consist of 4-8 base pair, palindromic sequences. After being cut with a restriction
enzyme, the ends of the cut DNA molecule can exist as a single-stranded tail (sticky end) or as a
blunt (flush) end. DNA ligase is an enzyme that catalyzes the reformation of the phosphodiester
bond between nucleotides in the presence of ATP.

9-7 – Southern blotting is a technique in which DNA fragments are separated by size in a gel and
then transferred to a sold support such as a nitrocellulose or nylon membrane. The DNA is fixed
to the nylon membrane and hybridized to a labeled DNA or RNA probe. The hybridized probe is
then detected by some technique such as autoradiography. Northern blotting is similar to
Southern, except that RNA is denatured and then separated on the gel instead of DNA. Southern
blotting can be used to identify a DNA fragment that contains a DNA sequence of interest.
Northern can be used to determine the steady-state levels of a specific RNA.

9-9 - Genomics is defined as the genome-wide analysis of the organization, structure and
expression of genes. Proteomics is the global analysis of the function and expression of proteins.
Computer searches for open reading frames are more useful for bacterial genomes because
bacterial genes are present as an uninterrupted stretch of nucleotides. IN contrast, in eukaryotes
many of the genes are divided into exons and introns which makes the search for genes more
complex. Paralogous genes are genes that have diverged as a result of a gene duplication, i.e.,
two genes an in organisms that have different functions by very similar nucleotide sequences.
Orthologous genes are genes that arose because of speciation, i.e., genes found in different
species that have very similar nucleotide sequences. Because of alternative splicing, a gene can
give rise to numerous protein products. Thus a small increase in gene number could result in a
very large increase in protein number. Thus the number of proteins and protein-protein
interactions could be much greater in the organism with the larger genome.

9-10 – A DNA microarray consists of hundreds or thousands of individual, closely packed gene-
specific sequences attached to the surface of a glass microscope slide. The expression of each of
these genes can be analyzed globally following hybridization of the array with labeled cDNA
prepared from RNA. Microarrays allow the simultaneous analysis of the expression of thousands
of genes, whereas Northern blotting allows the analysis of gene expression a single gene at a
time. Because northern blotting involves the separation of RNA on a gel, the different sizes of a
given mRNA can be observed, whereas microarray analysis does not allow the examination of
different mRNA sizes.

9-13 - Restriction fragment length polymorphisms (RFLP) result from mutations that create or
destroy restriction enzyme sites. As a result, DNA molecules with or without the restriction
enzyme sites are cleaved into different sized fragments. Single nucleotide polymorphisms
(SNPs) are changes in a single nucleotide between two individuals. Simple Sequence Repeats
(SSRs) also known as microsatellites, consist of a variable number of repeating one-, two-, or
three-base sequences. The number of these repeat units at a specific genetic locus varies between
individuals. All of these types of polymorphisms can be used as a molecular marker for mapping
studies. The recombination frequency between two polymorphisms can be determined and serve
as the basis for development of a genetic map. In general, the further two markers are separated
on a chromosome, the greater the recombination frequency between those two markers and vice
versa.

Analyze the Data parts A & B

a. The northern blot reveals the steady state mRNA levels for p24 (in the center) and p25 (on the
right). For p24, only the transfetion of siRNA-p24 reduces the amount of p24 mRNA; whereas
the control transfection without siRNA and transfection with siRNA-p25 did not affect p24
mRNA levels. Similarly, for p25, only siRNA-p25 reduced p25 RNA levels. These results
demonstrate that the siRNAs can cause specific degradation of their target RNAs.

b. In the cultured cells, transfection of either siRNA-p24 or siRNA-p25 yielded a viral titer that
was slightly lower than the control transfesction. This result indicates that reduction of either p24
mRNA or p25 mRNA and presumably p24 and p25 protein only minimally affects the ability of
the virus to infect the cells. Transfection, however, of both siRNA-p24 and siRNA-p25 did result
in a significant reduction in viral titer. In combination with the previous results, these results
indicate that either p24 or p25 can be used as a viral receptor.You can hypothesize that loss of
p24 and p25 mRNA would lead to a decrease in p24 and p25 protein, resulting in inhibition of
viral infection and replication. Transfection of siRNA to a viral protein resulted in an even
greater reduction of virus titer compared to transfection with siRNA to both p24 and p25. One
possible explanation is that transfection of siRNA to a viral protein directly inhibited viral
replication/assembly. A second possibility has to do with the half-life of p24 and p25 proteins. A
reduction of p24 and p25 mRNA may not lead to an immediate reduction in p24/p25 protein due
to the stability of the protein. The protein level is determined by the half-life of the protein and
the length of time after transfection of siRNA. For example, if the half lives of p24 and p25
proteins are greater then 24 hours, then during the 20 hour incubation after transfection with
siRNA there should still be more than half of the protein still present. IN this problem no
information was given, so a definitive conclusion cannot be made. But based on the results, it is
reasonable to conclude that the half-life of the p24 and p215 protein is relatively long, such that
20 hours after transfection with the siRNA, sufficient protein remains to act as a receptor for the
virus.

A) What does “export ready” mRNA mean, and what distinguishes “export ready” mRNA
from a bit of excised intron that needs to be degraded?
All introns are excised out and 5’ and 3’ caps are present. Cannot be bound to splicesome. This
only applies to eukaryotes since they are exported from the nucleus to cytoplasm.

B) DNA microarray analysis of the patterns of mRNA abundance in different human cell
types shows that the level of expression of almost every active gene is different. The
patterns of mRNA abundance are so characteristic of cell type that they can be used to type
human cancer cells of unknown tissue origin. By definition, however, cancer cells are
different from their non-cancerous precursor cells. How do you suppose then that patterns
of mRNA expression can be used to determine the tissue source of a human cancer?

Original cells cDNA would be diff from the mutated DNA by single NT base pair. The mRNA
would be similar in sequence to the proteins??

C) (From Molecular Biology of the Cell, 4th Edition, Wilson &

Hunt)

What if formation of the stem-loop IREs were inhibited? What would the consequence be
for production of both ferritin and transferrin receptor proteins?

IF you had no IRE then you could still make both ferritin and transferrin at high and low levels
of iron. Need IRE for regulation.

D) (Modified from Molecular Biology of the Cell, 4th Edition, Wilson & Hunt)
Internal ribosome entry sites (IRES; not to be confused with iron response elements) allow
some viral and eukaryotic mRNAs to be translated in the absence of 5’ cap-associated
proteins. However, you are skeptical that IRES really allow direct binding to the
eukaryotic translation machinery to the interior of a mRNA. As a critical test of this
notion, you prepare a set of linear and circular RNA molecules, with and without IRES (see
A and B in figure above). You translate these various RNAs in an in vitro system and
analyze the proteins by SDS-PAGE (see C in figure above). Do these results support or
refute the idea that IRESs allow ribosomes to initiate translation of mRNAs in a cap-
independent fashion? Explain your answer.

Linear+IRE= protein
Linear+ no IRE= protein
Circular+IRE=protein
Circular+ no IRE= no protein

IRE translation of 18kd protein is still made. For linear, with or w/o IRE protein but
circular needs IRE to get a protein because the IRE stabilizes the mRNA circular formation
to allow translation