Molecular Biology, Spring 2006

Problem Set #9 (Lectures 16, 17 & 18)

Lodish et al. Molecular Cell Biology “Review the Concepts” chapter end problems:
4-9 – In prokaryotes, the 8 nucleotide Shine-Dalgarno sequence located near the AUG
star codon binds to specific sequences in the 16S rRNA allowing for positioning of the
30S ribosomal subunit near the start site of translation. In eukaryotes, recognition of the
start site involves other factors such as eIF4, eIF3 proteins, and Kozak sequences near the
start site of the mRNA. eIF4 recognizes and binds to the 5' cap structure on eukaryotic
mRNAs, and eIF3 proteins are part of the preinitiation complex which is thought to scan
along the mRNA, most often stopping at the first AUG. The Kozak sequences facilitate
the preinitiation complex in choosing the proper start site. Poliovirus initiates translation
of its transcripts utilizing host cell machinery so it is identical to the eukaryotic host
except for the presence of internal ribosome entry sites (IRES)s, which are internal AUG
sites.

4-11 – Since poly (A)-binding protein I is involved in increasing the efficiency of

translation, a mutant in poly (A) binding protein I would have less efficient translation.
Polyribosomes from such a mutant would not contain circular structures of mRNAs
during translation because lack of the poly (A) binding protein I would eliminate the
3'binding site for eIF4G.

Analyze the Data

A) The restriction nucleases BamHI and

PstI cut their recognition sequences as
shown.
A) Indicate the 5’ and 3’ ends of the cut
DNA molecules.

B) How would the ends be modified if

you incubated the cut molecules with
DNA polymerase in the presence of all 4
dNTPs?
BamHI can be filled in 5’3’ but Pstl
cannot b/c that is not filled in 5’3’

C) After the reaction in part B, could

you still join the BamHI ends together
by incubation with T4 DNA ligase?
yes
Could you still join the PstI ends
together? (T4 DNA ligase will join blunt
ends together as well as cohesive ends)
no
D. will joining the ends in part C regenerate BamHI site? No because you added in
extra NT
Will it regenerate the Pstl site? Yes because the cleaved ends just join back together
and you get the same sequence

B)
There has been a colossal snafu in the maternity
ward of your local hospital. 4 sets of male twins,
born within hours of each other, were
inadvertently shuffled in the excitement of the
unlikely event. You have been called to set
things right. As a first step, you want the twins
matched up. To that end, you analyze a small
blood sample from each infant using a
hybridization probe that detects RFLPs located
in widely scattered regions of the genome. The
results are shown on the Southern blot to the
left.

A) Which infants are brothers?

3,6 1,7 2,8 4,5

B) how will u match brothers to correct parents?

Take blood sample from parents. Do a southern blot and hybridize with labled probe with
sequence only the twins possess

C) Almost all cells in an individual animal contain identical genomes. In an

experiment, a tissue composed of multiple cell types is fixed and subjected to in situ
hybridization with a DNA probe to a particular gene. To your surprise, the
hybridization signal is much stronger in some cells than in others. Explain this
result.
In Situ is mRNA expression in whole tissue. A DNA probe would get a signal everywhere
but using an mRNA probe would be more specific.