Mol Bio Ps-9

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.