Mol Bio Ps-8

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 genespecific 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 capindependent 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