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Berkeley MCELLBI 110 - MCELLBI 110 Problem set 3

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2007 1/2 MCB 110 Problem set 3. DNA packaging, analysis methods and bioinformatics - Answers 1. When human cell nuclei were treated with DNaseI, DNA fragments created a ladder comprised of fragments of ~150, ~350, ~550 and so on base pairs. Explain how a nonspecific, double-stranded-DNA endonuclease could produce DNA fragments of these defined lengths. Each nucleosome protects ~150 bp from nuclease digestion. When DNaseI cuts in the linker between adjacent nucleosomes, a fragment of ~150 bp is generated. When the linkers separated by 2 nucleosomes are cut, ~300-350 bps are protected. Each additional nucleosome between cuts in the linkers adds ~150-200 bp. 2 a) If you probe a Southern blot of this DNaseI-digested, nuclear DNA with a DNA probe for a specific, single-copy gene, would it hybridize to a specific set of bands? No. Why or why not? DNaseI cuts nonspecifically and most nucleosomes are placed nonspecifically on the DNA. b) Would the probe hybridize to a specific pattern of bands if you fully digested purified, protein-free, human DNA with a restriction enzyme instead of DNaseI? Yes. Why or why not? Restriction sites occur at specific positions relative to the probe gene. 3. How does the histone octamer recognize DNA without sequence specificity? Many histone DNA interactions occur via bound water or through the DNA backbone. 4. What is the function of the chromosome scaffold? Not well understood. One idea is that the scaffold defines the ends of loops. The scaffold also seems to mimic the shape of the metaphase chromosome, perhaps playing a key role in chromosome partitioning. 5. The E. coli chromosome contains ~4,000,000 base pairs. By what factor does it need to be compacted to fit into a 1 µm bacterial cell? 3.4 Å/bp x 4 x 106 bp/cell x 1 cell/10-6 m x 1 m/1010 Å = 13.6 x 102 = 1,360 or 1,360/2 since the DNA is a circle. 6. You clone the cDNA for human albumin (1800 bp) into the E. coli plasmid pBluescript (2961 bp) using the restriction enzymes BamH1 and EcoRI. The plasmid has the following structure: a) What is cDNA? A dsDNA copy of ssRNA. b) What are the recognition sites of EcoRI and BamHI? GAATTC and GGATCC b) Do these enzymes leave 5’ overhangs, blunt ends or 3’ overhangs? Both 5’ overhangs. c) Does the cDNA with EcoR1 and BamH1 ends go into the plasmid in a unique orientation? Why or why not? Yes, the overhangs are different, and they only hybridize with the corresponding overhang in the cut plasmid. d) What are the sizes of DNA fragments produced by digesting the plasmid with HindIII? ~1700 bp + ~3000 bp. HindIII and BamH1? ~1100 bp + ~600 bp + ~3000 bp. e) What are the functions of the Ampr and ori sequences in the plasmid? Ampr allows for selection for the plasmid by conferring ampicillin resistance, and ori mediates replication of the plasmid DNA.2007 2/2 7. How are synthetic DNA oligonucleotides used for site-directed mutagenesis? The oligonucleotides encode the mutation(s). They are used to prime synthesis of the entire mutated plasmid. 8. The most commonly mutated human disease gene is APC (adenomatous polyposis coli), which is altered in 75% of colon cancers. a) Using the web (e.g. Entrez-Protein), find the sequence for the human APC protein. What are the first 5 amino acids in the protein sequence? Met – Ala – Ala – Ala – Ser b) Find the nucleotide sequence of the APC gene (Hint: an accession number for the gene is gi:182396) and display it with the Graphics option in Entrez Nucleotide. Design two PCR primers to amplify the segment coding for the first 55 amino acids of the protein, starting with the initiator met (ATG) and ending with Ile55 (ATT). The primers should have calculated melting temperatures of ~56 ºC, using the formula, Tm=a(GC) x 4º + b(AT) x 2º, where a(GC) and b(AT) are the number of GC and AT base pairs, respectively. Forward: 5’- ATGGCTGCAG CTTCATATG – 3’, Reverse: 5’ – AATACTTCCT TGTAGTTGTT TA – 3’ c) Which region of the APC protein binds the EB1 protein (Hint: use BLAST, CDD or CDART to analyze the amino acid sequence. To insert the amino acid sequence into any of these programs, get the sequence in FASTA format with the Display pulldown menu on the Entrez Protein page. Pick FASTA and click Display.). The C-terminal 174 amino acids. d) Is the 3-dimensional structure of the EB1 binding domain of APC known? No. (Hint: BLAST the PDB (Protein Data Bank) database with the amino acid sequence of the EB1 binding region of APC (Accession # P25054). Insert this sequence in FASTA format on the BLAST page at NCBI and select pdb in the Database pulldown menu.) What is the closest match in the PDB to the EB1-binding domain of human APC? Depending on how you do it, you get either “no significant homology”, Pdz2 of Ptp-Bl or B. anthracis Soda-2. Is this quality of match statistically significant? No. the respective E-valued of 5.3 or 2.7 denote >5 or nearly 3 expected random hits of this quality. The accepted cut-off for a significant hit is E = 0.001. 9. The pedigree below is that of a family with Tay-Sachs disease: a) Is Tay-Sachs disease autosomal or sex-linked? Dominant or recessive? Autosomal recessive. b) Identify on the pedigree a female carrier, a male sufferer and a phenotypically normal male. I.1, II.4, II.2, respectively. c) What are the numerical probabilities that individuals II.2 and II.5 are carriers of the Tay-Sachs disease gene? 66.6 % d) How could you test the individuals in this family for the Tay-Sachs disease gene assuming that you know the sequence of the gene and the surrounding region in normal and affected individuals? Southern blot: Digest the DNA with a restriction enzyme that shows a RFLP linked with Tay- Sachs. Probe the blot with the DNA sequence that covers the RFLP. Or amplify the Tay-Sachs gene by PCR and sequence the


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Berkeley MCELLBI 110 - MCELLBI 110 Problem set 3

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