Name Section Problem Set 3

Transcription

1 Name Section Problem Set 3 The completed problem must be turned into the wood box outside by 4:40 pm, Thursday, March 13. Problem sets will not be accepted late. Question 1 Based upon your impressive performance in you were hired to work in a rare yeast lab where you immediately isolate an interesting new strain with a dominant mutation that causes the yeast to glow in the dark. You map the mutation to a gene you name ID1. Putting the genetics you learned in to good use, you were able to determine that ID1 is tightly linked to LE2 and HIS3. (LE2 and HIS3 mutants require leucine and histidine in their medium for growth, respectively). Recombination Frequency between ID1 and LE2 = 0.1% Recombination Frequency between ID1 and HIS3 = 0.3% Recombination Frequency between LE2 and HIS3 = 0.4% a) Draw the genetic map between ID1, LE2 and HIS3 labeling the distance between them in cm or map units. LE2 ID1 HIS3 0.1cM 0.3cM You are able to obtain the genomic sequence flanking the outside ends of the LE2 and HIS3 genes. See below. 5 TTT...TTTT 3 3 TTTTT...TTTTTTT 5 b) From the genome of your mutant glowing strain, you wish to amplify the region marked by dots in the sequence above which contains the ID1 mutant gene, and the wild type LE2 and HIS3 genes. Design primers each 20 nucleotides long to perform PR (polymerase chain reaction) that will amplify this center region. 5 TTT 3 3.TTTTTTT 5 1

2 Question 2 fter successfully amplifying your genome fragment containing the ID1 mutant gene, you plan to clone the gene that causes the glowing phenotype into a plasmid for further study. Your boss, Dr. Ngo gives you a vector pset3 with the following cloning sites,, and. Before setting out to clone the fragment into the plasmid you decide to hone up on your restriction mapping skills and check which of the restriction sites will work best for cloning. iven below are the results of several different single and multiple restriction digests of your PR product. The digests are loaded onto an agarose gel and subject to electrophoresis to separate the fragments according to size. Below is what the gel looks like after staining to see the DN fragments. MW Markers ndigested control a) Draw the restriction map of your PR product. Label the distance between each restriction site in bp (basepairs)

3 fter an arduous semester of cloning, you were able create 4 different plasmids each with a unique fragment isolated from the bands on the previous gel. One plasmid has the 1000 bp fragment insert, another has the 400 bp insert, another with the 600 bp band, and another with the 2000 bp insert. You observe that plasmids containing these fragments when transformed into a LE2, HIS3, ID1 haploid yeast strain confer the phenotypes shown below. (This strain requires leucine and histidine for growth, and it does not glow.) The chart below shows the phenotypes of the strain transformed with the plasmids carrying the different inserts. indicates the ability to grow without that amino acid present in the medium or the ability to glow in the dark. indicates that the strain is unable to grow without the amino acid or is unable to glow in the dark. ability to grow without added Leucine ability to grow without added histidine ability to glow in the dark Plasmid with a fragment insert whose size is B B b) Based on the results above, position the ID1, HIS3, and LE2 genes in your restriction map drawn in a). LE2 ID1 HIS c) Based on b), fill in the chart above with the predicted phenotype of the rest of the fragments (if they were individually cloned and transformed into the Leu His nonglowing strain). B LE2 ID1 HIS

4 Question 3 Now that you have narrowed the ID1 gene to a particular fragment, you wish to determine the DN sequence of the wild type ID1 gene with the help of the biosequencing center. You isolate the ID1 gene, send it to them, and they return the following sequence * * * * * 5...TTTTTTTTTTTTTTTTTTT 3...TTTTTTTTTTTTTT * * * * * * TTTTTTTTTTT TTTTTTTTTTTTTTTT * * * * TTTTTTTTTTTTTT...3 TTTTTTTTT...5 a) ssuming that transcription starts at the first nucleotide (position 1) and translation begins at first, what is the predicted amino acid sequence of the id1 protein. (SE the single letter code shown in the genetic code chart, and label the N and termini of your predicted protein.) NH 3 MERWLIHT OO 4

5 b) While you were busy cloning the mutant ID1 gene into pset3, your friend Bob, the biochemist, had purified the id1 protein from wild type cells and has determined that its amino acid sequence is... NH 3 MERWDRK OO Based on Bob s result, circle the intron(s) of ID1 in the sequence on the previous page. phe F ser S tyr Y cys phe F ser S tyr Y cys leu L ser S STOP STOP leu L ser S STOP try W leu L pro P his H arg R leu L pro P his H arg R leu L pro P gln Q arg R leu L pro P gln Q arg R ile I thr T asn N ser S ile I thr T asn N ser S ile I thr T lys K arg R met M thr T lys K arg R val V ala asp D gly val V ala asp D gly val V ala glu E gly val V ala glu E gly Question 4 rmed with the knowledge of the sequence of ID1, you decide to sequence the same region from 4 new mutant ID1 strains. a) ircle the primer that will allow you to sequence all the exons of the ID1 gene. 5 TTT3 5 T3 5 TTTTT3 5 T3 b) Since ID1 is so small, you decide to try your hands at sequencing. You sequence the beginning of the ID1 in your 4 mutants. Shown below is the start of the first exon. ID11 ID12 ID13 ID14 T T T T 5

6 In the table below write the DN and deduced protein sequence for the four mutants. ssume that the primer you chose will sequence the coding strand (not the template strand) and that translation will start at the first of the message. Label the 5 and 3 ends of the DN sequence and N and termini of peptide you translate. lassify the mutations as (S=silent, N=nonsense, M=missense, ND/or F=frameshift). ssuming the unsequenced regions have no mutations, indicate whether a strain with this mutation will glow in the dark or not. (Y= yes, N= no or D = can t be determined). Mutant DN sequence Protein Sequence (se single letter code) Type of mutation lows in the dark? ID11 T TT T MELW M Y ID12 T T T T ME N D ID13 T TT METL F D ID14 T T T MERW S N Wildtype T T T MERW none N Okay, we re told that the first mutant we were working with on page 1 had a dominant mutation. Further evidence of this was on page 3, where we introduce our cloned DN fragments derived from the mutant and transform ID1 strain (nonglowing) and see that the fragment carrying the mutant gene confers a glowing phenotype. So when we have one wildtype copy and one mutant copy the yeast glowsdominant mutation. What are the reasons for a dominant mutation? One possibility is that the wildtype protein is a multimer, like a dimer. In the previous complementation assay, the mutant protein binds to the wildtype protein and prevents it from functioning. That would mean that the role of the wildtype protein would be to block somehow the cell from glowing. (sually we don t work with dominant mutations unless we know they are in a gene of interest.) On this page we re looking at 4 different mutants. (We know they are different because the amino acids don t match the previous mutant.) We are given the information that ID11 glows in the dark. Okay maybe it doesn t fold properly and thus can t perform the wildtype function (to prevent glowing). If this is true than probably ID12 and ID13 mutants would glow as well. Mutant 2 is only 2 amino acids, almost like a null mutant. Mutant is a frameshift, so it doesn t look at all like the original protein. Mutant ID14 looks just like wildtype (silent mutation) so it should NOT glow. Okay now what if the mutants that glow do so because of a gainof function. That is, their mutant protein does something that the wildtype protein does not do. If this is the case, then mutants ID2, and ID 3 will probably NOT glow. Okay this is very complex and I assure that nothing this complex will ever rear its face on a quiz, but the writer of this problem set thought that you would benefit from the twists. 6