121 MCB Spring 2011 Problem Set 3 Problems 1-4 You are studying galactose metabolism in a new species of yeast. You have purified a novel protein important for galactose metabolism and have called it Gal400p. You have raised antibodies (Ab) against this protein and have carried out a Western blot experiment, where you have probed for the presence of this protein in cell extracts prepared from cells grown under different carbon sources (as indicated in the figure). Here is the data:
In an independent set of experiments, you have cloned what you believe to be the GAL400 gene by complementing the gal minus phenotype of a gal400 mutant with a plasmid from a genomic DNA library that contains a fragment of DNA prepared from a wild type strain of this yeast species. Below is a restriction map of this fragment of DNA that shows the location of several unique restriction sites (evenly spaced every 500 bp). Also shown on this map is the location of a large open reading frame (Gene X) that you believe is likely to correspond to the GAL400 gene.
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In a final set of experiments, you have carried out a Northern blot experiment to gain insight into the structure of the GAL400 mRNA as well as the nature of the regulation of the GAL400 gene. In this experiment, total mRNA was prepared from cells grown in the presence of either glucose or galactose, as indicated. Radioactive DNA probes were prepared using specific fragments of DNA prepared from by digestion of the plasmid with specific restriction enzymes, also as indicated. Below are the results of this experiment, where a dark band shows a positive signal on the Northern blot:
One final piece of information: other studies of this species of yeast have shown that all genes lack introns and that all mRNAs contain a 500 nucleotide 3' untranslated region (3' UTR) in the mRNA that follows each open reading frame (ORF). With all of this information at hand, answer the following questions (starting on the next page) about the gal400 mutant, the GAL400 gene, as well as the mRNA and protein encoded by this gene.
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1. Explain how the size of the protein that is recognized by the anti-Gal400p antibody and the size of the predicted ORF in the DNA fragment are consistent with your conclusion that the gene encoded in the DNA fragment is actually GAL400. Answer: The protein recognized by the antibody is 500 amino acids in length. This corresponds well to a 1.5 kb ORF sequence (1500 nucs 3 nucs/amino acid codon = 500 amino acids) 2. Given the results of both the Western and the Northern blots, what can you conclude about the nature of GAL400 gene regulation? In other words, under what carbon-source conditions is the Gal400p protein expressed? Do you think this regulation is likely to be at the step of RNA transcription or at the step of protein synthesis. Answer: The Gal400p protein is specifically expressed in the presence of galactose. This regulation is most likely to occur at the level of transcription since the results of the Northernblot indicate the that mRNA is present in samples prepared from cells grown in galactose but not in samples grown in glucose. Of course, there are other possibilities that we will learn about later in the quarter. For example the mRNA could be produced under both growth conditions but selectively degraded during growth in glucose. This is an example of what is termed "post-transcriptional regulation". 3. Given the results of the Northern blot experiment indicate on the DNA map below where the following features are encoded for the GAL400 gene: (1) The approximate locations of the translational start site (ATG codon), (2) the translational termination site (let us say that for this gene it is TAA), (3) the location of the 500 bp 3' UTR.
4. You and a friend are a hard working biochemists and have discovered a protein, which you call "Nodoze", that is expressed in the brains of former A+ students of MCB 121 (we won't go into the details of how you isolated this protein..). You decide that this would be a great product to market to other students, but that it would be much easier to express this protein in E. coli 3
using recombinant DNA technology, rather than continue your difficult biochemical preparation using native material. You have been able to obtain the sequence of two small peptide fragments of Nodoze: Sequence 1: Trp-Cys-Met-Trp-His Sequence 2: Met-Trp-Tyr-Gln-Trp a) Further experiments indicate that one of these sequences is likely to correspond to the extreme N-terminus of the protein while the other is likely to correspond to the extreme C-terminus of the protein. Which peptide is likely to be the N-terminal sequence? Sequence 2 as it has a methionine at the N-terminus b) You decide that you will take a PCR-based approach to clone the Nodoze gene. Your idea is to amplify the gene using human DNA and degenerate oligonucleotide primers. Your partner has used the peptide sequences and the genetic code to design four possible primers to use in PCR reactions. For each primer listed, nucleotides in parentheses mean that there is an equal mixture of those bases at that position in the primer; the total length of any given primer is 15 nucleotides. Primer 1: Primer 2: Primer 3: Primer 4: 5'-CCA(T/C)TG(G/A)TACCACAT-3' 5'-TGGTG(T/C)ATGTGGCA(T/C)-3' 5'-ATGTGGTA(T/C)CA(A/GTGG-3' 5-(G/A)TGCCACAT(G/A)CACCA-3'
Which set of primers has the best chance of amplifying your gene? Primer 3 + Primer 4 c) What source of human DNA is the best to use as a template with your primers in this PCR reaction, genomic DNA or cDNA? Why? cDNA would be best. Because of the likely presence of large introns in human genes, it would be unlikely that you would be able to PCR amplify the entire coding region if you used genomic DNA as a template. d) Amino acid analysis of your protein sample indicates that Nodoze is 300 amino acids in length. How many base pairs do you expect the product of your PCR reaction to be? 300 amino acids x 3 basepairs/amino acid codon = 900 base pairs
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