Molecular Biology-2013
Assignment #6
Exercise 9
Transcriptional control of Mel1 (Northern analysis)
1. Submit a figure of your Northern hybridizations with an appropriate figure legend.
2. Obtain densitometric data for the ribosomal RNA on your RNA gels as well as the
mRNA signals obtained at the different time points following the transfer to galactose
or glucose + galactose. Submit a table of the raw data obtained with Image J for the
Northerns and the corresponding RNA gel (exercise 7). Your table must include the
raw data (the values for each of the areas) , the normalized values (Mel1/rRNA) for
each of the time points and the relative expression as compared to the zero time point
levels of Mel1 at the different time points following the transfer. (Ex. Normalized
value time 1 hour/normalized value time 0).
Site directed mutagenesis of LacZ: Beta-galactosidase assay
3. Submit a table of the data and corresponding analysis of the beta-galactosidase
assays. Your table should include:
a. Average absorbencies and OD600 values for each of the assayed mutants for
each dilution assayed.
b. The calculated activity units for each mutant.
c. The relative activity levels as compared to the pUC control.
d. The type of amino acid substitution each mutant represents (ex. conserved,
semi conserved, etc.)
e. A caption indicating which mutant showed the greatest relative reduction in
activity.
Translational control of Mel1
4. Submit a table of the data and corresponding analysis of the alpha-galactosidase
assays. Your table should include:
a. Absorbencies and OD600 values for each of the assayed dilutions of the
respective cultures, obtained for the alpha galactosidase assay representing the
different time points following the transfer to galactose or glucose + galactose.
b. The calculated activity units for each time point.
c. The relative activity levels following the transfer as compared to time 0.
d. A caption indicating whether the differences in the activity levels observed are
consistent with the levels of transcript observed.
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Molecular Biology-2013
Bioinformatics:
1. Submit the annotated sequence (nucleotide with corresponding amino acids) of the
longest ORF found for the viral1 sequence.
2. Does the sequence submitted in the previous question represent the genome sequence
of the virus, the mRNA sequence or both?
3. Submit the following information for the longest ORFs from the viral1 and viral2
sequences:
 The definition
 The organism this gene comes from
 The name of the gene (Not the gene product)
 The name of the gene product
4. Submit the following information for the viral3 sequences:
 What SNPs (single nucleotide polymorphisms) has the viral3 sequence acquired?
 What is the percentage identity at the nucleotide level between the viral1 and
viral3 sequences?
 What is the percentage identity at the protein level between the two sequences?
 What type of amino acid changes have occurred: conserved, semi-conserved or
non-conserved?
5. Amongst the SNPs found in the viral 3 sequence, do all of them change the coded
amino acid? If not, give a specific example where this was not the case. Include the
original codon, the new codon, and the amino acid in both cases.
6. What three conserved protein domains are present in the unknown human sequence?
Bonus (2.5 points total on this assignment)
You should now be quite familiar with the NCBI site and be able to complete the
following exercise with relatively few directives. Consider this as a practice run for
the bioinformatics section on the final exam.
GGCTCGCTGCCTCGCATTGCCACAGGCTCCTGAGAGGTCGCGGGCAGTGCCTGCGGGGA
GGCGCGGGGCCCTGCTCTGTAGGGCTGAAGGCCGCCCGAGGTTCGCCAAGGCTCTGGGC
TCTCGAAAGGAAGCCAAGAAAA
1.
2.
3.
4.
5.
6.
7.
This sequence is from what gene?
What is the accession number of this gene?
This sequence is from what organism?
What is the accession number of this protein?
Give the accession number of a rat (Rattus Norvegicus) protein orthologue.
What is the percent identity between the human and rat protein orthologues?
How many times do BamHI and HincII cut within the sequence of the human gene?
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Molecular Biology-2013
8. Which of the following primers would hybridize to the mRNA of the human gene and
allow you to perform a reverse transcriptase reaction?
A.
B.
C.
D.
CAACCCATCACACAAAAC
GCATTGTAGCTTGTGTTGC
CTGACCGTTTTGTGTGATG
GCTTATTACGGTATCTCTG
9. The sequence on the following page was obtained from a patient. Does it contain one
or more SNPs as compared to the sequence obtained in question 1? If so, what are
they? Indicate the position as well as the base change (Ex. G218 to C)
10. Do these SNPs change the reading frame of the gene?
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Molecular Biology-2013
ATGAGGACTCTGAACACCTCTGCCATGGACGGGACTGGGCTGGTGGTGGAGA
GGGACTTCTCTGTTCGTATCCTCACTGCCTGTTTCCTGTCGCTGCTCATCCTGT
CCACGCTCCTGGGGAACACGCTGGTCTGTGCTGCCGTTATCAGGTTCCGACAC
CTGCGGTCCAAGGTGACCAACTTCTTTGTCATCTCCTTGGCTGTGTCAGATCT
CTTGGTGGCCGTCCTGGTCATGCCCTGGAAGGCAGTGGCTGAGATTGCTGGCT
TCTGGCCCTTTGGGTCCTTCTGTAACATCTGGGTGGCCTTTGACATCATGTGCT
CCACTGCATCCATCCTCAACCTCTGTGTGATCAGCGTGGACAGGTATTGGGCT
ATCTCCAGCCCTTTCCGGTATGAGAGAAAGATGACCCCCAAGGCAGCCTTCA
TCCTGATCAGTGTGGCATGGACCTTGTCTGTACTCATCTCCTTCATCCCAGTG
CAGCTCAGCTGGCACAAGGCAAAACCCACAAGCCCCTCTGATGGAAATGCCA
CTTCCCTGGCTGAGACCATAGACGACTGTGACTCCAGCCTCAGCAGGACATA
TGCCATCTCATCCTCTGTAATAAGCTTTTACATCCCTGTGGCCATCATGATTGT
CACCTACACCAGGATCTACAGGATTGCTCAGAAACAAATACGGCGCATTGCG
GCCTTGGAGAGGGCAGCAGTCCACGCCAAGAATTGCCAGACCACCACAGGT
AATGGAAAGCCTGTCGAATGTTCTCAACCGGAAAGTTCTTTTAAGATGTCCTT
CAAAAGAGAAACTAAAGTCCTGAAGACTCTGTCGGTGATCATGGGTGTGTTT
GTGTGCTGTTGGCTACCTTTCTTCATCTTGAACTGCATTTTGCCCTTCTGTGGG
TCTGGGGAGACGCAGCCCTTCTGCATTGATTCCAACACCTTTGACGTGTTTGT
GTGGTTTGGGTGGGCTAATTCATCCTTGAACCCCATCATTTATGCCTTTAATG
CTGATTTTCGGAAGGCATTTTCAACCCTCTTAGGATGCTACAGACTTTGCCCT
GCGACGAATAATGCCATAGAGACGGTGAGTATCAATAACAATGGGGCCGCG
ATGTTTTCCAGCCATCATGAGCCACGAGGCTCCATCTCCAAGGAGTGCAATCT
GGTTTACCTGATCCCACATGCTGTGGGCTCCTCTGAGGACCTGAAAAAGGAG
GAGGCAGCTGGCATCGCCAGACCCTTGGAGAAGCTGTCCCCAGCCCTATCAG
TCATATTGGACTATGACACTGACGTCTCTCTGGAGAAGATCCAACCCATCACA
CAAAACGGTCAGCACCCAACCTGA
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