Molecular Biology-2014
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 Mel1 and actin 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. Your table must include the raw
data (the values for each of the areas) , the normalized values (Mel1/actin) 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. (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. 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-2014
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 ORF 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 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?
 Indicate the number of conserved, semi-conserved and non-conserved amino
acid changes
5. What three conserved protein domains are present in the unknown human sequence?
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.
This sequence is from what gene?
What is the accession number of this gene?
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 the two orthologues?
How many times do BamHI and HaeIII cut within the gene sequence obtained in
question 1?
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Molecular Biology-2014
7. Which of the following primers would hybridize to the mRNA of the gene sequence
obtained in question 1 which would allow you to perform a reverse transcriptase
reaction?
A.
B.
C.
D.
CAACCCATCACACAAAAC
TGGCAATGCGAGGCAGCG
CCATGTTTGTAGTGTCAG
GGCAATTCTTGGCGTGGAC
8. The sequence on the following page represents the gene discussed which was
obtained from a patient. Does it contain one or more SNPs as compared to the
sequence corresponding to that obtained from the NCBI site? If so, what are they?
Indicate the position as well as the base change (Ex. G218 to C)
9. Do these SNPs change the reading frame of the gene?
10. How long is the protein encoded by the gene sequence obtained in question 1?
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Molecular Biology-2014
GGCTCGCTGCCTCGCATTGCCACAGGCTCCTGAGAGGTCGCGGGCAGTGCCTGCGGGGAGGCGCGGGGCC
CTGCTCTGTAGGGCTGAAGGCCGCCCGAGGTTCGCCAAGGCTCTGGGCTCTCGAAAGGAAGCCAAGAAAA
GAAGCTGCCCAGGTGACCAGTCCTGGGAGTGCTCTCTCTCAAGGAAGCTCCGAGCGCCCAGGAGCCCTTA
GCCGGGGTCCAGTGCCCTTTGAACAATCTCCAGCTGTTCAAGGAAGTGGGGCGCCGCCGCCTCTCTTGGG
ACCTGGCCTGGGATCCTTTCCCCAAACGCACCCCGGCGATTTTTGCGCACCGCGAGCCGAACCCCTGCTG
CGCGCAGCTGGCTGGGCTCAGGCGCGCTTCCTCAACGTTTCGGAGCCGCTGCCCCCAGCGAAGTCCACAT
TCCAAGCTCCAGGGGCTTTGAGAGAGACGACCCCAAGGCAAGGCGTTTGGAGAGCTGCTGAGGAGCCAGG
GGCTTGGAGGAGCGAGAAGACATGTATTTTCAGCTGAGTCTCAGAAGGGGAGAATCTCCTGTCACCACCA
GAAAAGCAACAGCCCCGAAATGTCATTGCAACTGACTAGCAGAGCAGAGGCCCAGGAGTCACTGGATTGA
TGATTTAGAATATGCTAAAAAGCCAGTGCTTTATTTGGGGAATTCAGGGGCTTTCTGGTGCCCAAGACAG
TGACCTGCAGCAAGGGAGTCAGAAGACAGATGTAGAAATCAAGAGTGACCATCCACGGGATTGACTTGGA
TTGCCACTCAAGCGGTCCTCTCATGGAATGTTGGTGAGGCCCTCTGCCAGGGAAGCAATCTGGCTGTGCA
AAGTGCTGCCTGGTGGGGAGGACTCCTGGAAATCTGACTGACCCCTATTCCCTGCTTAGGAACTTGAGGG
GTGTCAGAGCCCCTGATGTGCTTTCTCTTAGGAAGATGAGGACTCTGAACACCTCTGCCATGGACGGGAC
TGGGCTGGTGGTGGAGAGGGACTTCTCTGTTCGTATCCTCACTGCCTGTTTCCTGTCGCTGCTCATCCTG
TCCACGCTCCTGGGGAACACGCTGGTCTGTGCTGCCGTTATCAGGTTCCGACACCTGCGGTCCAAGGTGA
CCAACTTCTTTGTCATCTCCTTGGCTGTGTCAGATCTCTTGGTGGCCGTCCTGGTCATGCCCTGGAAGGC
AGTGGCTGAGATTGCTGGCTTCTGGCCCTTTGGGTCCTTCTGTAACATCTGGGTGGCCTTTGACATCATG
TGCTCCACTGCATCCATCCTCAACCTCTGTGTGATCAGCGTGGACAGGTATTGGGCTATCTCCAGCCCTT
TCCGGTATGAGAGAAAGATGACCCCCAAGGCAGCCTTCATCCTGATCAGTGTGGCATGGACCTTGTCTGT
ACTCATCTCCTTCATCCCAGTGCAGCTCAGCTGGCACAAGGCAAAACCCACAAGCCCCTCTGATGGAAAT
GCCACTTCCCTGGCTGAGACCATAGACAACTGTGACTCCAGCCTCAGCAGGACATATGCCATCTCATCCT
CTGTAATAAGCTTTTACATCCCTGTGGCCATCATGATTGTCACCTACACCAGGATCTACAGGATTGCTCA
GAAACAAATACGGCGCATTGCGGCCTTGGAGAGGGCAGCAGTCCACGCCAAGAATTGCCAGACCACCACA
GGTAATGGAAAGCCTGTCGAATGTTCTCAACCGGAAAGTTCTTTTAAGATGTCCTTCAAAAGAGAAACTA
AAGTCCTGAAGACTCTGTCGGTGATCATGGGTGTGTTTGTGTGCTGTTGGCTACCTTTCTTCATCTTGAA
CTGCATTTTGCCCTTCTGTGGGTCTGGGGAGACGCAGCCCTTCTGCATTGATTCCAACACCTTTGACGTG
TTTGTGTGGTTTGGGTGGGCTAATTCATCCTTGAACCCCATCATTTATGCCTTTAATGCTGATTTTCGGA
AGGCATTTTCAACCCTCTTAGGATGCTACAGACTTTGCCCTGCGACGAATAATGCCATAGAGACAGTGAG
TATCAATAACAATGGGGCCGCGATGTTTTCCAGCCATCATGAGCCACGAGGCTCCATCTCCAAGGAGTGC
AATCTGGTTTACCTGATCCCACATGCTGTGGGCTCCTCTGAGGACCTGAAAAAGGAGGAGGCAGCTGGCA
TCGCCAGACCCTTGGAGAAGCTGTCCCCAGCCCTATCGGTCATATTGGACTATGACACTGATGTCTCTCT
GGAGAAGATCCAACCCATCACACAAAACGGTCAGCACCCAACCTGAACTCGCAGATGAATCCTGCCACAC
ATGCTCATCCCAAAAGCTAGAGGAGATTGCTCTGGGTCTTGCTGTTAAGAAACTAAGGTACGGTGAGACT
CTGAGGTGTCAGGAGAGCCCTCTGCTGCTTTCCAACACACAACTAACTCTGTTTTCAAATACATTCCAGT
GTATTTTCTGTGTTGTTCATAGTCAATCAAACAGGGACACTACAAACATGGGGAGCCATAAGGGACATGT
CTTTGGCTTCAGAATTGTTTTTAGAAATGTATTCTTATCTTAGGATTTACCAAATAGGGCAAAGAATCAA
CAGTGAACAACTTCACTTAAAATCAAATTTTTCTGGGAAGAAAAGGAGATGGGTTGAGTTTGCTGTATAC
AAACAGGTGCTAACACTGTTCCCAGCAAAGTTTTCAGATTGTAAAGGTAGGTGCATGCCTTCATAAATTA
TTTCTAAAACATTAATTGAGGCTTACAGTAGGAATGAGAAATTTTTTTCCAGAATTGAGAGATGTTTTGT
TGATATTGGTTCTATTTATTTATTGTATATATGGATATTTTTAATTTATTATATAATAAATATATATTTA
TCATATTTAATAGGATAAATTAATGAGTTTTATCCAAGACCTTACAACCACATTTCTGGCCATTTAACTA
GCACTTTATAAGCCAATGAAGCAAACACACAGACTCTGTGAGATTCTAAATGTTCATGTATAACTTCTAG
AAACACAGCAGAAACTGATAAGGGAATAAAGTTGAAATGATTCCTTAAAATTCATGGACACAGATAAATG
CAAGGTGAGAATTGACAAATGCTATAAATGCTTTCTTTTTCTGAAAAGATTTTGAAAAATTTAAAAAAGT
GTAGCTACTGCTGTGTTCAAAACGTTTTAAATGACAAAAGACTTTCCCAGGGGAATTTGCAGTTCTGTAA
ATATCTTAAATAAAAGCCAACTTAAGAAGAACCCAGCATTAAATTTACGATCTTAGGTGGTAATGAAAAG
TATATGCTGCTTTGTATTTATGTAAAATAATTGGCCCTCTCCATCTTTTCTCATTTCATGTGTCAGGTAG
TTTTTCTGA
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