Bio lab 12 - Lab report PDF

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BIOL203- SPRING 2020 LAB12 LAB REPORT LINK TO THE WIKIPAGE EXERCISE FOR LAB 12: ht t p: / / di v e r g e . hun t e r . c un y . e du/ l a b wi ki / QuBi / mo dul e / bi o 203 l a b 12%E2%8 0%9 42 020

Exercise 1. Homology searching using BLAST

1. Species and strain: Mus musculus strain C57BL/6J (House

mouse) 2. Chromosome: 10 3. Length of your query sequence: 411 4. Percent identity, number of matched bases, and number of gaps between the matched sequences: 99% identity match, 402/403 bases matched, 0/403 gaps between the matched sequences.

1. Locus ID: NC_000076 2. Total length of the gene: 21884 bp 3. Number of exons: There are 11 exons. 4. How many mRNAs do you find for this sequence? How many exons are there for the smallest mRNA? Why are there several mRNAs shown for one gene? There are 4 mRNAs for this sequence. There are 7 exons in the smallest mRNA. There are multiple mRNA for 1 gene because each mRNA codes for different proteins which express the gene. Exercise 2. Explore the structure of human mdm2 gene

Gene name: MDM2 proto-oncogene Brief description of its function: Promotes tumor formation by targeting tumor suppressor proteins like p53, resulting in proteasomal degradation.

How many exons and introns does this transcript have? 11 exons, 10 introns How many nucleotides does the coding sequence contain? 1494 nt How many amino acids does it code for? 497 aa

Coordinates of exon 1: 1971, 2271 Coordinates of exon 2: 2988, 3072 Where does the highlighted sequence start, and in which exon? It starts from nucleotide 2992, and in exon 2. What does this point correspond to? This is the CDS portion of exon 2.

Gene structure from NCBI:

1.

Do all exon sequences code for proteins? Are there non-coding exons in mdm2, and if so which ones? No, not all exon sequences code for proteins. The very first exon of the mRNA sequence is a non-coding exon, as it is not on the CDS line.

2.

Copy/paste and align the first 5 bases of all introns. Which bases are conserved near intron start ("donor site")? It appears that g t a _ _ are the conserved bases of the intron start.

gtact gttag gtaag gtaat gtaag gtaag gtaat gtaat gtagt gtaag

3.

Do the same with the last 5 bases of all introns. Which bases are conserved near intron end ("acceptor site")? It appears that _ _ _ a g are the conserved bases near the intron end. tgtag tatag ttcag acaag ctcag cttag tccag tttag attag tgaag

Weblogo for donor site:

Weblogo for acceptor site:

Paste your amino acid sequences below- leave a blank line between them. >Human MDM2 >NP_002383.2 E3 ubiquitin-protein ligase Mdm2 isoform a [Homo sapiens] MVRSRQMCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTK RLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSENRCHLEGGSD QKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIRE ICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDE VYQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEK AKLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEF EREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPCPVCRQPIQM IVLTYFP

>Mouse MDM2 >NP_034916.1 E3 ubiquitin-protein ligase Mdm2 isoform 1 [Mus musculus] MCNTNMSVSTEGAASTSQIPASEQETLVRPKPLLLKLLKSVGAQNDTYTMKEIIFYIGQYIMTKRLYDEK QQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLSESRRQPEGGSDLKDPLQAPP EEKPSSSDLISRLSTSSRRRSISETEENTDELPGERHRKRRRSLSFDPSLGLCELREMCSGGSSSSSSSS SESTETPSHQDLDDGVSEHSGDCLDQDSVSDQFSVEFEVESLDSEDYSLSDEGHELSDEDDEVYRVTVYQ TGESDTDSFEGDPEISLADYWKCTSCNEMNPPLPSHCKRCWTLRENWLPDDKGKDKVEISEKAKLENSAQ AEEGLDVPDGKKLTENDAKEPCAEEDSEEKAEQTPLSQESDDYSQPSTSSSIVYSSQESVKELKEETQDK DESVESSFSLNAIEPCVICQGRPKNGCIVHGKTGHLMSCFTCAKKLKKRNKPCPVCRQPIQMIVLTYFN

>Zebrafish MDM2 >NP_571439.3 E3 ubiquitin-protein ligase Mdm2 isoform 2 [Danio rerio] MATESCLSSSQISKVDNEKLVRPKVQLKSLLEDAGADKDVFTMKEVMFYLGKYIMSKELYDKQQQHIVHC GEDPLGAVLGVKSFSVKEPRALFALINRNLVTVKNPESQSTFSEPRSQSEPDRGPGDTDSDSRSSTSQQQ RRRRRSSDPESSSAEDESRERRKRHKSDSFSLTFDDSLSWCVIGGLHRERGNSESSDANSNSDVGISRSE GSEESEDSDSDSDNFSVEFEVESINSDAYSENDVDSVPGENEIYEVTIFAEDEDSFDEDTEITEADYWKC PKCDQFNPPLPRHCKTCWTVRADWLPETHSNWENLSRNTRTNPEDTSVTTTPNTTFEKKLSKPSSPLPET DDGVDVPDGKCFPSPATTKDELPSSATITDSQTTSSQPSTSSGGGSSQEETPELERFNSLEACLPATCLE PCVICQSRPKNGCIVHGRTGHLMACYTCAKKLKNRNKLCPVCREPIQSVVLTYMS

Paste your alignment output on the next page:

1. Explain the BLAST term: “Expect” (e-value) Read this FAQ The e-value is a measurement that allows us to see how many hits are expected that are random/unrelated. It is best to have a smaller e-value, as that would cause more significant hits to be seen. 2. Which is a statistically more significant match by BLAST, a match with an e-value=1e-5 or a match with an e-value of 1? An e-value of 1e-5 is statistically more significant than an e-value of 1. 3. List and describe individual elements of a typical human gene based on mdm2. MDM2 is a protein that interacts with several individual elements in a typical human gene. MDM2 interacts with promoters, enhancers/silencers and other substrates. Activated oncogenes lead to the activation of ARF, which binds to MDM2. A tumor suppressor protein, p53, is inhibited by high concentrations of MDM2. This allows normal cellular growth and development.

4. What are two determinants that can lead to the production of isoforms for a specific locus? Two determinants that could lead to production of isoforms for a specific locus are the start sites for transcription, as well as differing protein coding sequences. These have the possibility to change the function of a given gene. 5. What is the "GT-AG" rule? Explain how to read the sequence logos. Explain the significance of sequence conservation at exon-intron junctions. The GT-AG rule is the common patter for intron junctions to start with GT, and finish with AG, at the donor/acceptor splice sites. This is significant because post-transcriptional mRNA modification is dependent upon splice sites that occur upon conserved regions for spliceosome recognition. 6. Look at your alignment from part III: what are the black boxes- the grey boxes? The black boxes are regions where the residues of the three genes are conserved. The gray boxes are regions where there are conserved mutations. 7. do you see many gaps/insertions? Do you think there is a pattern? There are multiple areas where there are gaps and insertions in the alignment, however there is not a visible pattern. The more conserved regions are the ones that are more vital in coding for their specific proteins, and as a result have less gaps/insertions....