Slide 1

The Next Three Weeks: Today: Sanger Sequencing Central Dogma Overview Mutation Unpacking Central Dogma Transcription (Mar 28) RNA Processing (Mar 28) Translation (Mar 28 / Apr 2) [+ Sculpting] Regulation of Gene Expression + Trivia (Apr 4) Tutorial (Apr 5) Review (Apr 9) EXAM 3 (Apr 11)

Slide 2

Sanger Sequencing

Slide 3

dideoxyNucleoside TriPhosphate

Slide 4

Slide 5

2011 Pearson Education, Inc.

Slide 6

Slide 7

Slide 8

Slide 9

Slide 10

Central Dogma Overview

Slide 11

2011 Pearson Education, Inc.

Slide 12

The Language of Genetics DNA Language vs. Protein Language Discuss: What does it mean to say that DNA is written in one language and Proteins are written in a different language?

Slide 13

DNA Language 3 GTGCA 5 5 CACGT 3 The language of DNA is written in nucleotides.

Slide 14

Protein Language A protein is a chain of amino acids.

Slide 15

Connecting the Languages Transcribing versus Translating -Transcribe means: to rewrite Transcription Example: 3 parts gravel, 2 parts sand, 1 part cement.

Slide 16

An Academic Transcript: A copy of your grades

Slide 17

Connecting the Languages Transcribing versus Translating -Transcribe means: to rewrite -Translating means: to change language Translation Example: 3 parts gravel, 2 parts sand, 1 part cement. 3 partes de grava, 2 partes de arena, 1 parte de cemento.

Slide 18

The Language of Genetics DNA Language vs. Protein Language DNA is written in the language of nucleotides. A message on DNA can be transcribed (copied) onto a piece of mRNA. A message on mRNA can be translated into a chain of amino acids. Proteins are written in the language of amino acids.

Slide 19

DNA mRNA Amino Acids Transcribing: Replace Thymine with Uracil. Transcribe from the template strand.

Slide 20

DNA mRNA Amino Acids 5 CCACTGATAGACCTT 3 3 GGTGACTATCTGGAA 5 mRNA is made using the template strand Template Strand non -Template Strand

Slide 21

DNA mRNA Amino Acids 5 CCACTGATAGACCTT 3 3 GGTGACTATCTGGAA 5 5 CCACUGAUAGACCUU 3 The mRNA sequence is nearly identical to the non-template strand. Transcribing complete. Template Strand non -Template Strand mRNA

Slide 22

2011 Pearson Education, Inc. The Translation Key:

Slide 23

DNA mRNA Amino Acids Non-Templ: 5 CCACTGATAGACCTT 3 Template: 3 GGTGACTATCTGGAA 5 mRNA: 5 CCACUGAUAGACCUU 3 Pro - Leu - Ile - Asp - Leu

Slide 24

Transcribe/Translate the Following Sequences: 5 CTGCGTGACTGCAAA 3 3 GACGCACTGACGTTT 5 3 CCGACTCACTGATGC 5 3 ATGTTTGAACTACAG 5 5 TACAAACTTGATGTC 3

Slide 25

Important Properties of the Code It is redundant : All amino acids except two are encoded by more than one codon. It is unambiguous: One codon never codes for more than one amino acid. It is nearly universal: With a few minor exceptions, all codons specify the same amino acids in all organisms. It is conservative: The first two bases are usually identical when multiple codons specify the same amino acid.

Slide 26

Mutations changes to the code

Slide 27

Whats a Mutation? A mutation is any permanent change in an organisms DNA. Mutations result in new alleles!

Slide 28

Point Mutations Point mutations occur when the DNA polymerase inserts the wrong base into the newly synthesized strand of DNA.

Slide 29

Point Mutations Point mutations may be as a result of a substitution or an insertion/deletion and may be: Silent mutations. Does not change the amino acid sequence of the gene product. Missense mutations. Result in changes in the amino acid sequence of the encoded protein. Nonsense mutations. Results in a stop codon.

Slide 30

Mutations and their impacts

Slide 31

Slide 32

Slide 33

Slide 34

Slide 35

Slide 36

Describing a Mutation: Three Categories: 1.What happened? Insertion, deletion, substitution. 2.What was the impact on the protein? Missense, nonsense, silent 3.What was the reading frame? Frameshift

Slide 37

Two Examples of mutation 1.Beach Mice (from the text) 2.Taste in peas

Slide 38

Peromyscus polionotus

Slide 39

Fact Sheet: Peromyscus polionotus Common names: Beach Mouse or Old Field Mouse. Location: Southeastern U.S.A. Habitat: Sand burrows in dunes or old fields. Home-range: ~1000 m 2 Breeding: Monogamous pair- bonding. Litters of 2-8 pups, every 30 days. Lifespan: 9-12 months.

Slide 40

Fur Color:

Slide 41

The mc1r Gene The mc1r gene is located in Chromosome #16 in mammals. It codes for the MC1R protein that aids in pigment synthesis.

Slide 42

2011 Pearson Education, Inc.

Slide 43

mc1r Gene Sequence 5TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAA CTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCA GACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCT TCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTC GTGATAGCCATCACCAAAAACCGCAACCTGCACTCGCCCATGTATT CCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAG CTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGG CCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTC ATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCC TTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTA CGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATC GTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCA CCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTT TCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTC ACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAG AGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGC CACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCC TTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCA CCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCAT CATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGC CAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTG GTGA 3

Slide 44

mc1r Gene Sequence 5TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAA CTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCA GACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCT TCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTC GTGATAGCCATCACCAAAAACTGCAACCTGCACTCGCCCATGTATT CCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAG CTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGG CCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTC ATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCC TTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTA CGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATC GTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCA CCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTT TCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTC ACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAG AGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGC CACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCC TTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCA CCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCAT CATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGC CAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTG GTGA 3

Slide 45

Single Substitution 5TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCC CTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGG CCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACC AAAAACTGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATG GTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGA CCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGT GTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGT TATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCA TCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTT CTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCA TGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAG GGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCAT CTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCT CTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGA GCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3 -Substituting 1 of 954 nucleotides -Cytosine to a Thymine (Pyrimidine Pyrimidine) -Changed the mRNA codon from CGC to UGC

Slide 46

2011 Pearson Education, Inc. The Translation Key:

Slide 47

Consequence of Mutation A single nucleotide mutation from a Cytosine to a Thymine leads to An amino acid change from an Arginine to a Cysteine Amino Acid Sequence Dark Fur: MPTQGPQKRLLGSLNSTSTATPHLGLATNQTGPWCLQVSIPDGLFLSLGLVSLVENVL VVIAITKN R NLHSPMYSFICCLALSDLMVSISLVLETAIILLLEAGALVTRAALVQQLDNVI DVLICGSMVSSLCFLGVIAIDRYISIFYALRYHSIVTLPRARRAIXGIWVASIFFSTLFITYY NHTAVLICLVTFFLAMLALMAXLYVHMLTRAYQHAQGIAQLQKRQGSTXQGFCLKGAX TLTIILGIFFLCWGPFFLHLTLIVLCPQHPTCSCIFKNFNLYLVLIIFSSIVDPLIYAFRSQEL RMTLREVLLCSW Amino Acid Sequence Light Fur: MPTQGPQKRLLGSLNSTSTATPHLGLATNQTGPWCLQVSVPDGLFLSLGLVSLVENVL VVIAITKN C NLHSPMYSFICCLALSDLMVSISLVLETAIILLLEAGALVTRAALVQQLDNVI DVLICGSMVSSLCFLGVIAIDRYISIFYALRYHSIVTLPRARRAIVGIWVASIFFSTLFITYY NHTAVLICLVTFFLAMLALMAILYVHMLTRAYQHAQGIAQLQKRQGSTRQGFCLKGAAT LTIILGIFFLCWGPFFLHLTLIVLCPQHPTCSCIFKNFNLYLVLIIFSSIVDPLIYAFRSQELR MTLREVLLCSW

Slide 48

Changing 1 amino acid: Arginine: Strongest + charge Very hydrophilic Cysteine: Not hydrophilic Forms disulfide bonds

Slide 49

Two Examples of mutation 1.Beach Mice -Missense substitution mutation of one nucleotide C T -Changes one amino acid: Arginine Cysteine -Changes the function of the MC1R protein 2.Taste in peas

Slide 50

Mendels Peas

Slide 51

Fact Sheet: Pisum sativum Common names: Field peas. Native to: Near East Iraq, Turkey, Israel, Palestine, etc. A key crop of the Neolithic Agricultural Revolution, oldest recorded samples date to 10,000 BCE.

Slide 52

The Evolution of Wrinkled Peas Wrinkled peas taste sweet. WrinkledRound

Slide 53

Molecular Biology: Central Dogma The R allele of the sbe1 gene codes for the SBE1 protein. The SBE1 protein is an enzyme that converts simple starch (amylose) to complex starch (amylopectin).

Slide 54

Inside the Pea Cell: Sugar and Starch

Slide 55

Slide 56

Slide 57

Slide 58

Sucrose

Slide 59

The Evolution of Wrinkled Peas Wrinkled peas arent just wrinkled they taste good too! Wrinkled (r allele of sbe1 gene) Round (R allele of sbe1 gene) G3P (Intermediates) Amylose Amylopectin G3P (Intermediates) Amylose non-functioning starch branching enzyme Sucrose functioning starch branching enzyme

Slide 60

The Genetics of Round and Wrinkled Peas

Slide 61

The sbe1 Gene The sbe1 gene has two alleles: Wrinkled Pea Codes for functional SBE1 protein. Round Pea Codes for non- functional SBE1 protein.

Slide 62

3549 nucleotides What does the R allele represent? Round Pea

Slide 63

The R Allele These nucleotides code for a polypeptide protein of about 920 amino acids in length:

Slide 64

What does the R allele do? How does the SBE protein lead to round seeds? It gives rise to highly branched starch (amylopectin). Highly branched starch in the seeds leads to seeds with low water content. When the seeds dry, they stay round.

Slide 65

The original 3549 nucleotides plus an additional 800 nucleotides What does the r allele represent? Round Pea

Slide 66

Unbranched starch in the seeds leads to seeds with high water content. They are sweet, but when the seeds dry, they wrinkle. How does the altered SBE protein lead to round seeds? It gives rise to unbranched starch only (amylose). What does the r allele do?

Slide 67

The R allele and the r Allele: 800 bp fragment of DNA inserted R allele genetic code r allele genetic code

Slide 68

Two Examples of mutation 1.Beach Mice -Missense substitution mutation of one nucleotide C T -Changes one amino acid: Arginine Cysteine -Changes the structure and function of the MC1R protein 2.Taste in peas -Nonsense insertion mutation of 800 nucleotides. -Disrupts amino acid production. -Changes the structure and function of the starch branching enzyme.

Slide 69

Mutations Insertion/Deletion/Substitution Frameshift or no Frameshift Silent/missense/nonsense

Slide 70

Coming Up: Thursday: Homework #5 StudyNotes 8a Transcription, RNA Processing, Translation Tuesday: Molecular Sculpting