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)

Sanger Sequencing

dideoxyNucleoside TriPhosphate

© 2011 Pearson Education, Inc.

© 2011 Pearson Education, Inc.

© 2011 Pearson Education, Inc.

© 2011 Pearson Education, Inc.

© 2011 Pearson Education, Inc.

Central Dogma Overview

© 2011 Pearson Education, Inc.

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?

DNA Language

3’ – GTGCA – 5’5’ – CACGT – 3’

The language of DNA is written in nucleotides.

Protein Language• A protein is a chain of amino acids.

Connecting the Languages

Transcribing versus Translating- Transcribe means: to rewrite

Transcription Example:3 parts gravel, 2 parts sand, 1 part cement.3 parts gravel, 2 parts sand, 1 part cement.

An Academic Transcript:

A copy of your grades

Connecting the Languages

Transcribing versus Translating- Transcribe means: to rewrite- Translating means: to change languageTranslation Example:3 parts gravel, 2 parts sand, 1 part cement. 3 partes de grava, 2 partes de arena, 1 parte de cemento. 三石 二砂 一水泥

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.

DNA mRNA Amino Acids

• Transcribing:– Replace Thymine with Uracil.– Transcribe from the template strand.

DNA mRNA Amino Acids

5’– CCACTGATAGACCTT –3’3’– GGTGACTATCTGGAA –5’

mRNA is made using the template strand…

Template Strand

non -Template Strand

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

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The Translation Key:

DNA mRNA Amino Acids

Non-Templ: 5’– CCACTGATAGACCTT –3’Template: 3’– GGTGACTATCTGGAA –5’

mRNA: 5’– CCACUGAUAGACCUU –3’Pro - Leu - Ile - Asp - Leu

Transcribe/Translate the Following Sequences:

5’– CTGCGTGACTGCAAA –3’3’– GACGCACTGACGTTT –5’

3’– CCGACTCACTGATGC –5’

3’– ATGTTTGAACTACAG –5’

5’– TACAAACTTGATGTC –3’

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.

Mutations… changes to the code

What’s a Mutation?

A mutation is any permanent change in an organism’s DNA.

Mutations result in new alleles!

Point Mutations

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

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.

Mutations and their impacts

Mutations and their impacts

Mutations and their impacts

Mutations and their impacts

Mutations and their impacts

Describing a Mutation:

Three Categories:1. What happened?

– Insertion, deletion, substitution.2. What was the impact on the protein?

– Missense, nonsense, silent3. What was the reading frame?

– Frameshift

Two Examples of mutation

1. Beach Mice (from the text)2. Taste in peas

Peromyscus polionotus

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 m2

• Breeding: Monogamous pair-bonding. Litters of 2-8 pups, every 30 days.

• Lifespan: 9-12 months.

Fur Color:

The mc1r Gene

• The mc1r gene is located in Chromosome #16 in mammals.

• It codes for the MC1R protein that aids in pigment synthesis.

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mc1r Gene Sequence5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACCGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

mc1r Gene Sequence5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACTGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

Single Substitution…5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACTGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

- Substituting 1 of 954 nucleotides- Cytosine to a Thymine

(Pyrimidine Pyrimidine)- Changed the mRNA codon

from CGC to UGC

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The Translation Key:

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:MPTQGPQKRLLGSLNSTSTATPHLGLATNQTGPWCLQVSIPDGLFLSLGLVSLVENVLVVIAITKNRNLHSPMYSFICCLALSDLMVSISLVLETAIILLLEAGALVTRAALVQQLDNVIDVLICGSMVSSLCFLGVIAIDRYISIFYALRYHSIVTLPRARRAIXGIWVASIFFSTLFITYYNHTAVLICLVTFFLAMLALMAXLYVHMLTRAYQHAQGIAQLQKRQGSTXQGFCLKGAXTLTIILGIFFLCWGPFFLHLTLIVLCPQHPTCSCIFKNFNLYLVLIIFSSIVDPLIYAFRSQELRMTLREVLLCSW

Amino Acid Sequence Light Fur:MPTQGPQKRLLGSLNSTSTATPHLGLATNQTGPWCLQVSVPDGLFLSLGLVSLVENVLVVIAITKNCNLHSPMYSFICCLALSDLMVSISLVLETAIILLLEAGALVTRAALVQQLDNVIDVLICGSMVSSLCFLGVIAIDRYISIFYALRYHSIVTLPRARRAIVGIWVASIFFSTLFITYYNHTAVLICLVTFFLAMLALMAILYVHMLTRAYQHAQGIAQLQKRQGSTRQGFCLKGAATLTIILGIFFLCWGPFFLHLTLIVLCPQHPTCSCIFKNFNLYLVLIIFSSIVDPLIYAFRSQELRMTLREVLLCSW

Changing 1 amino acid:

• Arginine:– Strongest +charge– Very hydrophilic

• Cysteine:– Not hydrophilic– Forms disulfide bonds

Two Examples of mutation1. Beach Mice

- Missense substitution mutation of one nucleotide CT

- Changes one amino acid: Arginine Cysteine

- Changes the function of the MC1R protein

2. Taste in peas

Mendel’s Peas

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.

The Evolution of Wrinkled Peas

Wrinkled peas taste sweet.

WrinkledRound

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).

Inside the Pea Cell: Sugar and Starch

Sucrose

The Evolution of Wrinkled PeasWrinkled peas aren’t just wrinkled…

they taste good too!

Wrinkled(r allele of sbe1 gene)

Round(R allele of sbe1 gene)

G3P

(Intermediates)

Amylose

Amylopectin

G3P

(Intermediates)

Amylosenon-functioning starch branching enzyme

Sucrose

functioning starch branching enzyme

The Genetics ofRound and Wrinkled Peas

The sbe1 Gene

The sbe1 gene has two alleles:

Wrinkled Pea

Codes for functional

SBE1 protein.

Round Pea

Codes for non-functional

SBE1 protein.

3549 nucleotidesWhat does the “R” allele represent?

Round Pea

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

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.

The original 3549 nucleotidesplus an additional 800 nucleotides

What does the “r” allele represent?

Round Pea

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?

The R allele and the r Allele:

800 bp fragment of DNA inserted

R allele genetic code

r allele genetic code

Two Examples of mutation1. Beach Mice

- Missense substitution mutation of one nucleotide CT

- 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.

Mutations

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

Coming Up:

• Thursday:– Homework #5– StudyNotes 8a– Transcription, RNA Processing, Translation

• Tuesday:– Molecular Sculpting