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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)

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the next three weeks
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)
the language of genetics
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
DNA Language

3’ – GTGCA – 5’5’ – CACGT – 3’

The language of DNA is written in nucleotides.

protein language
Protein Language
  • A protein is a chain of amino acids.
connecting the languages
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
An Academic Transcript:

A copy of your grades

connecting the languages1
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.

三石二砂一水泥

the language of genetics1
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
DNA  mRNA  Amino Acids
  • Transcribing:
    • Replace Thymine with Uracil.
    • Transcribe from the template strand.
dna mrna amino acids1
DNA  mRNA  Amino Acids

non -Template Strand

5’– CCACTGATAGACCTT –3’

3’– GGTGACTATCTGGAA –5’

mRNA is made using the template strand…

Template Strand

dna mrna amino acids2
DNA  mRNA  Amino Acids

non -Template Strand

5’– CCACTGATAGACCTT –3’

3’– GGTGACTATCTGGAA –5’

5’– CCACUGAUAGACCUU –3’

The mRNA sequence is nearly identical to the non-template strand.

Transcribing… complete.

Template Strand

mRNA

dna mrna amino acids3
DNA  mRNA  Amino Acids

Non-Templ: 5’– CCACTGATAGACCTT –3’

Template: 3’– GGTGACTATCTGGAA –5’

mRNA: 5’– CCACUGAUAGACCUU –3’

Leu

Asp -

Ile -

Pro -

Leu -

transcribe translate the following sequences
Transcribe/Translate the Following Sequences:

3’– ATGTTTGAACTACAG–5’

5’– TACAAACTTGATGTC–3’

5’– CTGCGTGACTGCAAA –3’

3’– GACGCACTGACGTTT –5’

3’– CCGACTCACTGATGC –5’

important properties of the code
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.
what s a mutation
What’s a Mutation?

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

Mutations result in new alleles!

point mutations
Point Mutations
  • Point mutations occur when the DNA polymerase inserts the wrong base into the newly synthesized strand of DNA.
point mutations1
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.
    • Missensemutations.
      • Result in changes in the amino acid sequence of the encoded protein.
    • Nonsense mutations.
      • Results in a stop codon.
describing a mutation
Describing a Mutation:

Three Categories:

  • What happened?
    • Insertion, deletion, substitution.
  • What was the impact on the protein?
    • Missense, nonsense, silent
  • What was the reading frame?
    • Frameshift
two examples of mutation
Two Examples of mutation
  • Beach Mice (from the text)
  • Taste in peas
fact sheet peromyscus polionotus
Fact Sheet: Peromyscuspolionotus
  • 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.
the mc1r gene
The mc1r Gene
  • The mc1r gene is located in Chromosome #16 in mammals.
  • It codes for the MC1R protein that aids in pigment synthesis.
m c1r gene sequence
mc1r Gene Sequence

5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACCGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

m c1r gene sequence1
mc1r Gene Sequence

5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACTGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

single substitution
Single Substitution…

5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACTGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

  • Substituting 1 of 954 nucleotides
  • Cytosine to a Thymine (Pyrimidine  Pyrimidine)
  • Changed the mRNA codon from CGC to UGC
consequence of mutation
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
Changing 1 amino acid:
  • Arginine:
    • Strongest +charge
    • Very hydrophilic
  • Cysteine:
    • Not hydrophilic
    • Forms disulfide bonds
two examples of mutation1
Two Examples of mutation
  • Taste in peas
  • Beach Mice
  • Missense substitution mutation of one nucleotide CT
  • Changes one amino acid: Arginine  Cysteine
  • Changes the function of the MC1R protein
fact sheet pisum sativum
Fact Sheet: Pisumsativum
  • 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
The Evolution of Wrinkled Peas

Wrinkled peas taste sweet.

Round

Wrinkled

molecular biology central dogma
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).

the evolution of wrinkled peas1
The Evolution of Wrinkled Peas

Wrinkled peas aren’t just wrinkled…they taste good too!

Round

(R allele of sbe1 gene)

Wrinkled

(r allele of sbe1 gene)

G3P

(Intermediates)AmyloseAmylopectin

G3P

(Intermediates)Amylose

 Sucrose

functioning starch branching enzyme

non-functioning starch branching enzyme

the sbe1 gene
The sbe1 Gene

The sbe1 gene has two alleles:

Codes for functional SBE1 protein.

Codes for non-functional SBE1 protein.

Round Pea

Wrinkled Pea

the r allele
The R Allele

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

what does the r allele do
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.

what does the r allele represent1
What does the “r”allele represent?

The original 3549 nucleotides

plus an additional 800 nucleotides

Round Pea

slide66

What does the “r” allele do?

How does the altered SBE protein lead to round seeds?

 It gives rise to unbranchedstarch only (amylose).

Unbranched starch in the seeds leads to seeds with high water content. They are sweet, but when the seeds dry, they wrinkle.

the r allele and the r allele
The R allele and the r Allele:

R allele genetic code

r allele genetic code

800 bp fragment of DNA inserted

two examples of mutation2
Two Examples of mutation
  • Taste in peas
  • Nonsense insertion mutation of 800 nucleotides.
  • Disrupts amino acid production.
  • Changes the structure and function of the starch branching enzyme.
  • 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
mutations
Mutations
  • Insertion/Deletion/Substitution
  • Frameshiftor no Frameshift
  • Silent/missense/nonsense
coming up
Coming Up:
  • Thursday:
    • Homework #5
    • StudyNotes 8a
    • Transcription, RNA Processing, Translation
  • Tuesday:
    • Molecular Sculpting