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The Next Three Weeks:

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  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)

  2. Sanger Sequencing

  3. dideoxyNucleosideTriPhosphate

  4. Central Dogma Overview

  5. 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?

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

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

  8. 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.

  9. An Academic Transcript: A copy of your grades

  10. 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

  11. 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.

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

  13. DNA  mRNA  Amino Acids non -Template Strand 5’– CCACTGATAGACCTT –3’ 3’– GGTGACTATCTGGAA –5’ mRNA is made using the template strand… Template Strand

  14. 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

  15. The Translation Key:

  16. DNA  mRNA  Amino Acids Non-Templ: 5’– CCACTGATAGACCTT –3’ Template: 3’– GGTGACTATCTGGAA –5’ mRNA: 5’– CCACUGAUAGACCUU –3’ Leu Asp - Ile - Pro - Leu -

  17. Transcribe/Translate the Following Sequences: 3’– ATGTTTGAACTACAG–5’ 5’– TACAAACTTGATGTC–3’ 5’– CTGCGTGACTGCAAA –3’ 3’– GACGCACTGACGTTT –5’ 3’– CCGACTCACTGATGC –5’

  18. 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.

  19. Mutations… changes to the code

  20. What’s a Mutation? A mutation is any permanent change in an organism’s DNA. Mutations result in new alleles!

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

  22. 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.

  23. Mutations and their impacts

  24. Mutations and their impacts

  25. Mutations and their impacts

  26. Mutations and their impacts

  27. Mutations and their impacts

  28. 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

  29. Two Examples of mutation • Beach Mice (from the text) • Taste in peas

  30. Peromyscuspolionotus

  31. 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.

  32. Fur Color:

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

  34. mc1r Gene Sequence 5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACCGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

  35. mc1r Gene Sequence 5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACTGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

  36. Single Substitution… 5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACTGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’ • Substituting 1 of 954 nucleotides • Cytosine to a Thymine (Pyrimidine  Pyrimidine) • Changed the mRNA codon from CGC to UGC

  37. The Translation Key:

  38. 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

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

  40. 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

  41. Mendel’s Peas