1. BEGR 424/Bio 324 Molecular Biology
William Terzaghi
Spring, 2013

2. BEGR424/BIO 324 - Resource and Policy Information
Instructor: Dr. William Terzaghi
Office: SLC 363
Office hours: MWF 10:00-12:00, or by appointment
Phone: (570)

3. BEGR424/BIO 324 - Resource and Policy Information
Instructor: Dr. William Terzaghi
Office: SLC 363
Office hours: MWF 10:00-12:00, or by appointment
Phone: (570)
Course webpage:

4. General considerations
What do you hope to learn?

5. General considerations
What do you hope to learn?
Graduate courses
learning about current literature

6. General considerations
What do you hope to learn?
Graduate courses
learning about current literature
Learning how to give presentations

7. General considerations
What do you hope to learn?
Graduate courses
learning about current literature
Learning current techniques

8. General considerations
What do you hope to learn?
Graduate courses
learning about current literature
Learning current techniques
Using them!

9. Plan A
Provide a genuine experience in using cell and molecular biology to learn about a fundamental problem in biology.
Rather than following a set series of lectures, study a problem and see where it leads us.
Lectures & presentations will relate to current status
Some class time will be spent in lab & vice-versa
we may need to come in at other times as well

10. Plan A
Pick a problem
Design some experiments

11. Plan A
Pick a problem
Design some experiments
See where they lead us

12. Plan A
Pick a problem
Design some experiments
See where they lead us
Grading?
Combination of papers and presentations

13. Plan A
Grading?
Combination of papers and presentations
First presentation:10 points
Research presentation: 10 points
Final presentation: 15 points
Assignments: 5 points each
Poster: 10 points
Intermediate report 10 points
Final report: 30 points

14. Plan A
Topics?
Bypassing Calvin cycle
Making vectors for Dr. Harms
Making vectors for Dr. Lucent
Cloning & sequencing antisense RNA
Studying ncRNA
Something else?

15. Plan A
Assignments?
identify a gene and design primers
presentation on new sequencing tech
designing a protocol to verify your clone
presentations on gene regulation
presentation on applying mol bio
Other work
draft of report on cloning & sequencing
poster for symposium
final gene report
draft of formal report
formal report

16. Plan B
Standard lecture course, except:
Last lectures will be chosen by you -> electives

17. Plan B
Standard lecture course, except:
Last lectures will be chosen by you -> electives
Last 4 labs will be an independent research project

18. Plan B
Standard lecture course, except:
Last lectures will be chosen by you -> electives
Last 4 labs will be an independent research project
20% of grade will be “elective”
Paper
Talk
Research proposal
Poster
Exam

19. Plan B schedule- Spring 2013
Date TOPIC
JAN 14 General Introduction
16 Genome organization
18 Cloning & libraries: why and how
21 DNA fingerprinting
23 DNA sequencing
25 Genome projects
28 Studying proteins
30 Meiosis & recombination
FEB 1 Recombination
4 Cell cycle
6 Mitosis
8 Exam 1
11 DNA replication
13 Transcription 1
15 Transcription 2
18 Transcription 3

20. 20 mRNA processing
22 Post-transcriptional regulation
25 Protein degradation
27 Epigenetics
MAR 1 Small RNA
4 Spring Recess
6 Spring Recess
8 Spring Recess
11 RNomics
13 Proteomics
15 Exam 2
18 Protein synthesis 1
20 Protein synthesis 2
22 Membrane structure/Protein targeting 1
25 Protein targeting 2
27 Organelle genomes
29 Easter
Apr 1 Easter

21. APR 3 Mitochondrial genomes and RNA editing
5 Nuclear:cytoplasmic genome interactions
8 Elective
10 Elective
12 Elective
15 Elective
17 Elective
19 Elective
22 Elective
24 Elective
26 Elective
29 Exam 3
May 1 Elective Last Class!
??? Final examination

22. Lab Schedule
Date TOPIC
Jan 16 DNA extraction and analysis
23 BLAST, etc, primer design
30 PCR
Feb 6 RNA extraction and analysis
13 RT-PCR
20 qRT-PCR
27 cloning PCR fragments
Mar 6 Spring Recess
13 DNA sequencing
20 Induced gene expression
27 Northern analysis
Apr 3 Independent project
10 Independent project
17 Independent project
24 Independent project

23. Genome Projects
Studying structure & function of genomes

24. Genome Projects
Studying structure & function of genomes
Sequence first

25. Genome Projects
Studying structure & function of genomes
Sequence first
Then location and function of every part

26. Genome Projects
How much DNA is there?
SV40 has 5000 base pairs
E. coli has 5 x 106
Yeast has 2 x 107
Arabidopsis has 108
Rice has 5 x 108
Humans have 3 x 109
Soybeans have 3 x 109
Toads have 3 x 109
Salamanders have 8 x 1010
Lilies have 1011

27. Genome Projects
C-value paradox: DNA content/haploid genome varies widely

28. Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp

29. Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp
mammals all have ~ 3 x 109 bp

30. Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp
mammals all have ~ 3 x 109 bp
Other phyla are all over:
insects and amphibians vary 100 x

31. Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp
mammals all have ~ 3 x 109 bp
Other phyla are all over:
insects and amphibians vary 100 x
flowering plants vary 1000x

32. C-value paradox
One cause = variations in chromosome numbers and ploidy
2C chromosome numbers vary widely
Haplopappus has 2

33. C-value paradox
One cause = variations in chromosome numbers and ploidy
2C chromosome numbers vary widely
Haplopappus has 2
Arabidopsis has 10

34. C-value paradox
One cause = variations in chromosome numbers and ploidy
2C chromosome numbers vary widely
Haplopappus has 2
Arabidopsis has 10
Rice has 24
Humans have 46
Tobacco (hexaploid) has 72
Kiwifruit (octaploid) have 196

35. C-value paradox
Chromosome numbers vary
So does chromosome size!
Reason = variation in amounts of repetitive DNA

36. C-value paradox
Chromosome numbers vary
So does chromosome size!
Reason = variation in amounts of repetitive DNA
first demonstrated using Cot curves

37. Cot curves
denature (melt) DNA by heating

38. Cot curves
denature (melt) DNA by heating
dissociates into two single strands

39. Cot curves
denature (melt) DNA by heating
Cool DNA

40. Cot curves
denature (melt) DNA by heating
Cool DNA: complementary strands find each other & anneal

41. Cot curves
denature (melt) DNA by heating
Cool DNA: complementary strands find each other & anneal
hybridize

42. Cot curves
denature (melt) DNA by heating
Cool DNA: complementary strands find each other & anneal
Hybridize: don't have to be the same strands

43. Cot curves
denature (melt) DNA by heating
Cool DNA: complementary strands find each other & anneal
Hybridize: don't have to be the same strands
Rate depends on [complementary strands]

44. Cot curves
1) denature DNA
2) cool DNA
3) at intervals measure
[single-stranded DNA]

45. Cot curves
viruses & bacteria show simple curves
Cot is inversely proportional to genome size

46. Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”

47. Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”
Step 2 is intermediate: “moderately repetitive”

48. Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”
Step 2 is intermediate: “moderately repetitive”
Step 3 is ”unique"

49. Molecular cloning
To identify the types of DNA sequences found within each class they must be cloned

50. Molecular cloning
To identify the types of DNA sequences found within each class they must be cloned
Force host to make millions of copies of a specific sequence

51. Molecular cloning
To identify the types of DNA sequences found within each class they must be cloned
Why?
To obtain enough copies of a specific sequence to work with!
typical genes are 1,000 bp cf haploid human genome is 3,000,000,000 bp
average gene is < 1/1,000,000 of total genome

52. Recombinant DNA
Arose from 2 key discoveries in the 1960's
1) Werner Arber: enzymes which cut DNA at specific sites
called "restriction enzymes” because restrict host range for certain bacteriophage

53. Recombinant DNA
Restriction enzymes cut DNA at specific sites
bacterial” immune system”: destroy “non-self” DNA

54. Recombinant DNA
Restriction enzymes cut DNA at specific sites
bacterial” immune system”: destroy “non-self” DNA
methylase recognizes same sequence & protects it by methylating it
Restriction/modification systems

55. Recombinant DNA
Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence

56. Recombinant DNA
Arose from 2 key discoveries in the 1960's
1) restriction enzymes
2) Weiss: DNA ligase
-> enzyme which glues
DNA strands together
seals "nicks" in DNA backbone

57. Molecular cloning
How?
1) introduce DNA sequence into a vector
Cut both DNA & vector with restriction enzymes, anneal & join with DNA ligase
create a recombinant DNA molecule

58. Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant
molecules into suitable host

59. Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant molecules into suitable host
3) identify hosts which have taken up your recombinant molecules

60. Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant molecules into suitable host
3) identify hosts which have taken up your recombinant molecules
4) Extract DNA