BEGR 424/Bio 324 Molecular BiologyWilliam TerzaghiSpring, 2013

BEGR424/BIO 324 - Resource and Policy Information

Instructor: Dr. William TerzaghiOffice: SLC 363Office hours: MWF 10:00-12:00, or by appointmentPhone: (570) 408-4762Email: terzaghi@wilkes.edu

BEGR424/BIO 324 - Resource and Policy Information

Instructor: Dr. William TerzaghiOffice: SLC 363Office hours: MWF 10:00-12:00, or by appointmentPhone: (570) 408-4762Email: terzaghi@wilkes.edu

Course webpage: http://staffweb.wilkes.edu/william.terzaghi/BIO324.html

General considerations

What do you hope to learn?

General considerations

What do you hope to learn?

Graduate courses

1. learning about current literature

General considerations

What do you hope to learn?

Graduate courses

1. learning about current literature

• Learning how to give presentations

General considerations

What do you hope to learn?

Graduate courses

1. learning about current literature

2. Learning current techniques

General considerations

What do you hope to learn?

Graduate courses

1. learning about current literature

2. Learning current techniques

• Using them!

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

Plan A

1.Pick a problem2.Design some experiments

Plan A

1.Pick a problem2.Design some experiments3.See where they lead us

Plan A

1.Pick a problem2.Design some experiments3.See where they lead us

Grading?Combination of papers and presentations

Plan AGrading?

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

Plan ATopics?

1.Bypassing Calvin cycle2.Making vectors for Dr. Harms3.Making vectors for Dr. Lucent4.Cloning & sequencing antisense RNA5.Studying ncRNA6.Something else?

Plan AAssignments?

1.identify a gene and design primers2.presentation on new sequencing tech3.designing a protocol to verify your clone4.presentations on gene regulation5.presentation on applying mol bio

Other work1.draft of report on cloning & sequencing2.poster for symposium3.final gene report4.draft of formal report 5.formal report

Plan BStandard lecture course, except:1.Last lectures will be chosen by you -> electives

Plan BStandard lecture course, except:1.Last lectures will be chosen by you -> electives2.Last 4 labs will be an independent research project

Plan BStandard lecture course, except:1.Last lectures will be chosen by you -> electives2.Last 4 labs will be an independent research project3.20% of grade will be “elective”• Paper• Talk• Research proposal• Poster• Exam

Plan B schedule- Spring 2013Date TOPIC

JAN 14 General Introduction16 Genome organization18 Cloning & libraries: why and how 21 DNA fingerprinting23 DNA sequencing25 Genome projects28 Studying proteins 30 Meiosis & recombination

FEB 1 Recombination 4 Cell cycle6 Mitosis8 Exam 111 DNA replication13 Transcription 115 Transcription 218 Transcription 3

20 mRNA processing22 Post-transcriptional regulation25 Protein degradation27 Epigenetics

MAR 1 Small RNA4 Spring Recess6 Spring Recess8 Spring Recess11 RNomics13 Proteomics15 Exam 218 Protein synthesis 120 Protein synthesis 222 Membrane structure/Protein targeting 125 Protein targeting 227 Organelle genomes29 Easter

Apr 1 Easter

APR 3 Mitochondrial genomes and RNA editing5 Nuclear:cytoplasmic genome interactions8 Elective10 Elective12 Elective15 Elective17 Elective19 Elective22 Elective24 Elective26 Elective29 Exam 3

May 1 Elective Last Class!

??? Final examination

Lab ScheduleDate TOPICJan 16 DNA extraction and analysis

23 BLAST, etc, primer design30 PCR

Feb 6 RNA extraction and analysis13 RT-PCR20 qRT-PCR27 cloning PCR fragments

Mar 6 Spring Recess13 DNA sequencing20 Induced gene expression27 Northern analysis

Apr 3 Independent project10 Independent project17 Independent project24 Independent project

Genome Projects

Studying structure & function of genomes

Genome Projects

Studying structure & function of genomes

• Sequence first

Genome Projects

Studying structure & function of genomes

• Sequence first

• Then location and function of every part

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

Genome Projects

C-value paradox: DNA content/haploid genome varies widely

Genome Projects

C-value paradox: DNA content/haploid genome varies widely

Some phyla show little variation:

birds all have ~109 bp

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

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

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

C-value paradox

One cause = variations in chromosome numbers and ploidy

2C chromosome numbers vary widely

Haplopappus has 2

C-value paradox

One cause = variations in chromosome numbers and ploidy

2C chromosome numbers vary widely

Haplopappus has 2

Arabidopsis has 10

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

C-value paradox

Chromosome numbers vary

So does chromosome size!

Reason = variation in amounts of repetitive DNA

C-value paradox

Chromosome numbers vary

So does chromosome size!

Reason = variation in amounts of repetitive DNA

first demonstrated using Cot curves

Cot curves

• denature (melt) DNA by heating

Cot curves

• denature (melt) DNA by heating

dissociates into two single strands

Cot curves

1. denature (melt) DNA by heating

2. Cool DNA

Cot curves

1. denature (melt) DNA by heating

2. Cool DNA: complementary strands find each other & anneal

Cot curves

1. denature (melt) DNA by heating

2. Cool DNA: complementary strands find each other & anneal

• hybridize

Cot curves

1. denature (melt) DNA by heating

2. Cool DNA: complementary strands find each other & anneal

• Hybridize: don't have to be the same strands

Cot curves

1. denature (melt) DNA by heating

2. Cool DNA: complementary strands find each other & anneal• Hybridize: don't have to be the same strands

3. Rate depends on [complementary strands]

Cot curves

1) denature DNA

2) cool DNA

3) at intervals measure

[single-stranded DNA]

Cot curves

viruses & bacteria show simple curves

Cot is inversely proportional to genome size

Cot curves

eucaryotes show 3 step curves

Step 1 renatures rapidly: “highly repetitive”

Cot curves

eucaryotes show 3 step curves

Step 1 renatures rapidly: “highly repetitive”Step 2 is intermediate: “moderately repetitive”

Cot curves

eucaryotes show 3 step curves

Step 1 renatures rapidly: “highly repetitive”Step 2 is intermediate: “moderately repetitive”Step 3 is ”unique"

Molecular cloning

To identify the types of DNA sequences found within each class they must be cloned

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

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

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

Recombinant DNA

Restriction enzymes cut DNA at specific sites

bacterial” immune system”: destroy “non-self” DNA

Recombinant DNARestriction enzymes cut DNA at specific sitesbacterial” immune system”: destroy “non-self” DNAmethylase recognizes same sequence & protects it by methylating it Restriction/modification systems

Recombinant DNA

Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence

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

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

Molecular cloning How?1) create recombinant DNA2) transform recombinant molecules into suitable host

Molecular cloning

How?

1) create recombinant DNA

2) transform recombinant molecules into suitable host

3) identify hosts which have taken up your recombinant molecules

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