BEGR 424 Molecular BiologyWilliam TerzaghiSpring, 2014

BEGR424- Resource and Policy Information

Instructor: Dr. William TerzaghiOffice: SLC 363/CSC228 Office hours: MWF 12:00-1:00, TR 1-2 or by appointmentPhone: (570) 408-4762Email: terzaghi@wilkes.edu

BEGR424- Resource and Policy Information

Instructor: Dr. William TerzaghiOffice: SLC 363/CSC228 Office hours: MWF 12:00-1:00, TR 1-2 or by appointmentPhone: (570) 408-4762Email: terzaghi@wilkes.edu

Course webpage: http://staffweb.wilkes.edu/william.terzaghi/BEGR424.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.Cloning and analyzing oxalate decarboxylase and/or oxalate oxidase to see if they dissolve kidney stones in collaboration with Dr. VanWert2.Making vectors for Dr. Harms3.Cloning & sequencing antisense RNA4.Studying ncRNA5.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 2014Date TOPIC

JAN 13 General Introduction15 Genome organization17 Cloning & libraries: why and how 20 DNA fingerprinting22 DNA sequencing24 Genome projects27 Studying proteins 29 Meiosis & recombination31 Recombination

FEB 3 Cell cycle5 Mitosis7 Exam 110 DNA replication12 Transcription 114 Transcription 217 Transcription 3

19 mRNA processing21 Post-transcriptional regulation24 Protein degradation26 Epigenetics28 Small RNA

MAR 3 Spring Recess5 Spring Recess7 Spring Recess10 RNomics12 Proteomics14 Exam 217 Protein synthesis 119 Protein synthesis 221 Membrane structure/Protein targeting 124 Protein targeting 226 Organelle genomes28 Mitochondrial genomes and RNA editing31 Nuclear:cytoplasmic genome interactions

APR 2 Elective4 Elective7 Elective9 Elective11 Elective14 Elective16 Elective18 Easter21 Easter23 Elective25 Elective28 Exam 330 Elective Last Class!

??? Final examination

Lab ScheduleDate TOPICJan 14 DNA extraction and analysis

21 BLAST, etc, primer design28 PCR

Feb 4 RNA extraction and analysis11 RT-PCR18 qRT-PCR25 cloning PCR fragments

Mar 4 Spring Recess11 DNA sequencing18 Induced gene expression25 Northern analysis

Apr 1 Independent project 8 Independent project15 Independent project22 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!

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