BEGR 424 Molecular Biology William Terzaghi Spring, 2015

BEGR424- Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC228 Office hours: MWF 12:00-1:00, TR 1-2 or by appointment Phone: (570)

BEGR424- Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC228 Office hours: MWF 12:00-1:00, TR 1-2 or by appointment Phone: (570) Course webpage: l

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 problem 2.Design some experiments

Plan A 1.Pick a problem 2.Design some experiments 3.See where they lead us

Plan A 1.Pick a problem 2.Design some experiments 3.See where they lead us Grading? Combination of papers and presentations

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

Plan A Topics? 1.Making a probiotic strain of E.coli that destroys oxalate to help treat kidney stones in collaboration with Dr. Lucent and Dr. VanWert 2.Making plants/algae that bypass Rubisco to fix CO 2 3.Making vectors for Teresa Wasiluk’s project 4.Making vectors for Dr. Harms 5.Cloning & sequencing antisense RNA 6.Studying ncRNA 7.Revisiting blue-green algae that generate electricity 8.Something else?

Plan A Assignments? 1.identify a gene and design primers 2.presentation on new sequencing tech 3.designing a protocol to verify your clone 4.presentations on gene regulation 5.presentation on applying mol bio Other work 1.draft of report on cloning & sequencing 2.poster for symposium 3.final gene report 4.draft of formal report 5.formal report

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

Plan B Standard lecture course, except: 1.Last lectures will be chosen by you -> electives 2.Last 4 labs will be an independent research project

Plan B Standard lecture course, except: 1.Last lectures will be chosen by you -> electives 2.Last 4 labs will be an independent research project 3.20% of grade will be “elective” Paper Talk Research proposal Poster Exam

Plan B schedule- Spring 2015 DateTOPIC JAN12General Introduction 14Genome organization 16Cloning & libraries: why and how 19DNA fingerprinting 21DNA sequencing 23Genome projects 26Studying proteins 28Meiosis & recombination 30Recombination FEB2Cell cycle 4Mitosis 6Exam 1 9DNA replication 11Transcription 1 13Transcription 2 16 Transcription 3

18mRNA processing 20Post-transcriptional regulation 23Protein degradation 25Epigenetics 27Small RNA MAR2Spring Recess 4Spring Recess 6Spring Recess 9RNomics 11Proteomics 13Exam 2 16Protein synthesis 1 18Protein synthesis 2 20Membrane structure/Protein targeting 1 23Protein targeting 2 25 Organelle genomes 27Mitochondrial genomes and RNA editing 30Nuclear:cytoplasmic genome interactions

APR1Elective 3Easter 6Easter 8Elective 10Elective 13Elective 15Elective 17Elective 20Elective 22Elective 24Elective 27Exam 3 29ElectiveLast Class! ???Final examination

Lab Schedule DateTOPIC Jan14DNA extraction and analysis 21BLAST, etc, primer design 28PCR Feb 4RNA extraction and analysis 11RT-PCR 18qRT-PCR 25cloning PCR fragments Mar 4Spring Recess 11DNA sequencing 18Induced gene expression 25Northern analysis Apr 1Independent project 8Independent project 15Independent project 22Independent 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 10 6 Yeast has 2 x 10 7 Arabidopsis has 10 8 Rice has 5 x 10 8 Humans have 3 x 10 9 Soybeans have 3 x 10 9 Toads have 3 x 10 9 Salamanders have 8 x Lilies have 10 11

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 ~10 9 bp

Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~10 9 bp mammals all have ~ 3 x 10 9 bp

Genome Projects C-value paradox: DNA content/haploid genome varies widely Some phyla show little variation: birds all have ~10 9 bp mammals all have ~ 3 x 10 9 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 ~10 9 bp mammals all have ~ 3 x 10 9 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 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

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