jeff newman:
TRANSCRIPT
- 1.Integrating Genomics Throughout the Curriculum, with an Emphasis on Prokaryotes Jeffrey D. Newman Lycoming College May 20, 2002
2. The Context for Change
- Lycoming College Very Traditional Small National Liberal Arts College 1500 students
- Our Biology Major highly proscriptive
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- 2 semester intro bio series
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- Genetics
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- Microbiology
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- Human Physiology
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- Plant Science
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- Ecology
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- At least 1 upper level course
3. Incorporation of Molecular Biology, Bioinformatics, Genomics
- Phase I (97-99) Intro and core course labs
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- Intro. Biology DNA sequence analysis, plasmid prep, transformation, restriction digest, gel.
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- Genetics PCR from cheek cell DNA, cloning into pBS
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- Microbiology PCR of unknowns rRNA gene, sequencing.
- Phase II (99-02) Genomics added to many courses
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- Intro. Biology shotgun sequencing, HGP conclusions.
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- Genetics discussion of microarrays
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- Microbiology Microbial Genome Papers
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- Molecular Biology Microarrays (thanks to GCAT)
- Project assessment survey Spring 01, GCAT Spring 02
- Phase III (03 - ?) New course Genome Analysis
4. Genomics in Intro Biology
- ReplicationPCRDNA sequencingshotgun strategycontig assembly demo.
- In lab, students identify ORFs in pGLO sequence, translate to protein, BLAST search to ID genes.
- Model Organisms
- Human Genome Project
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- Gene number
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- Gene complexity
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- Types of gene products
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- Protein Families!
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- Disease genes
Venter et al., 2001 5. Genomics in Microbiology
- Students learn DNA sequencing details in lab (for rRNA gene fragment), use of BLAST search, multiple sequence alignment, construction of phylogenetic trees
- Shotgun sequencing method discussed, contig assembly, identification of ORFs demonstrated.
6. The Genomics Revolution
- Genome sequences allow the following questions to be asked:
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- How many genes/proteins do we still know nothing about?
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- What are the minimal requirements for a living organism?
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- How has evolution streamlined microbial genomes?
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- How are microbes related to each other?
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- What are the genomic differences between:
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- Archaea and Bacteria?
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- obligate parasites and free-living organisms?
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- Phototrophic and chemotrophic organisms?
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- Organotrophic and lithotrophic organisms?
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- Mesophiles and Thermophiles?
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- Pathogenic and non-pathogenic strains?
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7. Applications of Microbial Genome Data
- Gene chips/microarrays can detect tens of thousands of specific DNA or RNA sequences
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- pathogen identification in tissue sample
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- virulence genes used for prognosis
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- antibiotic resistance genes for determining best treatment
- Identification of genes required for pathogenesis will allow targeted drug/vaccine development
- Determination of gene function in simple organisms will help understand function of genes in eukaryotes.
- What enzymes might have industrial applications?
8. Completed Genomes in GenBank
- Aeropyrum pernix
- Aquifex aeolicus
- Archaeoglobus fulgidus
- Bacillus subtilis
- Borrelia burgdorferi
- Campylobacter jejuni
- Chlamydia pneumoniae CWL029
- Chlamydia pneumoniae AR39
- Chlamydia muridarum
- Chlamydia trachomatis D/UW-3/CX
- Deinococcus radiodurans
- Escherichia coli
- Haemophilus influenzae
- Helicobacter pylori26695
- Helicobacter pyloriJ99
- Methanobacterium thermoautotrophicum
- Methanococcus jannaschii
- Mycobacterium tuberculosis
- Mycoplasma genitalium
- Mycoplasma pneumoniae
- Neisseria meningitidis MC58
- Pyrococcus abyssi
- Pyrococcus horikoshii
- Rickettsia prowazekii
- Synechocystis PCC6803
- Thermotoga maritima
- Treponema pallidum
- Ureaplasma urealyticum
9. Annotation, sequencing in progress
- Bordetella pertussis
- Clostridium acetobutylicum
- Clostridium tetani
- Lactococcus lactis
- Mycobacterium tuberculosis CSU#93
- Neisseria gonorrhoeae
- Neisseria meningitidis Z2491
- Pasteurella multocida
- Pyrobaculum aerophilum
- Pyrococcus furiosus
- Rhodobacter capsulatus
- Sulfolobus tokodaii
- Streptococcus pyogenes
- Vibrio cholerae
- Xylella fastidiosa
- Actinobacillus actinomycetemcomitans
- Aquifex aeolicus strain VF5
- Bacillus anthracis
- Bacillus halodurans C-125
- Bacillus stearothermophilus C-125
- Bartonella henselae
- Bordetella bronchiseptica
- Bordetella parapertussis
- Buchnera aphidicola
- Burkholderia pseudomallei
- Caulobacter crescentus
- Chlorobium tepidum
- Clostridium difficile
- Clostridium sp. BC1
- Corynebacterium Glutamicum
10. Sequencing in progress
- Corynebacterium diphtheriae
- Dehalococcoides ethenogenes
- Desulfovibrio vulgaris
- Ehrlichia species HGE agent
- Enterococcus faecalis V583
- Francisella tularensis
- Geobacter sulfurreducens
- Halobacterium salinarium
- Halobacterium sp.
- Haemophilus ducreyi
- Klebsiella pneumoniae
- Lactobacillus acidophilus
- Legionella pneumophila
- Listeria monocytogenes
- Listeria innocua
- Methanococcus maripaludis
- Methanosarcina mazei
- Methylobacterium extorquens
- Mycobacterium avium
- Mycobacterium bovis (spoligotype 9)
- Mycobacterium bovis BCG
- Mycobacterium leprae
- Mycoplasma capricolum
- Mycoplasma mycoides subsp. mycoides SC
- Mycoplasma pulmonis
- Nitrosomonas europaea
- Nostoc punctiforme
- Photorhabdus luminescens
- Porphyromonas gingivalis
- Prochlorococcus marinus
11. Sequencing in progress
- Pseudomonas aeruginosa
- Pseudomonas putida
- Ralstonia solanacearum
- Rickettsia conorii
- Rhodobacter sphaeroides
- Rhodopseudomonas palustris
- Salmonella typhi
- Salmonella typhimurium
- Salmonella paratyphi A
- Shewanella putrefaciens
- Sinorhizobium meliloti
- Shigella flexneri 2a
- Staphylococcus aureus NCTC 8325
- Staphylococcus aureus COL
- Streptococcus mutans
- Streptomyces coelicolor
- Streptococcus pneumoniae
- Sulfolobus solfataricus
- Thermoplasma acidophilum
- Thermoplasma volcanium GSS1
- Thermus thermophilus
- Thiobacillus ferrooxidans
- Treponema denticola
- Vibrio cholerae
- Xanthomonas citri
- Yersinia pestis
12. Haemophilus influenzaeThe first genome
- Proof of principle
- 1.8 Mbp chromosome, encodes 1703 proteins
- Metabolic capability deduced from genes, not biochemistry
13. 14. Mycoplasma genitaliumthe smallest genome
- Obligate parasite obtains nutrients from host, lacking many metabolic pathways
- 580 kbp chromosome (many bacteria have larger plasmids)
- Only 470 protein-coding genes
15. Mycoplasma mutated265-350 genes are essential 16. Minimal GenomeEthical issues
- Microbial engineering - design of custom bacteria for specific tasks
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- will they spread?
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- Biological Weapons?
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- Are we playing God?, if so
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- is it wrong?
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- where do we draw the line?
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- Answers question What is life? from a reductionist perspective
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- is life now less special?
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- when does life begin?
17. Methanococcus jannaschii The first Archaeon sequenced
- 1.66 Mbp chromosome + 2 plasmids
- 62% of 1738 genes are of unknown function.
- metabolic genes most similar to bacteria
- information flow genes most similar to eukaryotes
18. 19. Escherichia coli - 38% of genes are of unknown function
- 4.64 Mbp chromosome, 4288 protein-coding genes
- despite amount of study, 38% of genes are of unknown function
- evidence for acquisition of substantial amount of DNA from viruses and other organisms
20. Genomics in Molecular Biology
- Yeast Gene Expression Lab (7 weeks)
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- student teams choose conditions, predict genes to be differentially regulated, design PCR primers
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- RT-PCR
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- Northern Blot
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- Microarray (GCAT)
- Yeast cell cycle microarraypaper discussed in class
- Students presented microarray papers for final exam
21. Genes Induced in Rich Medium mRNA splicing unknown SLU7 29 DNA replication, RNA processing ribonuclease H RNH70 31 Actin cortical patch assembly, Establishment of Cell polarity actin binding ABP1 33 Cell growth/maintenance, repression of transcription protein phosphatase type I REG1 33 ER to Golgi transport, IntraGolgi transport, Retrograde transport unknown SEC34 46 DNA dependent, DNA replication exit from mitosis protein phosphatase CDC14 47 DNA replication & Chromosome Cycle unknown HTL1 56.5 Function Protein Name Gene Name Ratio 22. Genes Repressed By Treatment With Ergosterol 23. The Assessment Survey
- Conducted April & May, 2001
- Concert recordings (legal) offered as incentive!!
- 40 Surveys completed
- Survey Sections
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- Assessment of Experience
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- Assessment of Content Knowledge
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- Assessment of Skills
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- Assessment of Attitudes/ Opinions
24. Significant Results
- Of students who had taken Microbiology (n=27)
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- 56% identified the source of a DNA sequence
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- 52% identified a protein from its amino acid sequence
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- 52% retrieved a the cyclin cDNA sequence from Genbank
- 0% of students who had not taken Microbiology (n=13) successfully completed the BLAST search, 15% successfully retrieved a sequence from the database
- Of students who had taken Microbiology but no upper level courses and had not done molecular research (n=11)
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- 45% identified the source of a DNA sequence
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- 45% identified a protein from its amino acid sequence
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- 36% retrieved a the cyclin cDNA sequence from Genbank
25. Significant Results - Genomics
- Of students with hands-on use of microarrays (Molecular Biology, Medical Genetics n=9) more students knew
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- microarrays are used to analyze many genes at once(89% vs 29%) (P=.02)
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- the shotgun method is used to sequence genomes(56% vs 13%) (P=.02)
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- how to perform a BLAST search (78% vs 26%) (P=.03)
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- How to translate a nucleic acid sequence (56% vs 10%) (P