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

• 2 semester intro bio series

• Genetics

• Microbiology

• Human Physiology

• Plant Science

• Ecology

• At least 1 upper level course

3. Incorporation of Molecular Biology, Bioinformatics, Genomics

Phase I (97-99) Intro and core course labs

• Intro. Biology DNA sequence analysis, plasmid prep, transformation, restriction digest, gel.

• Genetics PCR from cheek cell DNA, cloning into pBS

• Microbiology PCR of unknowns rRNA gene, sequencing.

Phase II (99-02) Genomics added to many courses

• Intro. Biology shotgun sequencing, HGP conclusions.

• Genetics discussion of microarrays

• Microbiology Microbial Genome Papers

• 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

• Gene number

• Gene complexity

• Types of gene products

• • Protein Families!

• 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:

• How many genes/proteins do we still know nothing about?

• What are the minimal requirements for a living organism?

• How has evolution streamlined microbial genomes?

• How are microbes related to each other?

• What are the genomic differences between:

• • Archaea and Bacteria?

• • obligate parasites and free-living organisms?

• • Phototrophic and chemotrophic organisms?

• • Organotrophic and lithotrophic organisms?

• • Mesophiles and Thermophiles?

• • Pathogenic and non-pathogenic strains?

7. Applications of Microbial Genome Data

Gene chips/microarrays can detect tens of thousands of specific DNA or RNA sequences

• pathogen identification in tissue sample

• virulence genes used for prognosis

• 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

• will they spread?

• Biological Weapons?

• Are we playing God?, if so

• • is it wrong?

• • where do we draw the line?

Answers question What is life? from a reductionist perspective

• is life now less special?

• 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)

• student teams choose conditions, predict genes to be differentially regulated, design PCR primers

• RT-PCR

• Northern Blot

• 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

• Assessment of Experience

• Assessment of Content Knowledge

• Assessment of Skills

• Assessment of Attitudes/ Opinions

24. Significant Results

Of students who had taken Microbiology (n=27)

• 56% identified the source of a DNA sequence

• 52% identified a protein from its amino acid sequence

• 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)

• 45% identified the source of a DNA sequence

• 45% identified a protein from its amino acid sequence

• 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

• microarrays are used to analyze many genes at once(89% vs 29%) (P=.02)

• the shotgun method is used to sequence genomes(56% vs 13%) (P=.02)

• how to perform a BLAST search (78% vs 26%) (P=.03)

• How to translate a nucleic acid sequence (56% vs 10%) (P