Integrating Genomics Throughout the Curriculum, with an Emphasis on Prokaryotes

1
Integrating Genomics Throughout the Curriculum,
with an Emphasis on Prokaryotes

• Jeffrey D. Newman
• Lycoming College
• May 20, 2002

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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

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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

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Genomics in Intro Biology

• Replication ? PCR ? DNA sequencing ? shotgun
  strategy ? contig 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
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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.

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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?

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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?

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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

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Annotation, sequencing in progress

• Bordetella pertussis
• Clostridium acetobutylicum
• Clostridium tetani
• Lactococcus lactis
• Mycobacterium tuberculosis CSU93
• 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

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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

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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

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Haemophilus influenzae The first genome

• Proof of principle
• 1.8 Mbp chromosome, encodes 1703 proteins
• Metabolic capability deduced from genes, not
  biochemistry

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Mycoplasma genitalium the 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

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Mycoplasma mutated 265-350 genes are essential
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Minimal Genome Ethical 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?

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Methanococcus jannaschiiThe 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

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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

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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 microarray paper discussed in
  class
• Students presented microarraypapers for final
  exam

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Genes Induced in Rich Medium
Ratio Gene Name Protein Name Function
56.5 HTL1 unknown DNA replication Chromosome Cycle
47 CDC14 protein phosphatase DNA dependent, DNA replication exit from mitosis
46 SEC34 unknown ER to Golgi transport, IntraGolgi transport, Retrograde transport
33 REG1 protein phosphatase type I Cell growth/maintenance, repression of transcription
33 ABP1 actin binding Actin cortical patch assembly, Establishment of Cell polarity
31 RNH70 ribonuclease H DNA replication, RNA processing
29 SLU7 unknown mRNA splicing
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Genes Repressed By Treatment With Ergosterol
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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

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Significant Results

• Of students who had taken Microbiology (n27)
• 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
  (n13) 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 (n11)
• 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

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Significant Results - Genomics

• Of students with hands-on use of microarrays
  (Molecular Biology, Medical Genetics n9) 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) (Plt.01)

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Survey question

• Microbial genome sequences are useful because...
• 8 understanding of human genes/proteins
• 7 clues to how organisms cause disease
• 6 define evolutionary relationships,
  adaptations
• 3 antibiotic development
• 3 identification of microbes
• 3 prep for human genome

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Good specific comments

• Connie Wilson figure out relationships between
  different species - two species in same
  environment both adapted to the conditions but in
  different ways.
• Jen Leader They can be compared to eukaryotes
  which will aid in structural and functional
  identification of proteins/genes.
• Justin Jay they provide us with a dictionary
  of the different genes a microbe has. With this
  information we can cut and paste different genes
  into different organisms.
• Amy Allen if people know the sequence for
  specific microbes they can better determine how
  those microbes interact with their surroundings
  ie bacteria interacting with other bacteria in
  biofilms.
• Kim Murray They are finding new ways to treat
  all different kinds of diseases by using genome
  sequences and they are also establishing new
  evolutionary relationships. They are also
  important because they are finding things they
  thought they never would that will be beneficial
  in many areas of biology.

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Conclusions

• Exposure to genomics has led to improved
  understanding of this field
• Students successfully used the NCBI website to
  perform a BLAST search or retrieve a sequence
  from the database.
• Students with little to moderate experience
  using Lasergene did not retain skills.
• New bioinformatics exercises will be based on
  web-based sources, or downloadable software

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Visit the Project Web Page athttp//www.lycoming.
edu/newman/models.html
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Thank You to.

• Malcolm Campbell for organizing GCAT
• Other GCAT members for protocols, advice via
  listserv
• DNAstar for Lasergene software
• Lycoming College Biology Department for
  encouragement, cooperation, financial support of
  the Molecular Biology and Bioinformatics
  Project.
• My students as we participate in thegenomics
  revolution together!