Transcript Jeff Newman - Davidson College

Integrating Genomics Throughout
the Curriculum, with an Emphasis on
Prokaryotes
Jeffrey D. Newman
Lycoming College
May 20, 2002
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
Genetics
Microbiology
Human Physiology
Plant Science
Ecology
At least 1 upper level course
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 unknown’s rRNA gene, sequencing.
• Phase II (’99-’02) Genomics added to many courses
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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
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
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.
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?
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:
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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?
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?
Completed Genomes in
GenBank
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Aeropyrum pernix
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Aquifex aeolicus
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Archaeoglobus fulgidus
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Bacillus subtilis
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Borrelia burgdorferi
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Campylobacter jejuni
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Chlamydia pneumoniae CWL029 •
Chlamydia pneumoniae AR39
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Chlamydia muridarum
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Chlamydia trachomatis D/UW-3/CX•
Deinococcus radiodurans
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Escherichia coli
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Haemophilus influenzae
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Helicobacter pylori26695
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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
Annotation, sequencing in
progress
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Bordetella pertussis
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Clostridium acetobutylicum
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Clostridium tetani
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Lactococcus lactis
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Mycobacterium tuberculosis CSU#93 •
Neisseria gonorrhoeae
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Neisseria meningitidis Z2491
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Pasteurella multocida
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Pyrobaculum aerophilum
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Pyrococcus furiosus
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Rhodobacter capsulatus
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Sulfolobus tokodaii
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Streptococcus pyogenes
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Vibrio cholerae
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Xylella fastidiosa
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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
Sequencing in progress
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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
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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
Sequencing in progress
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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
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Streptomyces coelicolor
Streptococcus pneumoniae
Sulfolobus solfataricus
Thermoplasma acidophilum
Thermoplasma volcanium GSS1
Thermus thermophilus
Thiobacillus ferrooxidans
Treponema denticola
Vibrio cholerae
Xanthomonas citri
Yersinia pestis
Haemophilus influenzae
The first genome
• Proof of principle
• 1.8 Mbp chromosome, encodes 1703 proteins
• Metabolic capability deduced from genes, not
biochemistry
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
Mycoplasma mutated
265-350 genes are essential
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?
Methanococcus jannaschii
The first Archaeon sequenced
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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
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
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 microarray
papers for “final exam”
Genes Induced in Rich Medium
Ratio
Gene
Name
Protein Name
Function
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HTL1
unknown
DNA replication & Chromosome Cycle
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CDC14
protein
phosphatase
DNA dependent, DNA replication exit from mitosis
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SEC34
unknown
ER to Golgi transport, IntraGolgi transport, Retrograde
transport
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REG1
protein
phosphatase
type I
Cell growth/maintenance, repression of transcription
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ABP1
actin binding
Actin cortical patch assembly, Establishment of Cell
polarity
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RNH70
ribonuclease H
DNA replication, RNA processing
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SLU7
unknown
mRNA splicing
Genes Repressed By Treatment
With Ergosterol
The Assessment Survey
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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
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
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<.01)
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
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.”
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
Visit the Project Web Page at
http://www.lycoming.edu/~newman/models.html
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 the
genomics revolution together!