Transcript Mycoplasma genitalium

From Mendel to Genomics
• Historically
– Identify or create mutations,
follow inheritance
• Determine linkage, create
maps
• Genomics: use of recombinant
DNA methods
– Focus: entire genome, not
individual genes
– Methodology in place for
sequencing entire genomes
www.bastardidentro.com
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Bioinformatics
• Sequencing creates
huge amount of
information that must be
stored and analyzed
• Bioinformatics is the
science of methods for
storing and analyzing
that information
– Melding of computer
science and molecular
biology
http://www.swbic.org/products/clipart/images/bioinformatics.jpg
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Proteomics
• Proteome: all the proteins an organism makes
• Proteomics: the study of those proteins
– Timing of gene expression
– Regulation of gene expression
– Modifications made to proteins
– Functions of the proteins
– Subcellular location of proteins
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/3_14d.jpg
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Sequencing the Human Genome
• Publicly funded consortium
– Clone-by-clone method
– Create library of clones of entire genome
– Order clones using various DNA markers
– Then sequence each clone
• Craig Venter and private enterprise
– Shotgun method
– Create library of clones of entire genome
– Sequence all the clones
– Use supercomputer to determine order
• Sequencing done multiple times to get it right.
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Clone-by-clone
www.yourgenome.org/ intermediate/all/
Shotgun approach
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Annotation: making sense of the sequence
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• Looking for regulatory regions, RNA genes,
repetitive regions, and protein genes.
• Finding protein genes
– Look for ORFs (open reading frames)
• Start codon (ATG), stop codon.
• Codons must be “in frame”, distance long enough
– Problems: 3 reading frames x 2 strands, widely
spaced genes, introns.
– Help: new software finds TATA box and other
elements; codon bias can help
• Different codons not used equally in organisms
Where is the reading frame?
Could start in one of 3 different places.
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Functional Genomics
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• OK you have a sequence. What does the gene
do? What is the function of the protein?
– Search database for similar sequences
– How does sequence compare to sequences for
proteins of known function?
– Use computer to search for functional motifs.
• Various proteins that do the same thing have
similar structural elements.
• Example: transcription factors like lecuine zippers
Fundamental questions
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• Questions can be asked using whole genome
information that couldn’t before.
– How did genomes evolve?
– What is the minimum number of genes necessary
for a free-living organism?
• Much can be learned about the ecology of an
organism by genomics and proteomics.
– First bacterium sequenced: Mycoplasma genitalium
– Lives a parasitic existence, evident from genes.
Protein function
Amino acid biosynthesis
Purine, pyrimidine, nucleoside and nucleotide
metabolism
Fatty acid and phospholipid metabolism
Biosynthesis of co-factors, prosthetic groups
and carriers
Central intermediary metabolism
Energy metabolism
Transport and binding proteins
DNA metabolism
Transcription
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# of genes
0
19
8
4
7
33
33
29
13
11
Protein synthesis
Protein fate
Regulatory functions
Cell envelope
Cellular processes
Other categories
Unknown
Hypothetical
Database match
No database match
90
21
5
29
6
0
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Total number
483
168
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Advances in understanding genomes
• Prokaryotic- eubacterial
• not all genomes are circular
• not all genomes are in one piece
• when is a plasmid not a plasmid but a
chromosome?
• not all genomes are small
• very little wasted space, very few with introns
• Significant quantity of genes organized into
operons
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Understanding-2
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• Archaeal genomes similar to eubacteria but
• have histones, sequence similarities to
eukaryotes, and introns in tRNA genes
• Eukaryotic genomes -wide variations
• low gene density, that is few genes per amount of
DNA
• introns, more in some (humans) than others
• repetitive sequences
Proteomics: study of proteins
• Proteomics
– 35,000 genes, 100,000 different proteins
• must be lots of post translational modifications
–>100 different ways of modifying proteins
–addition of groups, crosslinking, inteins
• many genes code for proteins of unknown
function
– methods of study
• 2D gel electrophoresis
• Peptide fragments generated with trypsin,
studied by MS
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2D gel electrophoresis of proteins
Blue and green
arrows mark
proteins of interest.
Proteins of
Halobacterium.
Left to right: pH
Vertical: MW
Spots digested w/
trypsin then studied
using mass spec.
http://www.biochem.mpg.de/en/research/rd/oesterhelt/web_page_list/Proteome_Hasal_cytosolic/absatz_3_bild.gif
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Biotechnology
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• What is Biotechnology?
– Use of organisms, especially microbes, to produce
useful products?
• Beer, wine, bread, organic solvents, antibiotics
• By this definition, very very old.
– Use of recombinant DNA techniques to harness the
power organisms to make use products.
• Very new technology
• Includes herbicide-resistant plants, human
proteins produced in yeasts, new vaccines.
Biotechnology has several applications:
overview
• Agriculture
– Herbicide resistant plants
– Improved nutritional qualities
• Pharmaceuticals
– Production of human proteins as drugs
– Production of vaccines
• Medical, legal, biological
– Screening for, treatment of genetic disease
– DNA fingerprinting, biological conservation
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Herbicide resistance
• Example: glyphosate resistant plants
– More than 2/3 of US soybeans and cotton
– Glyphosate inhibits EPSP synthase gene
• Engineered plants have extra copies of gene,
make more enzyme, so are more resistant.
• Steps in engineering:
– Gene from E. coli. Put next to strong promoter
– Cloned into Ti plasmid, plasmid put back into
A. tumefaciens which carries plasmid to plant cell.
– Grow whole plant from engineered plant cell
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Why and why not?
• Use of herbicide-resistant plants means less
herbicide use, no-till farming.
– less erosion and less non-point source pollution.
• Safe to eat? Why not?
– Proteins not automatically destroyed during
digestion; allergies possible. Otherwise, what’s
the problem?
• Environmental concerns
– Toxic pollen? Herbicide resistant weeds?
• Biotech: same only more targeted and
quicker.
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Ag-2: improved nutrition
• Not every food product has complete nutrition
– Corn very low in the amino acid lysine
– Countries relying on rice have low intake of betacarotene
– Some plants have health-improving chemicals
• Transgenic plants can provide relief
– Daffodil gene inserted into rice to make betacarotene, precursor to Vitamin A = golden rice
• Critics say: not enough to make a difference.
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Pharmaceuticals
• Dwarfism, diabetes, cancer can be treated
using human proteins
– Obtained with difficulty
– Insulin from slaughterhouse animals
• Recombinant insulin first from E. coli
– Required combination of cloning, chemical
treatment
– Starting point: mRNA, reverse transcriptase, then
insertion into plasmid vector
– E. coli or yeast cells used.
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Future directions
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• “pharming”: growing of protein drugs in farm
plants and animals
– Cloning into sheep (etc.) with mammary specific
promoter, only expressed in that tissue.
• Released in, collected from milk.
– Using tobacco plants, especially for vaccines
• Tobacco easy to grow, easy to engineer, easy to
harvest
• Years of agricultural experience
Vaccines
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• Exposing host to antigens found on pathogen
– Whole, live, weakened pathogen
• Strong immunity, but risk of live pathogen
– Whole, dead pathogen
• Nucleic acid not “dead”;
• cancer or toxic reaction
– Subunit vaccine: using a molecule from pathogen
• Host reacts, then protects against later exposure
to entire pathogen
Vaccines-2
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• Recombinant vaccines
– Clone gene for surface antigen of pathogen
– Express gene i.e. get antigenic proteins made
• Collect proteins, process into vaccine
– Express proteins in food
• Because there are food allergies, proteins taken
orally can result in immune reactions
• Eliminates worries about sterilization, storage,
needle-phobia
Transgenic vaccine
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Medical diagnosis
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• Sickle cell anemia
– Fetal cell samples
– CVS or amniocentesis
– Gene obtained from fetal DNA
• Sickle cell anemia caused by a single nucleotide
base substitution that removes a MstII site.
– Different banding pattern on gel indicates whether
fetus will be a carrier or have disease
(homozygous)
Medical diagnosis -2
• Cystic fibrosis
– Most cases causes by a specific deletion of DNA
– PCR used to make allele-specific oligonucleotides
• This DNA hybridizes to region in normal gene
that is deleted in faulty allele
• Absence of hybridization means deletion is
present, person has the Cf allele.
• Huntington disease
– Because of variable number of trinucleotide
repeats, probably PCR or VNTR-type test looking
for varying lengths of DNA fragments.
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Ethics!
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• Genetic engineering, medical tests opens up
wide range of issues and questions
– Environmental and global economic issues
– Stem cell research and cloning
– Who owns the data? Can someone else patent your
genes? Privacy issues.
• Should your boss, insurance company,
government have access to your data?
– We can tell you that you have the disease, but
• We can’t do anything about it!