Transcript ppt - eweb.furman.edu

Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
- once you have the sequence data, you really have just started.
- The goals are then:
- identify where genes are (Open Reading Frames)
- find promoters and regulatory elements to confirm this is a gene (and not a
pseudogene).
- in eukaryotes, find splice sites, introns and exons
- identify structural sequences like telomeres and centromeres
- convert the DNA sequence into the predicted AA sequence of the protein
- predict protein structure and function by identifying ‘domains’ and ‘motifs’
- These goals are attained by computer analyses of gene/AA sequence data, and
comparison with known described genes. This is:
BIOINFORMATICS
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
1. NCBI – BLAST search compares sequence to other sequences in the
database
1 ggggcacccc tacccactgg ttagcccacg ccatcctgag gacccagctg cacccctacc
61 acagcacctc gggcctaggc tgggcggggg gctggggagg cagagctgcg aagaggggag
121 atgtggggtg gactcccttc cctcctcctc cccctctcca ttccaactcc caaattgggg
181 gccgggccag gcagctctga ttggctgggg cacgggcggc cggctccccc tctccgaggg
241 gcagggttcc tccctgctct ccatcaggac agtataaaag gggcccgggc cagtcgtcgg
301 agcagacggg agtttctcct cggggtcgga gcaggaggca cgcggagtgt gaggccacgc
361 atgagcggac gctaaccccc tccccagcca caaagagtct acatgtctag ggtctagaca
421 tgttcagctt tgtggacctc cggctcctgc tcctcttagc ggccaccgcc ctcctgacgc
481 acggccaaga ggaaggccaa gtcgagggcc aagacgaaga cagtaagtcc caaacttttg
541 ggagtgcaag gatactctat atcgcgcctt gcgcttggtc ccgggggccg cggcttaaaa
601 cgagacgtgg atgatccgga gactcgggaa tggaagggag atgatgaggg ctcttcctcg
661 gcgccctgag acaggaggga gctcaccctg gggcgaggtt ggggttgaac gcgccccggg
721 agcgggaggt gagggtggag cgccccgtga gttggtgcaa gagagaatcc cgagagcgca
781 accggggaag tggggatcag ggtgcagagt gaggaaagta cgtcgaagat gggatggggg
841 cgccgagcgg ggcatttgaa gcccaagatg tagaagcaat caggaaggcc gtgggatgat
901 tcataaggaa agattgccct ctctgcgggc tagagtgttg ctgggccgtg ggggtgctgg
961 gcagccgcgg gaagggggtg cggagcgtgg gcgggtggag gatgagaaac tttggcgcgg
1021 actcggcggg gcggggtcct tgcgccccct gctgaccgat gctgagcact gcgtctcccg
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
1. NCBI – BLAST search compares sequence to other sequences in the
database
2. Open Reading Frames: base sequences which would code for long
stretches of AA’s before a stop codon would be reached. Typically,
these are found by looking for [5’ – ATG…-3’] sequences that follow a
promoter (TATA, CAAT, GGGCGG). The complement would be [3’ –
TAC..-5’], which would encode a start codon in RNA [5’- AUG…3’]
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
1. NCBI – BLAST search compares sequence to other sequences in the
database
2. Open Reading Frames: base sequences which would code for long
stretches of AA’s before a stop codon would be reached. Typically,
these are found by looking for [5’ – ATG…-3’] sequences that follow a
promoter (TATA, CAAT, GGGCGG). The complement would be [3’ –
TAC..-5’], which would encode a start codon in RNA [5’- AUG…3’]
3. Regulatory regions and splicing sites (GT-AG):
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
- Sequence Homology: search libraries for similar sequences already described in
other proteins with known function, in other species…..
Arabidopsis thalia
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
- Sequence Homology: search libraries for similar sequences already described in
other proteins with known function, in other species… or the same species
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
- Sequence Homology: search libraries for similar sequences already described in
other proteins with known function, even in other species.
- Domain / Motif Analysis: Certain AA sequences are known to have a certain
structure (‘motif’ like “helix-turn-helix”) or function (‘domain’ like an ion channel
sequence, DNA binding region).
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
- Sequence Homology: search libraries for similar sequences already described in
other proteins with known function, even in other species.
- Domain / Motif Analysis: Certain AA sequences are known to have a certain
structure (‘motif’ like “helix-turn-helix”) or function (‘domain’ like an ion channel
sequence, DNA binding region).
- Mutant Analysis: Mutate the gene (insert a non-functional sequence) in vitro, then
insert in cells and observe effects of “knocking out” function in different tissues or
the whole organism.
Capecchi, Evans, and Smithies were awarded the 2007 Nobel Prize for
their technique for inserting a gene into embryonic cells…this gene can be
a mutant, non-functional gene (“knock-out”) or a functional gene (“knockin”).
Typically, you would then screen mice for those who, by luck, had transformed cells
end up in their gonads. These mice will pass the mutation to their gametes; so if you
mate a male and female, you will create offspring that are homozygous for this
mutation across their entire genome….and you can see it’s effects.
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
E. Comparing Protein Expression
- Construction of a microarray – ‘gene chip’
Can create a chip with unique sequence DNA from every gene in a
genome (‘probe’).
Take a tissue sample
Isolate m-RNA
Make labeled c-DNA
Expose to chip and allow
complementation
Wash
Analyze florescence at
each point; binding
denotes that this tissue
has this gene on at this
point in development
Take a tissue sample
Isolate m-RNA
Make labeled c-DNA
Expose to chip and allow
complementation
Wash
Analyze florescence at
each point; binding
denotes that this tissue
has this gene on at this
point in development
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
E. Comparing Protein Expression
F. Phylogenetic Analyses: Comparative Genomics
- DNA or AA sequences can be compared across species
For example… download the sequence for cytochrome-c from different organisms:
>Arabidopsis
MASFDEAPPGNPKAGEKIFRTKCAQCHTVEKGAGHKQGPNLNGLFGRQSGTTPGYSYSAA
NKSMAVNWEEKTLYDYLLNPKKYIPGTKMVFPGLKKPQDRADLIAYLKEGTA
>Euglena
GDAERGKKLFESRAGQCHSSQKGVNSTGPALYGVYGRTSGTVPGYAYSNANKNAAIVWED
ESLNKFLENPKKYVPGTKMAFAGIKAKKDRLDIIAYMKTLKD
>Hippo
GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQSPGFSYTDANKNKGITWG
EETLMEYLENPKKYIPGTKMIFAGIKKKGERADLIAYLKQATNE
>Mosquito
MGVPAGDVEKGKKLFVQRCAQCHTVEAGGKHKVGPNLHGLFGRKTGQAAGFSYTDANKAK
GITWNEDTLFEYLENPKKYIPGTKMVFAGLKKPQERGDLIAYLKSATK
>Rice
MASFSEAPPGNPKAGEKIFKTKCAQCHTVDKGAGHKQGPNLNGLFGRQSGTTPGYSYSTA
NKNMAVIWEENTLYDYLLNPKKYIPGTKMVFPGLKKPQERADLISYLKEATS
Use clustalX to align sequences and resolve a phylogeny
Use n-j plot to see the plot
0.02
Eu glena
Mosquit o
Hippo
Ri ce
Arabidopsis
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
E. Comparing Protein Expression
F. Phylogenetic Analyses: Comparative Genomics
G. Conclusions from Genomic Studies:
- there is remarkable homology in protein/gene sequence between species
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
E. Comparing Protein Expression
F. Phylogenetic Analyses: Comparative Genomics
G. Conclusions from Genomic Studies:
- there is remarkable homology in protein/gene sequence between species
- physiological/developmental complexity is not correlated with genome size
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
E. Comparing Protein Expression
F. Phylogenetic Analyses: Comparative Genomics
G. Conclusions from Genomic Studies:
- there is remarkable homology in protein/gene sequence between species
- physiological/developmental complexity is not correlated with genome size
- only 2-5% of human genome codes for proteins
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
E. Comparing Protein Expression
F. Phylogenetic Analyses: Comparative Genomics
G. Conclusions from Genomic Studies:
- there is remarkable homology in protein/gene sequence between species
- physiological/developmental complexity is not correlated with genome size
- only 2-5% of human genome codes for proteins
- although there are 100,000 proteins, there are only 20,000 genes…
suggesting that most genes encode multiple proteins, produced through transcript and
post-translational processing.
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
E. Comparing Protein Expression
F. Phylogenetic Analyses: Comparative Genomics
G. Conclusions from Genomic Studies:
- there is remarkable homology in protein/gene sequence between species
- physiological/developmental complexity is not correlated with genome size
- only 2-5% of human genome codes for proteins
- although there are 100,000 proteins, there are only 20,000 genes…
suggesting that most genes encode multiple proteins, produced through transcript and
post-translational processing.
- Most of the genome does NOT encode protein. However, large fractions of
DNA do encode nc-RNA’s… “non-coding RNA’s” which are not translated but are
produced by transcription and then exert a regulatory function (mi-RNA’s and others).
Genomics
A. Overview:
B. Sequencing:
C. Finding Genes – structural genomics and ‘annotation’:
D. Identifying Gene Function – functional genomics:
E. Comparing Protein Expression
F. Phylogenetic Analyses: Comparative Genomics
G. Conclusions from Genomic Studies:
- there is remarkable homology in protein/gene sequence between species
- physiological/developmental complexity is not correlated with genome size
- only 2-5% of human genome codes for proteins
- although there are 100,000 proteins, there are only 20,000 genes…
suggesting that most genes encode multiple proteins, produced through transcript and
post-translational processing.
- Most of the genome does NOT encode protein. However, large fractions of
DNA do encode nc-RNA’s… “non-coding RNA’s” which are not translated but are
produced by transcription and then exert a regulatory function (mi-RNA’s and others).
- So, organisms with similarities in coding genes can be remarkably
different…as a consequence of how the production of those proteins is regulated in
different cell types and at different developmental periods.
PHEW!!!!
Recombinant DNA Technology combines DNA from different sources
– usually different species
Utility:
this is done to study DNA sequences
to mass-produce proteins
to give recipient species new characteristics
as a therapy/curative for genetic disorders (‘gene therapy’)
Human insulin, created in bacteria
Corn damaged by
corn borer and fungi
“bt-corn”, with a
bacterial gene
Genomics
Genetic Engineering
A. To mass-produce proteins
Genomics
Genetic Engineering
A. To mass-produce proteins
Making human insulin
Genomics
Genetic Engineering
A. To mass-produce proteins
A1-antitrypsin was
the first;
antithrombin is the
first transgenic
protein produced
in animals to be
approved by FDA
for human use.
Eukaryote genes may not be read properly by bacterial
hosts because of introns and regulatory elements. In
addition, the protein may not be processed correctly or
fold correctly. Using a eukaryotic host solves these
problems… but tissue expression is the problem.
Genomics
Genetic Engineering
A. To mass-produce proteins
Vaccines (HPV vaccine – ‘Gardasil’ ) are being
synthesized that consist of only a few proteins
that initiate the immune response, rather then
the entire virus (or bacterium). The genes for
these proteins could be put in food, to intiate an
immune response.
Genomics
Genetic Engineering
A. To mass-produce proteins
B. To give species new characteristics
The EPSP synthase gene in E. coli confers
resistance to glyphosate – the primary
ingredient in herbicides like Round-Up©.
Genomics
Genetic Engineering
A. To mass-produce proteins
B. To give species new characteristics
Agrobacterium is a plant pathogen that
inserts Ti plasmids into host cells. These
plasmids have been used as vectors for
introducing the gene into plant tissues,
which grow into new plants.
Genomics
Genetic Engineering
A. To mass-produce proteins
B. To give species new characteristics
Bacillus thuringiensis is a bacterium that produces a protein that crystallizes in insect guts,
killing the insect.
Since the 1930’s, the bacteria were sprayed on crops to reduce insect damage. The
treatment was very short term, as the bacteria died quickly.
Genomics
Genetic Engineering
A. To mass-produce proteins
B. To give species new characteristics
Same process – splice to an Agrobacterium
plasmid, with tissue-specific promoters.
Genomics
Genetic Engineering
A. To mass-produce proteins
B. To give species new characteristics
Issues:
- genetic homogeneity of crop plants
- 2011 study – toxin present in 93% of
pregnant women in a town in Canada,
and increases in immunological
responses.
- used as feed for animal stock
- patterns of use and the evolution of
resistance
Genomics
Genetic Engineering
A. To mass-produce proteins
B. To give species new characteristics
Place gene for growth hormone from chinook
salmon into Atlantic salmon, next to a
constitutive promoter (gene always on, right?)
Grow 10x faster, to same mature size
Models suggest it would outcompete native
species if released into the wild
Genomics
Genetic Engineering
A. To mass-produce proteins
B. To give species new characteristics
C. Gene Therapy
- Create a viral vector with a functional human allele – adenosine
deaminase
- Infect target tissue
- Probably need to repeat unless you can transform stem cells
1990-first trial of
gene therapy –
Ashanti DeSilva.
40 treated since
then with 100%
efficacy.
OTC - ornithine transcarbamylase deficiency syndrome.
An X-linked disorder resulting in the inability to bind and
convert ammonia to urea. Total loss of this protein is
usually fatal shortly after birth.
Jesse Gelsinger – died in
1999 at age 18, as a
consequence of a gene
therapy trial involving an
adenovirus vector. He has
an immunological reaction
to the virus and died.
OTC - ornithine transcarbamylase deficiency syndrome.
“First, although Gelsinger and his family were under the impression
that the pre-clinical animal studies had affirmed the trial's safety, two
monkeys had actually died. This information appeared on the consent
form submitted to the National Institutes of Health review board, but
did not appear on the form signed by Jesse.
Moreover, the Penn researchers did not disclose to either the
Gelsingers or federal regulators that human volunteers in the same
study had suffered adverse reactions - side effects serious enough to
have halted the trials had they been reported. Not reporting adverse
events in gene therapy clinical trials is clearly wrong, but it seems to
have been par for the course in the 1990s: evidence collected shortly
after Gelsinger's death showed that fewer than six percent of adverse
events associated with gene therapy were properly reported at this
time.
Lastly, the lead researcher in the Penn study - James Wilson - did not
disclose to the Gelsingers that he was conducting the clinical trial with a
private company in which he had a stake. Wilson had a direct financial
interest - not merely an academic one - in the trial's successful
outcome.” From Center for Genetics and Society http://www.geneticsandsociety.org/article.php?id=4955
Genomics
Genetic Engineering
Bioethics
A. GMO’s – Genetically Modified Organisms
Should the consumer know?
If content is < 5%, should it be labeled GMO-free?
Required in Europe and Asia… why not in U.S., which produces
65% of GM food worldwide?
Genomics
Genetic Engineering
Bioethics
A. GMO’s – Genetically Modified Organisms
B. Genetic Testing
- 2008 – Genetic Information Nondiscrimination Act
“prohibits the improper use of genetic information in health insurance and
employment”
?
Genomics
Genetic Engineering
Bioethics
A. GMO’s – Genetically Modified Organisms
B. Genetic Testing
- 2008 – Genetic Information Nondiscrimination Act
“prohibits the improper use of genetic information in health insurance and
employment”
Lily
Ledbetter
XX
XY
?
Genomics
Genetic Engineering
Bioethics
A. GMO’s – Genetically Modified Organisms
B. Genetic Testing
- 2008 – Genetic Information Nondiscrimination Act
“GINA does not cover an individual's manifested disease or condition--a condition from
which an individual is experiencing symptoms, being treated for, or that has been
diagnosed.”
Sex discrimination
in the workplace
was not
prohibited under
GINA… so “Lily”
was needed as a
separate “equal
pay for equal
work” act.
Lily
Ledbetter
XX
XY
?
Genomics
Genetic Engineering
Bioethics
A. GMO’s – Genetically Modified Organisms
B. Genetic Testing
- GINA
- Genetic screening and embryo selection.
“Preimplantation Genetic Diagnosis” – used in in vitro fertilization,
screening early embryos for genetic abnormalities… or other traits?
Suppose a child needs
a bone marrow
transplant… should
parents be allowed to
select among embryos
to make a sibling
capable of transfer?
Genomics
Genetic Engineering
Bioethics
A. GMO’s – Genetically Modified Organisms
B. Genetic Testing
- GINA
- PGD
- Germline engineering
Genomics
Genetic Engineering
Bioethics
A. GMO’s – Genetically Modified Organisms
B. Genetic Testing
- GINA
- PGD
- Germline engineering
- Enhancement Gene Therapy
Why not insert “better” genes? For Youth? Strength? Health?
Genomics
Genetic Engineering
Bioethics
A. GMO’s – Genetically Modified Organisms
B. Genetic Testing
- GINA
- PGD
- Germline engineering
- Enhancement Gene Therapy