Finding Genes – structural genomics and - Welcome to eweb.furman ...

roachavocadoBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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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…..







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 (“knock
-
in”).






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








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’)

Corn damaged by
corn borer and fungi

“bt
-
corn”, with a
bacterial gene

Human insulin, created in bacteria

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











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.

A1
-
antitrypsin was
the first;
antithrombin is the
first transgenic
protein produced
in animals to be
approved by FDA
for human use.

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 crystalizes 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 from salmon
farms