Questions'''agree: green disagree: red

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Questions...agree greendisagree red

• A cis-element is a short DNA sequence in the
  promoter that allows transcription factors to
  bind
• A transcription factor plays a role in activating
  gene expression
• HATs are receptors on the surface of the cell
• MAP kinases are part of a signal transduction
  pathway that allows the plant to respond to
  external signals by activating the appropriate
  genes
• Reporter genes can only be activated by their
  native promoter
• In order for a gene to become active, histone
  repositioning is a necessary step
• Peptidyl transferases are enzymes that attach
  amino acids to tRNA molecules.
• The Kozak sequence allows the ribosome to
  terminate translation.

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Figures from Yamamoto et al. (1991)
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Post-transcriptional processing Nonsense
mediated mRNA degradation Once an mRNA has been
synthesized, it is thought to undergo a pioneer
round of translation. This is to make sure that
there are no premature stop codons present in the
mRNA as a result of mutations or transcription
mistakes. If there were, the cell would be
wasting resources synthesizing dysfunctional
proteins. The ribosomes scan the mRNA. If a stop
codon is found within a window of 50 nt upstream
of the 3 exon-exon junction (which may be marked
by proteins remaining there from the splicing
reaction), the mRNA is going to get tagged for
degradation. This model may not cover all cases
where reduced expression is observed as a result
of stop codons.
RT-PCR of COMT cDNA from sorghum
cDNA of interest
Control cDNA
TAA
(1,3,5)
wild type
TAA
TAG
(2)
bmr12
TAA
TGA
(4)
bmr18
TAA
TGA
(6)
bmr26
200 bp
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Post-translational processing

• The newly synthesized peptide is typically
  inactive and needs to undergo some modifications
• protein folding to achieve the correct tertiary
  structure. Folding is often mediated by
  chaperones, proteins that help the protein find
  its correct folding.
• proteolytic cleavage to remove parts of the
  protein. This is a mechanism to prevent a
  protein from being active too early or in the
  wrong spot.
• chemical modification of individual amino acids.
  The following modifications can occur
• SMALL CHEMICAL SUBSTITUTIONS
• Acetylation of lysine
• Methylation of lysine
• Phosphorylation of serine, threonine and tyrosine
• Hydroxylation of proline and lysine
• N-formylation of N-terminal glycine
• SUGARS
• O-linked glycosylation of serione and threonine
• N-linked glycosylation of asparagine
• LIPIDS
• Acylation of serine, threonine and cysteine
• N-myristoylation of N-terminal glycine

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This is in bacteria similar mechanisms found in
eukaryotes
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Protein degradation Proteins are degraded after
they become conjugated to ubiquitin. Ubiquitin is
a 76 aa protein which is transferred to lysine
residues of the protein to be degraded. The
degradation signal is not quite understood, but
certain motifs seem to play a role in the binding
of the E3 protein. Three proteins are involved
in the conjugation with ubiquitin and subsequent
degradation E1 carries the ubiquitin protein.
Ubiquitin is then transferred to E2, while E3 is
linked to the substrate. E2 is then transferred
to a lysine in the substrate. Conjugation of
ubiquitin leads to the degradation of the protein
in proteasomes, large cylindrical structures with
a sedimentation coefficient of 26S (made up of a
20S cylinder and tow 19S caps). The protein first
needs to unfold to fit in the cylinder, and the
degradation results in the production of short
peptide of 4-10 aa long. These are subsequently
broken down into individual amino acids that are
recycled.
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(No Transcript)
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• Cloning plant genes
• This topic will cover three lectures
• I. the targeted approach cloning of a
  specific gene
• II. the random approach cloning of a number of
  genes involved in one process
• III. the genomics approach identification of
  all of the genes of an organism and their
  function
• What is cloning?
• Isolation of (typically uncharacterized) DNA so
  that it can be studied in detail
• sequencing
• deduction of amino acid sequence
• gene expression studies
• in vitro transcription or translation
• promoter analysis
• constructs for the generation of transgenic
  plants
• Basically, in order to fully understand the
  molecular basis of many processes or traits, it
  is very helpful (if not required) to have DNA
  sequence information of key genes.

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• The targeted approach
• In this case we know of the existence of a
  particular gene (from genetic studies) and we are
  interested in cloning it.
• The choice of the cloning technique depends on
• the organism
• the gene of interest dowe have any prior
  information on it?
• genetic data are there mutants and is a map
  location available?
• Choices
• PCR
• RT-PCR
• T-DNA tagging
• Tranposon tagging
• Map-based cloning

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• PCR
• Polymerase Chain Reaction
• Is like a DNA copy machine
• Requires
• DNA template,
• thermostable DNA polymerase,
• oligomer primers,
• Mg and buffer,
• a thermal cycler
• Small volume (10-100 µl)
• Fast (1-3 hrs)
• Sensitive (small amounts (i.e. pg-ng quantities)
  of DNA are sufficient)
• Cloning by PCR
• Requires partial sequence information
• DNA or amino acid
• Works well for conserved genes

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PCR
RT-PCR
mRNA
5
3
AAAAAn
TTTTTn
denaturation (94 oC)
first-strand cDNA synthesis by RT
AAAAAn
5
3
TTTTTn
3
5
RNase treatment primer annealing (50-70 oC)
primer annealing (50-70 oC)
5
TTTTTn
3
5
primer extension (72 oC)
3
AAAAAn
5
primer extension (72 oC)
TTTTTn
Next round..
5
3
AAAAAn
5
5
3
TTTTTn
5
TTTTTn
3
AAAAAn
Next round..
An example of cloning based on RT-PCR is
described in the article by Halpin et al. (1998).
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Figure 4 from Halpin et al. (1998). This shows
an alignment of CAD amino acid sequences from
different species. Degenerate PCR primers were
designed based on sequences conserved in most
species, and these primers were used on maize
genomic DNA. The resulting PCR product was then
used to screen a maize cDNA library. Full length
CAD cDNA sequence was obtained via Inverse PCR.
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T-DNA tagging

• T-DNA is transfer DNA from the pathogenic
  bacteria Agrobacterium tumefaciens
• Part of the Ti- ( tumor inducing) plasmid
• Induction of crown gall, a growth on the stem of
  the plant that ends up synthesizing opines for
  the bacteria
• Can be engineered to remove disease symptoms
• A. tumefaciens transfers the T-DNA into the plant
  where it stably integrates into the plant genome
• In the lab tissues are dipped in a suspension of
  A. tumefaciens harboring recombinant Ti-plasmids.
  The transformed tissue is regenerated in vitro
  and results in transgenic plants.
• - Introduction of foreign DNA (overexpression,
  antisense)
• Opportunity for insertional mutagenesis

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An example of cloning based on T-DNA tagging is
described in the article by Meyer et al. (1996).
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Transposon-tagging

• Transposons are mobile genetic elements first
  described by Barbara McClintock in the1940s.
• Maize Ac/Ds
• Spm(En)/ dSpm(I)
• Mutator
• Anthirrinum (snapdragon) Tam
• Transposons are two-element systems.
• One element encodes a transposase
• The other element transposes due to the action of
  the transposase
• In some cases there are families of transposable
  elements
• Mutation rates vary typically 10-6 10-4
• Upon transformation, the maize elements are
  active in tobacco, Arabidopsis and tomato
• Independent mutant alleles are required to use
  transposon-tagging for cloning purposes, unless
  transformation and complementation is possible.

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An example of cloning based on transposon-tagging
is described in the article by Xu et al. (1996).
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Map-based cloning or chromsome walking

• Facilitated by large insert clones (YAC, BAC)
• Mutant locus is mapped to a chromosome arm and
  then fine-mapped with the use of molecular and/or
  visual markers
• Create recombinants from an F2 population, i.e. a
  set of lines in which the allele of interest is
  flanked by DNA from a different source
• Map the recombinants. The mutants will all have
  the allele of interest but the flanking regions
  will show variation.
• The locus of interest can now be narrowed down to
  a small region defined by a distal and a proximal
  marker.
• Start to chromosome walk with a probe derived
  from a nearby marker

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An example of map-based cloning is described in
the article by Franke et al. (2002).
YAC A distal end maps near M2 we know that
locus Y is distal of M2 YAC B distal end
co-segregates with locus Y. YAC B may contain
Y YAC C distal end maps near M4 we know that
locus Y is proximal of M4, so that we have now
gone too far. Investigate YAC C in more detail
(cosmids, transformation).
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An overview of gene cloning strategies
Is the identity of the gene known?
Clone via T-DNA tagging
Is T-DNA tagging possible?
Is there a mutant in which the gene is deficient?
Clone via transposon tagging
Is transposon-tagging possible?
Has the gene been cloned from a different species?
Is there partial amino acid sequence available?
Are there BAC or YAC clones available?
Can the gene be mapped?
Is this a closely related species?
Map-based cloning
Does the gene enclode a protein that can be
assayed?
Can the protein be purified?
Are there candidate genes in the databases?
Screen a genomic of cDNA library with a
heterologous probe
RT-PCR with degenerate primers
Obtain cDNA, check expression patterns,
functional assays..
Postpone or abandon project
LD-PCR on genomic or cDNA