GENE30001 Lecture Notes - Lecture 3: Lac Repressor, Tetraodontidae, Deoxyribonuclease I
14 Jun 2018
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Professor
ENCODE: Incorrectly over-interpreted biochemical function
DNAse1 Footprinting.
DNAse1 can't to a short fragment due to an added protein, it can be
seen
Phylogenetic footprinting is a technique used to identify transcription factor
binding sites (TFBS) within a non-coding region of DNA of interest by comparing it
to the orthologous sequence in different species.
From <https://www.google.com.au/webhp?sourceid=chrome-instant&ion=1
&espv=2&ie=UTF-8#q=phylogenetic+footprinting>
Scenario: ApoA
Get divergence by sequencing lots of closely related species (ortho/paralogs)
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ApoA-seq in 18 old world monkeys and it can be seen that the 'conserved
pats of the troughs while the variable parts of the peaks.
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High plasma levels risk indicator for cardiovas disease , gene only found in O.W.
Monkey and hominids
Ultra-conserved seq (UCS) : often clustered and in gene deserts where there
aren't many gene, many near genes involves wih regulation of transcription
and development.
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Conserved non-coding elements - regulatory modules?
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Conserved non-genic sequences
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Extreme levels of conservation Non-coding Conserved Sequences (NCS)
Reporter can see if the CNS are enhance by introducing reporter construction and
recapitaulate a component of the pattern
Scenario: Eye development
Tested 7 CNS to see if they drive GFP expression in fish
Found that it was conserved in puffer fish and humans
In saying this, alternative splicing can determine that there is a possibility of
functional conservation without seq. Conservation.
Has 1 sripe 2 enhancer with a regulatory modeil with multiple TF binding
sites but these binding sites diverged in sequence.
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Yet adding the pseudobscura enhancer-reporter contrust in melanogaster
and expression is driven the same way.
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Scenario: Eve (counter-example)
That's to say chimeric enhancers can't do this
There's a distinction b/w what a seq element does and its function
Clone a piece of DNA that contains the operator site to which the repressor binds.
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Label one end of the DNA molecules with a radioactive molecule, e.g. radioactive ATP.
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DNase I cuts DNA molecules randomly (in contrast to restriction enzymes that cut where they find a particular
sequence)
○
Choose such gentle conditions that most molecules will be cut only once.
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Digest the DNA with DNase I.
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The result will be a mixture of radioactive fragments of varying length, with the smallest increment in length represented by
a single nucleotide.
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Separate the fragments by electrophoresis.
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Binding of the lac repressor to the sequence of 24 base pairs in the operator prevents DNase I from attacking that region of
the molecule.
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When the fragments are separated by electrophoresis, those representing the lengths covered by the repressor will be
missing from the autoradiogram.
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The resulting gap is the "footprint".
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The same sample of DNA (unprotected by the repressor) is subjected to normal DNA sequencing and the resulting ladder
aligned with the footprint autoradiogram.
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The exact sequence of bases in the lac operator can then be read directly because they represent the rungs of the ladder
missing in the footprint.
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DNAse footprinting The procedure:
From <http://www.biology-pages.info/F/Footprinting.html>
Lecture 3 : Negative Selection and selective constraint
Wednesday, 22 June 2016
11:58 AM
Lecture Notes 1- 12 Molecular Evolution and Population Genetics Page 1
Document Summary
Lecture 3 : negative selection and selective constraint. Dnase1 can"t to a short fragment due to an added protein, it can be seen. Phylogenetic footprinting is a technique used to identify transcription factor binding sites (tfbs) within a non-coding region of dna of interest by comparing it to the orthologous sequence in different species. High plasma levels risk indicator for cardiovas disease , gene only found in o. w. Get divergence by sequencing lots of closely related species (ortho/paralogs) Apoa-seq in 18 old world monkeys and it can be seen that the "conserved pats of the troughs while the variable parts of the peaks. Extreme levels of conservation non-coding conserved sequences (ncs) Ultra-conserved seq (ucs) : often clustered and in gene deserts where there aren"t many gene, many near genes involves wih regulation of transcription and development. Reporter can see if the cns are enhance by introducing reporter construction and recapitaulate a component of the pattern.
