Transcript Lecture 15 Biol302 Spring 2011

Blue/White Selection
Alpha complementation Trick
omega
alpha
Blue/White Selection
Bicistronic strategy:Labels Neurons
gal
gal
two proteins/same mRNA
Bicistronic strategy uses IRES
two proteins/same mRNA
gal
gal
IRES: Internal Ribosome Entry Site
M71-IRES-TaulacZ
M71
M71
Fe
add Fe+2
FRV
add FastREDViolet
M72 -IRES-GFP(Green Fluorescent Protein)
M71-IRES-LacZ (FRV-Xgal)
Foundations of a replicative organism
DNA Discovery by
Friedrich Miescher (Swiss, 1844-1895)
He discovered a substance
containing both phosphorus
and nitrogen, made up of molecules
that were apparently very large,
in the nuclei of white blood cells
Named the substance nuclein
because it seemed to come
from cell nuclei. In 1874 when
Miescher separated it into a protein
and an acid molecule. It is now known
as deoxyribonucleic acid (DNA)
Phoebus Levene
He worked with Albrecht Kossel
and Emil Fischer, the nucleic acid
and protein experts at the
turn of the 20th. century
He conducted experiments that in 1931
suggested that the four components of DNA
occur in approximately equal ratios
He suggested the possibly that DNA was
made of a repeating tetramer
If so, the implication was that the structure of
DNA was too simple and too regular to
contribute to genetic variation: attention
thereafter focused on protein as the
probable hereditary substance
Not only did Levene identify the components of DNA,
he also showed that the components were linked together
in the order phosphate-sugar-base to form units.
He called each of these units a nucleotide,
and stated that the DNA molecule consisted of a
string of nucleotide units linked together through the
phosphate groups, which are the 'backbone' of the molecule.
Scientist thought that Proteins (made from 20 aa) were
Needed to encode life, not the 4(?) forms of Nucleotides
The Tetranucleotide Hypothesis
Erwin Schrödinger published in 1945 a book titled What is Life?
that planted the idea for searching “the secret of life”
Chargaff noted the publication of Avery, MacCleod and McCarty
paper and realized that DNA was the key to life
and set out to prove Levene wrong
In 1944 Consden et al. showed that it
was possible to separate individual amino acids and
to determine the amino acid composition of protein
hydrolysates by partition chromatography
on paper strips. The method was, in principle, readily
adapted for the separation and identification of a
large number of other substances, including
the purines and pyrimidines of the nucleic acids
(Figure 1), a task carried out in Chargaff’s laboratory
by the Swiss post-doctoral fellow Ernst Vischer [12], and
independently at the Rockefeller Institute by Rollin Hotchkiss
Note C not always equal to G
Apart from not demonstrating equal amounts of the four
bases and thus casting doubt on the validity of the tetranucleotide
hypothesis, certain other unexpected patterns
also emerged: the amounts of purines seemed always to
equal those of pyrimidines (that is, A + G = C + T, or
(A + G)/(C + T) = 1). This had been found by Alfred Mirsky
in 1943, but seems to have been overlooked by the
Chargaff laboratory. More curiously, the ratios of A:G and
T:C were always similar to each other whether they were
greater or less than 1
The significance of these relationships was puzzling and
a constant source of comment. At the end of 1949 Chargaff
noted that ‘‘A comparison of the molar proportions [of the
bases] reveals certain striking, but perhaps meaningless,
regularities’’. Early in 1950, he wrote ‘‘It is noteworthy,
although possibly no more than accidental, that in all
desoxypentose nucleic acids examined thus far the molar
ratios of total purines to total pyrimidines were not far
from 1. More should not be read into these figures.’’
Later in 1950, apparently as a last-minute insertion in the
paper, Chargaff wrote ‘‘It is noteworthy – whether this is
more than accidental, cannot yet be said – that in all
desoxypentose nucleic acids examined thus far the molar
ratios of total purines and total pyrimidines, and also of
adenine to thymine and of guanine to cytosine [ratios
curiously not actually presented], were not far from 1’’
[2]. The following year, he wrote ‘‘As the number of
examples of such regularity increases, the question will
become pertinent whether it is merely accidental or
whether it is an expression of certain structural principles
that are shared by many desoxypentose nucleic acids,
despite far-reaching differences in their individual composition
and the absence of a recognizable periodicity in their
nucleotide sequence’’. He then added ‘‘It is believed
that the time has not yet come to attempt an answer’’,
although clearly the subject was very much on his mind.
Why didn’t Chargaff predict
The Double Helix?
-He didn’t want to be wrong like Levene!
Two reasons why he would have been wrong
1)
2) What if it was some other reason that
A/T G/C ratio was the same????
3) H and X nucleotides?
Accounting in Saccharomyces cerevisiae
From Davidson:
The state of
dNTP in the day.
From Chargaff:
Model Building of DNA
Early efforts to make a DNA model
Watson’s
Early attempt
To create Double Helix
Jerry Donohue:
Worked on dNTPs
Told Watson that
It was the Keto form
Found under physiological
conditions
B-DNA
Chapter 11
Transcription and RNA Processing
RNA Synthesis And Transport
in Eukaryotes
Method: PulseChase Labeling
At first, labeled RNA
is exclusively in the
nucleus.
Later, the labeled
RNA is found in the
cytoplasm.
Awful representation
Correct Representation of DNA
RNA make a new “top strand”
Modifications to Eukaryotic
pre-mRNAs
A 7-Methyl guanosine cap is added to the 5’
end of the primary transcript by a 5’-5’
phosphate linkage.
A poly(A) tail (a 20-200 nucleotide
polyadenosine tract) is added to the 3’ end of
the transcript. The 3’ end is generated by
cleavage rather than by termination.
When present, intron sequences are spliced
out of the transcript.
Eukaryotes Have Three RNA
Polymerases
Pol II is the only Polymerase that is routinely studied.
Pol I and Pol III are very complicated.
A Typical RNA Polymerase II
Promoter
What does the word Promoter mean?
It is the place at which RNA Pol II binds.
But the word is incorrectly used to describe
Enhancers plus Promoter.
Initiation by RNA Polymerase II
TFIID recognition site is TATAA
How often is this site found in the
genome? 1/45
Once every 1000 nucleotides 109 nucleotides or 106 times
Transient transfection
More Cells
But on a per cell
Basis expression levels of -gal
is about the same
Stable transfection
The 7-Methyl Guanosine
(7-MG) Cap
The 3’ Poly(A) Tail
AATAAA
Interrupted Genes in Eukaryotes:
Exons and Introns
Most eukaryotic genes contain noncoding
sequences called introns that interrupt the
coding sequences, or exons. The introns are
excised from the RNA transcripts prior to their
transport to the cytoplasm.
Removal of Intron Sequences
by RNA Splicing
The noncoding introns are excised
from gene transcripts by several
different mechanisms.
Excision of Intron Sequences
Splicing
Removal of introns must be very precise.
Conserved sequences for removal of the
introns of nuclear mRNA genes are minimal.
– Dinucleotide sequences at the 5’ and 3’ ends of
introns.
– An A residue about 30 nucleotides upstream from
the 3’ splice site is needed for lariat formation.
Types of Intron Excision
The introns of tRNA precursors are excised by
precise endonucleolytic cleavage and ligation
reactions catalyzed by special splicing
endonuclease and ligase activities.
The introns of nuclear pre-mRNA (hnRNA)
transcripts are spliced out in two-step reactions
carried out by spliceosomes.
The Spliceosome
 Five snRNAs: U1, U2,
U4, U5, and U6
 Some snRNAs associate
with proteins to form
snRNAs (small nuclear
ribonucleoproteins)
What are Logo plots?
Logo for
a) Splice acceptor
b) Splice Donor
c) Initiator Met