Blue/White Selection

Alpha complementation Trick

alpha

omega

Blue/White Selection

two proteins/same mRNA

Bicistronic strategy:Labels Neurons

gal

gal

two proteins/same mRNA

Bicistronic strategy uses IRES

gal

gal

IRES: Internal Ribosome Entry Site

M71-IRES-TaulacZ

M71

M71

add Fe+2

Fe

add FastREDViolet

FRV

M71-IRES-LacZ (FRV-Xgal)

M72 -IRES-GFP(Green Fluorescent Protein)

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)

He worked with Albrecht Kossel and Emil Fischer, the nucleic acid and protein experts at the turn of the 20th. century

Phoebus Levene

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) wereNeeded 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, includingthe 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], andindependently at the Rockefeller Institute by Rollin Hotchkiss

Note C not always equal to G

Apart from not demonstrating equal amounts of the fourbases and thus casting doubt on the validity of the tetranucleotidehypothesis, certain other unexpected patternsalso emerged: the amounts of purines seemed always toequal those of pyrimidines (that is, A + G = C + T, or(A + G)/(C + T) = 1). This had been found by Alfred Mirskyin 1943, but seems to have been overlooked by theChargaff laboratory. More curiously, the ratios of A:G andT:C were always similar to each other whether they weregreater or less than 1

The significance of these relationships was puzzling anda constant source of comment. At the end of 1949 Chargaffnoted that ‘‘A comparison of the molar proportions [of thebases] reveals certain striking, but perhaps meaningless,regularities’’. Early in 1950, he wrote ‘‘It is noteworthy,although possibly no more than accidental, that in alldesoxypentose nucleic acids examined thus far the molarratios of total purines to total pyrimidines were not farfrom 1. More should not be read into these figures.’’Later in 1950, apparently as a last-minute insertion in thepaper, Chargaff wrote ‘‘It is noteworthy – whether this ismore than accidental, cannot yet be said – that in alldesoxypentose nucleic acids examined thus far the molarratios of total purines and total pyrimidines, and also ofadenine to thymine and of guanine to cytosine [ratioscuriously not actually presented], were not far from 1’’[2]. The following year, he wrote ‘‘As the number ofexamples of such regularity increases, the question willbecome pertinent whether it is merely accidental orwhether it is an expression of certain structural principlesthat are shared by many desoxypentose nucleic acids,despite far-reaching differences in their individual compositionand the absence of a recognizable periodicity in theirnucleotide sequence’’. He then added ‘‘It is believedthat the time has not yet come to attempt an answer’’,although clearly the subject was very much on his mind.

Why didn’t Chargaff predictThe 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 thatA/T G/C ratio was the same????

3) H and X nucleotides?

Accounting in Saccharomyces cerevisiae

From Davidson:The state ofdNTP in the day.

From Chargaff:

Model Building of DNA

Early efforts to make a DNA model

Watson’s Early attemptTo create Double Helix

Jerry Donohue:Worked on dNTPsTold Watson that It was the Keto formFound under physiological conditions

B-DNA

Chapter 11Transcription and RNA Processing

RNA Synthesis And Transport in Eukaryotes

Method: Pulse-Chase 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 describeEnhancers 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

More CellsBut on a per cellBasis expression levels of -gal is about the same

Transient transfection

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 fora) Splice acceptor

b) Splice Donor

c) Initiator Met