BlueWhite Selection Alpha complementation Trick omega alpha BlueWhite

Blue/White Selection

Alpha complementation Trick omega alpha

Blue/White Selection

Bicistronic strategy: Labels Neurons gal two proteins/same m. RNA

Bicistronic strategy uses IRES two proteins/same m. RNA gal IRES: Internal Ribosome Entry Site

M 71 -IRES-Taulac. Z M 71

Fe add Fe+2

FRV add Fast. REDViolet

M 72 -IRES-GFP(Green Fluorescent Protein) M 71 -IRES-Lac. Z (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 20 th. 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, Mac. Cleod and Mc. Carty 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 d. NTP 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 d. NTPs 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 4 Method: Pulse. Chase Labeling 4 At first, labeled RNA is exclusively in the nucleus. 4 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 -m. RNAs 4 A 7 -Methyl guanosine cap is added to the 5’ end of the primary transcript by a 5’-5’ phosphate linkage. 4 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. 4 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 4 Removal of introns must be very precise. 4 Conserved sequences for removal of the introns of nuclear m. RNA 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 4 The introns of t. RNA precursors are excised by precise endonucleolytic cleavage and ligation reactions catalyzed by special splicing endonuclease and ligase activities. 4 The introns of nuclear pre-m. RNA (hn. RNA) transcripts are spliced out in two-step reactions carried out by spliceosomes.

The Spliceosome 4 Five sn. RNAs: U 1, U 2, U 4, U 5, and U 6 4 Some sn. RNAs associate with proteins to form sn. RNAs (small nuclear ribonucleoproteins)

What are Logo plots?

Logo for a) Splice acceptor b) Splice Donor c) Initiator Met