Presentation on theme: "The History Of DNA. Meischer and DNA The history of deoxyribonucleic acid (DNA) research begins with Friedrich Miescher, a Swiss biologist who in 1868."— Presentation transcript:
Meischer and DNA The history of deoxyribonucleic acid (DNA) research begins with Friedrich Miescher, a Swiss biologist who in 1868 carried out the first carefully thought out chemical studies on the nuclei of cells. Using the nuclei of pus cells obtained from discarded surgical bandages, Miescher detected a phosphorus-containing substance that he named nuclein. He showed that nuclein consists of an acidic portion, which we know today as DNA, and a basic protein portion now recognized as histones, a class of proteins responsible for the packaging of DNA. Later he found a similar substance in the heads of salmon sperm cells. Although he separated the nucleic acid fraction and studied its properties, the covalent structure of DNA did not become known with certainty until the late 194Os.
Griffiths Experiment Rough Pneumococcus are harmless. They lack a gel capsule that would protect them from a host organism's immune system attack. Smooth Pneumococcus are pathogenic (they cause disease), and when injected, give a mouse fatal pneumonia. Griffiths injected several combinations of rough and smooth into hapless mice, and found...
Experimental Results –l–live rough --> mice okay –l–live smooth --> mice pushing up daisies –k–killed (boiled) rough --> mice okay –k–killed (boiled) smooth --> mice okay –l–live smooth + killed rough --> mice kick the bucket –l–live rough + killed smooth --> MICE CROAK! This was a surprise!
The Puzzle When Griffiths autopsied the mice, he found LIVE SMOOTH PNEUMOCOCCUS!! He replicated this many times, in case there had been an accidental injection of some live smooth bacteria into the mice, but there was no mistake. Somehow, the harmless live, rough bacteria had been TRANSFORMED into deadly smooth bacteria. But how?!
Conclusions Whatever the culprit, it had to have several properties in order to fit the bill: It had to be duplicated whenever a cell divided, so it could be passed on unchanged. It had to be in the form of an informational code It had to be (mostly) stable and resistant to change
Avery’s Experiment Oswald Avery (1944) demonstrated that the substance responsible for the transformation of harmless bacteria into disease-causing monsters was DNA: He exposed the extract of boiled bacteria Griffiths had used to various substances that would destroy one of the compounds (gel capsule, proteins or nucleic acids), one at a time. He found that DNase (an enzyme which breaks down DNA) would stop the transformation process. Boiled DNase (destroyed by heat) did not stop transformation
Luria and Delbruck at Cold Spring Harbor Worked on the life cycle of lysogenic and lytic bacteriophages Bacteriophages- viruses that attack bacteria And paved the way for
Bacteriophages and Genetics Scientists began to wonder what the differences were in the life cycle of the lytic and lysogenic phages
The Blender Experiment Martha Chase and Albert Hershey
Side by side experiments are performed with separate bacteriophage (virus) cultures in which either the protein capsule is labeled with radioactive sulfur or the DNA core is labeled with radioactive phosphorus. The radioactively labeled phages are allowed to infect bacteria. Agitation in a blender dislodges phage particles from bacterial cells. Centrifugation concentrates cells, separating them from the phage particles left in the supernatant. Results: Radioactive sulfur is found predominantly in the supernatant. Radioactive phosphorus is found predominantly in the cell fraction, from which a new generation of infective phage can be isolated. Conclusion: The active component of the bacteriophage that transmits the infective characteristic is the DNA. There is a clear correlation between DNA and genetic information.
Joshua Lederberg and Edward L. Tatum publish on conjugation in bacteria. The proof is based on the generation of daughter cells able to grow in media that cannot support growth of either of the parent cells. Their experiments showed that this type of gene exchange requires direct contact between bacteria. At the time Lederberg began studying with Tatum, scientists believed that bacteria reproduced asexually, but from the work of Beadle and Tatum, Lederberg knew that fungi reproduced sexually and he suspected that bacteria did as well. Conjugation in Bacteria- Bacterial Sex
Rosalind Franklin The elegant and comprehensive X-ray diffraction studies of Rosalind Franklin and Maurice Wilkins at King's College, (London, England) yielded a characteristic diffraction pattern from which it was deduced that DNA fibers have two periodicities along their long ans: a major one of 0.34 nm and a secondary one of 3.4 nm.Rosalind Franklin
Rosalind’s accomplishments The technique with which Rosalind Franklin set out to do this is called X-ray crystallography. With this technique, the locations of atoms in any crystal can be precisely mapped by looking at the image of the crystal under an X-ray beam. By the early 1950s, scientists were just learning how to use this technique to study biological molecules. Rosalind Franklin applied her chemist's expertise to the unwieldy DNA molecule. After complicated analysis, she discovered (and was the first to state) that the sugar-phosphate backbone of DNA lies on the outside of the molecule. She also elucidated the basic helical structure of the molecule.
The second species-invariant observation was that Chargaff's first parity rule also applies, to a close approximation, to single- stranded DNA (his "second parity rule"). If the individual strands of a DNA duplex are isolated and their base compositions determined, then %A = %T, and %C = %G (Rudner et al., 1968). Thus it was noted that there is an: A=T C=G
Chargaff’s conclusions 1) A + G = T + C; (2) A = T; (3) G = C; and as a logical consequence of these three equations: (4) A + C = G + T, i.e., the sum of the 6- amino compounds equals that of the 6-oxo derivatives.
He also studied the orientation of purines and pyrimidines in tobacco mosaic virus and in nucleic acids, by measuring the ultraviolet dichroism of oriented specimens, and he studied, with the visible-light polarizing microscope, the arrangement of virus particles in crystals of TMV and measured dry mass in cells with interference microscopes. He then began X-ray diffraction studies of DNA and sperm heads. Maurice Wilkins
a.Ribo-oligonucleotides - Khorana developed methods to synthesize short RNA molecules of specific sequence UUUUUUUUUUU,AAAAAAAAAAAA b. Cell-free extracts - simply the stuff inside cells after they are broken open; these extracts contain the protein synthesis machinery c. 14C-labeled amino acids The idea was to give the cell-free extracts RNAs of known sequence to translate. The amino acids that were incorporated into protein in response to a specific RNA could be identified by providing radiolabeled amino acids in each experiment then purifying and sequencing the radiolabeled peptides. Nirenberg and Khorana
UUGUUGUUG..., the cells synthesized poly-leucine, poly-cysteine and poly-valine; UGUGUGUGU... gave one peptide with alternating valines and cysteines; GGUGGUGGU... gave poly-glycine, poly-valine, and poly-tryptophan.
The small subunit of the ribosome binds to a site "upstream" (on the 5' side) of the start of the message. It proceeds downstream (5' -> 3') until it encounters the start codon AUG. Here it is joined by the large subunit and a special initiator tRNA. The initiator tRNA binds to the P site (shown in pink) on the ribosome. In eukaryotes, initiator tRNA carries methionine (Met). (Bacteria use a modified methionine designated fMet.)methionine Initiation
An aminoacyl-tRNA (a tRNA covalently bound to its amino acid) able to base pair with the next codon on the mRNA arrives at the A site (green) associated with: an elongation factor (called EF-Tu in bacteria) GTP (the source of the needed energy) The preceding amino acid (Met at the start of translation) is covalently linked to the incoming amino acid with a peptide bond (shown in red).peptide bond The initiator tRNA is released from the P site. The ribosome moves one codon downstream. This shifts the more recently-arrived tRNA, with its attached peptide, to the P site and opens the A site for the arrival of a new aminoacyl-tRNA. This last step is promoted by another protein elongation factor (named EF- G) and the energy of another molecule of GTP. Elongation
The end of the message is marked by one or more STOP codons (UAA, UAG, UGA). There are no tRNA molecules with anticodons for STOP codons. However, a protein release factor recognizes these codons when they arrive at the A site. Binding of this protein releases the polypeptide from the ribosome. The ribosome splits into its subunits, which can later be reassembled for another round of protein synthesis. Polysomes Termination