DNA: The Genetic Material Dr. Henry O. Ogedegbe Department of EHMCS

The Hammerling Experiment: Cells Store Hereditary Information in the Nucleus Where is hereditary information stored in the cell? The Danish biologist Hammerling cut cells into pieces to see which were able to express hereditary information He chose the green alga Acetabularia which grows up to 5 cm as a model organism for his experiments The genus Acetabularia have distinct foot, stalk and cap regions and the nucleus is located in the foot He amputated the stalk of some cells and the feet of others He found that when he amputated the cap, a new cap regenerated from the remaining portions of the cell

The Hammerling Experiment: Cells Store Hereditary Information in the Nucleus When the foot was amputated however, no new foot regenerated from the cap or the stalk He therefore hypothesized that the hereditary information resided within the foot of Acetabularia His hypothesis was tested by selecting individuals from two species of the genius Acetabularia which had different caps: A. mediterranea has a disc shaped cap A. crenulata has a branched flower-like cap He grafted a stalk from A. crenulata to a foot from A mediterranea

The Hammerling Experiment: Cells Store Hereditary Information in the Nucleus The cap regenerated looked somewhat like the cap of A. crenulata He then cut the regenerated stalk and all subsequent caps were disc shaped like like A. mediterranea The experiment supported Hammerling’s hypothesis

Transplantation Experiments: each Cell Contains s Full Set of Genetic Instructions Robert Briggs and Thomas King tested the hypothesis that that the nucleus is the repository of hereditary information They removed the nucleus from frog eggs and found out that without the nucleus, the egg did not develop When the nucleus was replaced, with one from a frog embryo cell, the egg developed into an adult frog While this experiment produced abnormal frogs modifications of the experiment by other workers produced satisfactory results

The Griffith Experiment: hereditary Information Can Pass between organisms Discovery of Transformation: Griffith performed experiments in which he injected mice with virulent strain of streptococcus pneumoniae The infected mice all died. When he infected similar mice with mutant strains of S. pneumoniae that lacked the virulence factor the mice showed no ill effect When he infected mice with dead mutant strain of S. pneumoniae the mice remained healthy Similarly, infection of the mice with the R form of the bacteria produced no ill effect in the mice

The Griffith Experiment: hereditary Information Can Pass between organisms When he infected similar mice with a mixture of dead virulent bacteria and the R form some of the mice died The virulence factor had been transferred to the R form which transformed the coatless form to the virulent form

The Avery Experiment: The Transforming Principle Is DNA Avery and co-workers characterized the transforming principle They prepared a mixture of dead and coatless S. pneumoniae similar to what Griffith had done Then they removed all the proteins from the mixture Despite the removal of the proteins, the transforming activity of the mixture was not reduced The properties of the transforming principle resembled those of DNA in many respects

The Avery Experiment: The Transforming Principle Is DNA Analysis of the purified principle produced elements which agreed closely with DNA In an ultracentrifuge the transforming principle migrated like DNA Removal of lipids and proteins from the principle did not diminish its activity Protein digesting enzymes did not affect the principle The DNA digesting enzyme DNase destroyed all the transforming principle

The Hershey-Chase Experiment: Some Viruses Direct Their Heredity with DNA Hershey-Chase experiment involved bacteriophages, viruses that attack bacteria They employed the bacteriophage T2 which is a DNA virus They labeled the viral DNA with radioactive isotope of phosphorus 32P and the protein coat with radioactive sulfur 35S After the labeled viruses were allowed to infect the bacteria the bacterial cells were agitated violently This was designed to remove the protein coats of the infecting viruses from the surface of the bacteria

The Hershey-Chase Experiment: Some Viruses Direct Their Heredity with DNA The 32P label had transferred to the interior of the bacteria and viruses released subsequently contained the 32P label The hereditary information injected into the bacteria that specified new generation of viruses was DNA Thus the DNA is clearly the repository of hereditary information

The Frankel-Conrat Experiment: Other Viruses Direct Their Heredity with RNA Fraenkel-Conrat experimented with RNA viruses to determine how they reproduce They employed the tobacco mosaic virus and the Holmes ribgrass virus. They separated the RNA from the proteins and discovered that the RNA molecules were still infective whereas the protein molecules were not

The Chemical Nature of Nucleic Acid The DNA was discovered in 1969 by Friedrich Miescher four years after the publication of Mendel’s work He extracted a white substance from human cells and fish sperm The proportion of nitrogen and phosphorus in the substance was different from any previous substances This convinced him that he was dealing with a new substance Due to its slight acidity, it came to be known as nucleic acid

The Chemical Nature of Nucleic Acid The primary structure was elucidated in the 1920s by the biochemist P.A. Levene DNA contains three components which include the phosphate group, five carbon sugars, and nitrogenous bases The nitrogenous bases are purines; adenine guanine and pyramidines; thymine, and cytosine RNA contains uracil instead of thymine A nucleotide consist of a sugar attached to a phosphate group and a base

The Chemical Nature of Nucleic Acid The four carbon atoms and the oxygen atom form a five membered ring The carbon atoms are numbered 1’ to 5’ proceeding clockwise from the oxygen atom The prime symbol indicates that the carbon refers to a carbon in a sugar rather than a base The subunits are linked together by phosphodiester bonds The resultant two-unit polymer still has a free 5’ phosphate group at one end and a free 3’ hydroxyl group at the other end

The Chemical Nature of Nucleic Acid Chargaff’s Analysis showed that the nucleotide composition of DNA molecules varies in complex ways This led to Chargaff’s rules: The proportion of A always equals that of T and the proportion of G always equals that of C There is always an equal proportion of purines (A and G) and pyramidines (C and T)

The three-Dimensional Structure of DNA The work of Rosalind Franklin involved X-ray crystallographic analysis of DNA This involved bombarding the DNA molecules with beams of X-rays Rosalind used DNA in the form of fibers in the laboratory of Maurice Wilkins The work of Rosalind led to the discovery of the double helix by Crick and Watson The double helix is stabilized by antiparallel strands one chain running 3’ to 5’ the other 5’ to 3’

The Meselson-Stahl Experiment: DNA Replication Is semiconservative The basis for copying the genetic information is complementarity If the DNA molecule is unzipped one would need only to assemble the appropriate complementary nucleotides This would produce two daughter duplexes with the same sequence This form of DNA replication is called semiconservative because the sequence of the original duplex is conserved Each strand of the duplex becomes part of another duplex

The Replication Complex The DNA polymerase III plays a very essential part in gene DNA replication The polymerase III is a complex of 10 different kinds of polypeptide chains The enzyme is a dimer with two similar multisubunit complexes Polymerase III threads the DNA through the complex at the rate of 1000 nucleotides per second

The Replication Complex The two strand of DNA are assembled differently The polymerase III can add nucleotide only to the 3’ end of a DNA strand That means that replication occurs in the 5’ to 3’ direction on a growing DNA strand The leading strand is built up by adding nucleotides continuously to it growing 3’ end The lagging strand which elongates away from the replication fork is synthesized discontinuously as short segments

The Replication Complex These discontinuous segments are called the Okasaki fragments They are about 100 to 200 nucleotides long in eukaryotes and about 1000 to 2000 nucleotides long in prokaryotes The Okasaki fragment is synthesized by DNA polymerase III in the 5’ to 3’ direction The overall replication of the DNA is said to be semidiscontinuous

The Replication Process The replication of the DNA molecule takes place in five interlocking steps: Opening of the DNA double helix Initiation replication Unwinding the duplex Stabilizing the single strand Relieving the torque generated by unwinding Building a primer Assembling complementary strands Removing the primer Joining the Okasaki fragments

The One-Gene/One-Polypeptide Hypothesis The discovery that certain types of inherited diseases were prevalent in particular families led to the conclusion that: These diseases were Mendelian traits which had resulted from changes in the hereditary information in an ancestor An example is alkaptonuria in which patients produce urine that contained homogentisic acid (alkapton) Such patients lacked the enzyme necessary to catalyze the breakdown of alkaptonuria Invariably it was concluded that genes specify particular enzymes This knowledge was clearly elucidated by Beadle and Tatum in their experiments involving the bread mold

The One-Gene/One-Polypeptide Hypothesis Beadle and Tatum were able to isolate mutant strains with defective form of that enzyme The mutations were always located at specific chromosmal sites and each enzyme had a different site Each mutant had a defect in a single enzyme caused by a mutation at a single site on the chromosome They concluded that genes produce their effects by specifying the structure of enzymes and each gene encodes the structure of one enzyme This relationship was termed by them the one-gene/one-enzyme hypothesis or one-gene/one-polypeptide