DNA The Genetic Material Dr Henry O Ogedegbe

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 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 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 T 2 which is a DNA virus • They labeled the viral DNA with radioactive isotope of phosphorus 32 P and the protein coat with radioactive sulfur 35 S • After the labeled viruses were allowed to infect 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 32 P label had transferred to the interior of the bacteria and viruses released subsequently contained the 32 P 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 1920 s 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 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/oneenzyme hypothesis or one-gene/one-polypeptide