DNA, the Genetic Material The story of the discovery of the Structure and Function of DNA. This correlates with chapter 16 in your textbook

Big Picture For most of the last century, no one knew what Mendel’s “factors” were. – Not Mendel – Not Darwin – The major principals of Genetics and Evolution were worked out, but the “factors” remained unknown. Sutton first developed chromosomal theory in 1902, but there was only circumstantial evidence that factors were genes on chromosomes.

This is a story... About the process of science, as well as the structure and function of DNA.

For many years scientists thought that ___ was the heritable material, but of course we now know that it is DNA.

What is the genetic material? The Hammerling Experiment: Cells Store Hereditary Information in the Nucleus Joachim Hammerling discovered that hereditary information in a green alga resided in the foot region, which is also the location of the nucleus. How did he know? By amputating different areas, grafting to other alga, and seeing what parts would grow. The form that was found in the foot region is the one that would influence new growth.

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nucleus in base determines type of cap regenerated A. crenulata A. mediterranea

Transplantation Experiments: Each Cell Contains a Full Set of Genetic Instructions Later experiments in the mid-1950s showed that the nucleus of eukaryotic cells includes a full set of genetic information. –What happens if you transplant the nucleus of Frog A to the egg of Frog B? Which frog will develop? Totipotent- nucleus of adult cell carries instructions to create entire organism

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Egg Tadpole or frog embryo UV light destroys nucleus, or it is removed with micropipette. Differentiated cells are isolated from tadpole or frog embryo. Nucleus is removed in micropipette. Differentiated cell nucleus is inserted into enucleate egg. No growth Embryo Tadpole Abnormal embryo Occasionally, an adult frog develops A B

The Griffith Experiment: Hereditary Information Can Pass Between Organisms 1928 Frederick Griffith - Non-pathogenic S. pneumoniae was transformed by dead pathogenic S. pneumoniae. Information specifiying the virulent surface protein had been delivered to the safe live form.

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Mice die; their blood contains live pathogenic strain of S. pneumoniae Mixture of heat-killed pathogenic and live nonpathogenic strains of S. pneumoniae + Heat-killed pathogenic strain of S. pneumoniae Live pathogenic strain of S. pneumoniae Live nonpathogenic strain of S. pneumoniae Polysaccharide coat Mice liveMice die (1)(3)(4) Mice live (2)

Transformation Griffith didn’t know the factor that was transmitted was DNA. He hypothesized it was protein. This is called transformation. We know DNA from the deadly strain was taken up by the non-deadly strain. We will transform E. coli with a glowing gene, and the bacteria should glow.

The Avery and Hershey-Chase Experiments: The Active Principle Is DNA Oswald Avery, Colin MacLeod and Maclyn McCarty provided conclusive evidence that DNA is the hereditary material for the transformation under investigation. –Repeated experiments similar to Griffith’s, but removed as much protein and other material as possible, but still non- pathogenic cells were transformed, so protein, fat, nor carb responsible.

Alfred Hershey and Martha Chase - Bacteriophage: virus with a simple nucleic acid and a protein coat. Identified DNA by marking phosphorous with 32 P (only DNA contains phosphorous) Identified protein by marking sulfur with 35 S (only protein contains sulfer). Found that it was the DNA, marked with 32 P, that was injected into the bacteria that caused disease.

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Protein coat labeled with 35 S DNA labeled with 32 P Bacteriophages infect bacterial cells. T2 bacteriophages are labeled with radioactive isotopes. Bacterial cells are agitated to remove protein coats. 35 S radioactivity found in the medium 32 P radioactivity found in the bacterial cells

What is the structure of DNA? The Chemical Nature of Nucleic Acids Both DNA and RNA are formed of nucleotides joined together in series. Each nucleotide is composed of a five- carbon sugar, a phosphate group, and a nitrogen-containing base.

Chargaff's Rule - there are always equal proportions of purines and pyrimidines. A-T G-C Pure AGgie (purines are A & G) –Purines have a double ring structure. –Pyrimidines have a single ring structure. Single always pairs with double..

-Do you remember how we numbered carbons in sugar? Start from right side. -Base attached to 1’ phosphate attached to 5’. This comes up when we talk about replication, transcription, and translation. (We read from 5’ to 3’) Phosphodiester bond between nucleotides.

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. OH CH 2 O PO 4 Base

Figure nm 1 nm 0.34 nm Hydrogen bond (a) Key features of DNA structure Space-filling model (c) (b) Partial chemical structure 3 end 5 end 3 end 5 end T T A A G G C C C C C C C C C C C G G G G G G G G G T T T T T T A A A A A A

The Three-Dimensional Structure of DNA (page 309) Rosalyn Franklin was able to obtain the first glimpse of DNA using X-ray diffraction in 1953, while Watson and Crick theorized that DNA exists in a double-helical, antiparallel configuration. Famous example of woman being scr’d. Could you guess the pattern by looking at the x-ray?

How does DNA replicate? The Meselson-Stahl Experiment: DNA Replication Is Semiconservative Matthew Meselson and Franklin Stahl demonstrated that DNA replication is semiconservative because each strand of the original duplex becomes one of the two strands in each new duplex. (p. 312) Also used isotopes, this time 15 N, a heavy isotope of Nitrogen

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4. The DNA was suspended in a cesium chloride solution. Centrifugation 1234 Control group (unlabeled DNA) Labeled parent DNA (both strands heavy) F 1 generation DNA (one heavy/light hybrid molecule) F 2 generation DNA (one unlabeled molecule, one heavy/light hybrid molecule)

What would the F 1 generation look like if completely new DNA were synthesized (conservative model), rather than semi- conservative replication? The new piece would be completely light, and the old piece would be completely heavy. There would be two separate bands of DNA.

The Replication Process (PAGE ) Replication of E. coli begins at a specific origin, proceeds bidirectionally, and ends at a specific terminus. OriC is beginning point. Contains many A-T pairs, which are double bonded, easy to open. Leading and lagging strand, why so named? One side is synthesized continuously, but the other limited to short segments (Okizaki fragments).

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA polymerase III DNA double helix Lagging strand Primer Okazaki fragment Leading strand

Figure Page 315 Leading strand Lagging strand Overview Origin of replication Lagging strand Leading strand Primer Overall directions of replication Origin of replication RNA primer Sliding clamp DNA pol III Parental DNA

DNA primase -creates a short RNA primer complementary to a DNA template DNA helicase unwinds the helix DNA polymerase, which then synthesizes new DNA by adding nucleotides to the growing strands DNA ligase creates phosphodiester bonds between adjacent Okazaki fragments Each of these has a name that gives away its job.

Replication fork- Open area of DNA where replication takes place. Topoisomerase- Relieves tension in the area ahead of replication fork Replication can be divided into three stages: initiation, elongation, and termination. –Initiation- there are two OriC’s, one on each strand.

Figure PAG PAGE 317 Overview Leading strand Origin of replication Lagging strand Leading strand Lagging strand Overall directions of replication Leading strand DNA pol III Lagging strand DNA pol I DNA ligase Primer Primase Parental DNA Page 317

DNA REPLICATION DNA Replication is directional. Synthesis of DNA and RNA occurs in the 5’ to 3’ direction. We are studying replication. What part of the cell cycle is replication?

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. OH CH 2 O PO 4 Base

Eukaryotic DNA Replication The major difference between prokaryotic and eukaryotic replication is that eukaryotic chromosomes have multiple replication origins, whereas prokaryotic chromosomes have a single point of origin.

What is a gene? The One-Gene/One-Polypeptide Hypothesis Beadle and Tatum concluded that genes produce their effects by specifying the structure of enzymes, and that each gene encodes the structure of one enzyme. Today, this is commonly referred to as the one- gene/one-polypeptide relationship.

Nutritional Mutants in Neurospora: Scientific Inquiry George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal media Using crosses, they and their coworkers identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme © 2011 Pearson Education, Inc.

View Discovery Video: How did researchers make the connection between DNA and proteins-- E8886C221312&blnFromSearch=1&productcode=US

Figure 17.2 Minimal medium No growth: Mutant cells cannot grow and divide Growth: Wild-type cells growing and dividing EXPERIMENT RESULTS CONCLUSION Classes of Neurospora crassa Wild type Class I mutants Class II mutants Class III mutants Minimal medium (MM) (control) MM  ornithine MM  citrulline Condition MM  arginine (control) Summary of results Can grow with or without any supplements Can grow on ornithine, citrulline, or arginine Can grow only on citrulline or arginine Require arginine to grow Wild type Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Gene (codes for enzyme) Gene A Gene B Gene C Precursor Enzyme A Enzyme B Enzyme C Ornithine Citrulline Arginine

Figure 17.2c CONCLUSION Wild type Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Gene (codes for enzyme) Gene A Gene B Gene C Precursor Enzyme A Enzyme B Ornithine Enzyme C Citrulline Arginine

How DNA Encodes Protein Structure Sanger sequenced the amino acids of insulin, first time to sequence a protein. Then Ingram found that a single aa substitution Valine instead of Glutamic acid, caused sickle cell anemia.

Over 50 years of research has yielded clear evidence that DNA is the molecule responsible for the inheritance of traits from one generation to the next, and that DNA is divided into functional subunits, or genes, located on chromosomes The question is answered