The Perpetuation of Life

Study of Genetics

• Genetics is the study of:
• How genetic information as a whole is duplicated and transferred to each new cell
• somatic cells and germ cells (gametes)
• Genes which are defined as the sequence of DNA that encodes proteins
• Genes are organized into chromosomes and the collection of chromosomes and plastid DNA is referred to as the genome

The Structure and Replication of DNA

• Molecular genetics begins with the study of DNA structure and mechanisms of replication
• Protein-digesting enzymes destroy nearly everything in the cell except the nuclear content
• Nucleus contains large amounts of nucleic acids
• Fuelgen stain binds specifically with nucleic acids
• Demonstrated that nuclear DNA is restricted to the chromosomes

Genes: DNA or Protein?

• Physician Fred Griffith (1928) while studying the epidemilogy of pneumonia identified a transforming agent
• Studied two stains of pneumococci
• Smooth strain (virulent) and rough strain (avirulent)
• Injection of smooth strain resulted in the disease, while the rough strain did not
• Smooth strain was heat labile (killed), if inject following heat treatment did not produce the disease
• Killed smooth injected with avirulent rough resulted in pneumonia symptoms
• Avery, McLeod and McCarty (1943) suggested the transforming agent was DNA
• Not a proof because the heat killed virulent DNA might have activated a protein based system
• Hershey and Chase (1952) studied a bacteriophage
• Bacteriophage destroy bacteria cells and are composed of a protein capsid and a nucleic acid core
• Radioisotopic studies demonstrated that nucleic acids are passed to the next generation, not proteins

Molecular Structure of DNA

• DNA is composed of nucleotides which are composed a nitrogeneous base, a five carbon carbohydrate and phosphate group
• There are four types of nucleotides in DNA each differ only in the nitrogenous bases
• Adenine and Guanine are purines
• Cytosine and Thymine are pyrimidines
• Nucleotides are arranged in a linear fashion; the carbohydrates of adjacent nucleotides are held together through a phosphodiester bond
• Linkage of the 3' carbon of one sugar to the 5' carbon of the next
• The nitrogenous bases are arranged as side-chains off of this carbohydrate chain

Double Stranded Structure of DNA

• DNA ordinarily exists as a double stranded structure
• Held together by hydrogen bonds between the nitrogenous bases
• Complementary nitrogenous bases can form stabilizing hydrogen bond
• The two strands of a DNA molecule are antiparallel to each other

Discovery of the Double Helix

• Erwin Chargaff analyzed the DNA of different organisms and found that DNA composition was species-specific
• In any organism the amount of the four nitrogenous bases differed among species
• Particular regularity of in base ratios
• Purines equaled the number of pyrimidines
• A=T and G=C
• Linus Pauling, Maurice Wilkins and Roslind Franklin
• Studied DNA structure using X-ray defraction
• James Watson and Francis Crick
• Crick developed mathematic models fro the interpreting X-ray defraction patterns
• Watson and Crick modeled the structure by combining:
• Known chemical content
• X-ray defraction patterns
• Known bond distances, angles and size of atoms
• Built scale models of the DNA components and attempted to fit them together in acordance with the other data
• The structure that fitted best was a double helix

How DNA is Replicated

• Watson and Crick model
• The double helix nature of DNA with complementary base hydrogen bonding suggested a mechanism for replication
• Separation of the two strands could produce templates for the replication of the complementary strand
• Authur Kornberg isolated DNA polymerase
• An enzyme that was capable of replicating DNA in a test tube if supplied with the four nucleotides and ATP

Models of DNA Replication

• Conservative model
• The two template strands separate, serve as templates for the new strands and the reaneal
• Old-old and new-new arrangement
• Dispersive model
• Template fragment resulting in all replicated strands being a mixture of old-new
• Semi-conservative (Watson Crick model)
• Template strands separate are replicated with new complementary strands
• Old-new and old new arrangement

Experimental Evidence for the Semi-conservative Model

• Messelson and Stahl experiments
• Grew bacteria on a medium for several generation that contained 15N
• Labeling all the DNA with 15N
• Transferred the bacteria to an 14N medium and allowed bacterial growth
• Isolated the DNA and employed density gradient centrifugation
• First generation DNA was a hybrid of 15N and 14N
• Disproving the conservative model
• Second generation had 15N- 14N and 14N- 14N molecules disproving the dispersive model

Difficulties of DNA Replication

• DNA replication is similar in both procaryotes and eucaryotes
• Originates at discrete sites on the replication (origins of replication)
• The enzyme which catalyzes this process is DNA polymerase
• DNA polymerase III (E. coli) adds new nucleotides to a template strand in the 5' to 3' direction
• This results in a continuously synthesized strand and a discontinuously synthesized strand

DNA Polymerase

• Catalyzes the addition of complimentary nucleotides to the template strand
• Initiation of nucleotide addition requires an RNA primer
• Continuously synthesized strand (Leading)
• Discontinuously synthesized strand (Lagging) is composed of a series of Okazaki fragments
• Short RNA primers and short DNA pieces

Synthesis of DNA

• Synthesis of Leading Strand (Continuous)
• Priming (primase)
• Elongation (polymerase)
• Synthesis of Lagging Strand (Discontinuous)
• Priming of Okazaki fragments (primase)
• Elongation of fragments (polymerase)
• RNA primers replaced by DNA (polymerase)
• Fragments are joined together (ligase)

Replication of Organelle DNA

• Occurs out of phase with the chromosomal replication
• Organelle DNA is circular
• Organelles contain several copies of their chromosomes
• Similar to bacterial chromosomes
• Differ from bacterial genomes in the number of proteins encoded
• Bacterial chromosomes encode 3000 products, while mitochondria encode about 40 gene products

Repair of Errors

• Precision in DNA replication is essential for normal cellular function
• Random changes (mutations) in the nucleotide sequence of a gene is more likely to disrupt metabolic function than it is to improve it
• Specialized enzymes have evolved to recognize and repair errors

Proofreading

• Error rate in DNA replication is about 3 nucleotides per 100,000 bases pairs
• DNA polymerase complex contains enzymes that recognize these mistakes and removes them
• This reduces the error rate to about 1in a billion
• Repair of mutations induced by environment
• Misalignment Deletion
• Thymine dimerization
• Methylated cytosine

• This page is maintained by James C. Pushnik; jpushnik@ecst.csuchico.edu
• Last modified 11/6/96