CHAPTER 16

THE MOLECULAR BASIS OF INHERITANCE


OBJECTIVES

After reading this chapter and attending lecture, the student should be able to:

1. Explain why researchers originally thought protein was the genetic material.
2. Summarize experiments performed scientists, which provided evidence that DNA is the genetic material:
3. List the three components of a nucleotide.
4. Distinguish between deoxyribose and ribose.
5. List the nitrogen bases found in DNA.
6. Explain the "base-pairing rule".
7. Describe the structure of DNA and explain what kind of chemical bond connects the nucleotides of each strand and what type of bond holds the two strands together.
8. Explain the semiconservative replication of DNA.

KEY TERMS

deoxyribose semiconservative replication nitrogen base
ribose DNA polymerase origin of replication
adenine thymine guanine
cytosine nucleotide replication fork
replication bubble antiparallel complimentary

LECTURE NOTES

Deoxyribonucleic acid or DNA is the genetic material – Mendel's heritable factors and Morgan's genes on chromosomes. Inheritance has its molecular basis in the precise replication and transmission of DNA from parent to offspring.

I. The search for the genetic material led to DNA: science as a process

By the 1940's, scientists knew that chromosomes carry hereditary material and consist of DNA and protein. Most researchers thought protein was the genetic material because:

A. Evidence That DNA Can Transform Bacteria

In 1928, Frederick Griffith performed experiments which provided evidence that genetic material is a specific molecule.

Griffith was trying to find a vaccine against Streptococcus pneumoniae, a bacterium that causes pneumonia in mammals. He knew that:

Griffith performed four sets of experiments: (See Campbell, Figure 15.1)

  1. Experiment: Griffith injected live S strain of Streptococcus pneumoniae into mice.
  2. Results: Mice died of pneumonia.

    Conclusions: Encapsulated strain is pathogenic.

  3. Experiment: Mice were injected with live R strain.
  4. Results: Mice survived and were healthy.

    Conclusions: The bacterial strain lacking the polysaccharide coat was non-pathogenic.

  5. Experiment: Mice were injected with heat-killed S strain of pneumococcus.
  6. Results: Mice survived and were healthy.

    Conclusions: Polysaccharide coat does not cause pneumonia because it is still present in heat-killed bacteria which proved to be non-pathogenic.

  7. Experiment: Heat-killed S cells mixed with live R cells were injected into mice.

Results: Mice developed pneumonia and died. Blood samples from dead mice contained live S cells.

Conclusions: R cells had acquired from the dead S cells the ability to make polysaccharide coats. Griffith cultured S cells from the dead mice. Since the dividing bacteria produced encapsulated daughter cells, he concluded that this newly acquired trait was inheritable.

The cells that aquired the phenomenon had undergone transformation.

Transformation = The assimilation of external genetic material by a cell.

B. What was the chemical nature of the transforming agent?

Griffith was unable to answer this question, but other scientists continued the search.

Griffith's experiments hinted that protein is not the genetic material. Heat denatures protein, yet it did not destroy the transforming ability of the genetic material in the heat-killed S cells.

In 1944, after a decade of research, Oswald Avery, Maclyn McCarty and Colin MacLeod discovered that the transforming agent had to be DNA.

The discovery by Avery and his coworkers was met with skepticism by other scientists, because they still believed protein was a better candidate for the genetic material and so little was known about DNA.

1. Evidence That Viral DNA Can Program Cells

More evidence that DNA is the genetic material came from studies of bacteriophages.

Bacteriophage (phage) = Virus that infects bacteria.

In 1952, Alfred Hershey and Martha Chase discovered that DNA is the genetic material of a phage known as T2. They knew that T2:

What Hershey and Chase did not know is which viral component – DNA or protein – was responsible for reprogramming the host bacterial cell. They answered this question by performing the following experiment: (See Campbell, Figure 15.2)

Experiment:

 

Results:

Conclusions:

These data provided evidence that nucleic acids rather than proteins are the hereditary material.

2. Additional Evidence That DNA Is the Genetic Material of Cells

Hershey and Chase's experiments provided evidence that DNA is the hereditary material in viruses. Additional evidence pointed to DNA as the genetic material in eukaryotes as well.

Some circumstantial evidence was:

Experimental evidence for DNA as the hereditary material in eukaryotes came from the laboratory of Erwin Chargaff. In 1947, he analyzed the DNA composition of different organisms.

Using paper chromatography to separate nitrogenous bases, Chargaff reported the following:

This source of molecular diversity made it more credible that DNA is the genetic material.

In every species he studied, there was a regularity in base ratios.

The number of adenine (A) residues approximately equaled the number of thymines (T), and the number of guanines (G) equaled the number of cytosines (C).

The A=T and G=C equalities became known later as Chargaff's rules. The explanation for these rules came with Watson and Crick's structural model for DNA.

II. Watson and Crick discovered the double helix by building models to conform to X-ray data: science as a process

By the 1950's, DNA was accepted as the genetic material, and the covalent arrangement in a nucleic acid polymer was well established. The three dimensional structure of DNA, however, was yet to be discovered. (See Campbell, Figure 15.3)

Among scientists working on the problem were:

James Watson went to Cambridge to work with Francis Crick who was studying protein structure with X-ray crystallography.

Watson saw an X-ray photo of DNA produced by Rosalind Franklin at King's College, London. Watson and Crick deduced from Franklin's X-ray data that:

Watson and Crick built scale models of a double helix that would conform to the X-ray data and the known chemistry of DNA.

One of their unsuccessful attempts placed the sugar-phosphate chains inside the molecule.

Watson next put the sugar-phosphate chains on the outside which allowed the more hydrophobic nitrogenous bases to swivel to the interior away from the aqueous medium.

Their proposed structure is a ladder-like molecule twisted into a spiral, with sugar-phosphate backbones as uprights and pairs of nitrogenous bases as rungs.

The two sugar-phosphate backbones of the helix are antiparallel; that is, they run in opposite directions.

Watson and Crick finally solved the problem of DNA structure by proposing that there is a specific pairing between nitrogenous bases. After considering several arrangements, they concluded:

To be consistent with a 2 nm width, a purine on one strand must pair (by hydrogen bonding) with a pyrimidine on the other.

Base structure dictates which pairs of bases can hydrogen bond. The base pairing rule is that adenine can only pair with thymine, and guanine with cytosine.

Purines Pyrimidines Possible

Base Pairs

Number of

Hydrogen Bonds

Adenine (A) Thymine (T) A – T 2
Guanine (G) Cytosine (C) G – C 3

 

The base-pairing rule is significant because:

Though hydrogen bonds between paired bases are weak bonds, collectively they stabilize the DNA molecule. Van der Waals forces between stacked bases also help stabilize DNA.

III. During DNA replication, base-pairing enables existing DNA strands to serve as templates for new complementary strands

In April 1953, Watson and Crick's new model for DNA structure, the double helix, was published in the British journal Nature. This model of DNA structure suggested a template mechanism for DNA replication.

Watson and Crick proposed that genes on the original DNA strand are copied by a specific pairing of complementary bases, which creates a complementary DNA strand.

The complementary strand can then function as a template to produce a copy of the original strand.

In a second paper, Watson and Crick proposed that during DNA replication:

Watson and Crick's model is a semiconservative model for DNA replication.

They predicted that when a double helix replicates, each of the two daughter molecules will have one old or conserved strand from the parent molecule and one newly created strand.

In the late 1950's, Matthew Meselson and Franklin Stahl provided the experimental evidence to support the semiconservative model of DNA replication. A brief description of the experimental steps follows. (See Campbell, Figure 15.8.)

Hypotheses:

There were three alternate hypotheses for the pattern of DNA replication:

Experiment:

Results:

DNA from E. coli grown with 15N was heavier than DNA containing the more common, lighter isotope, 14N. First-generation DNA after one generation of bacterial growth, was all of intermediate density. Second generation DNA after two generations of bacterial growth in light medium was of intermediate and light density.

 

Conclusions:

IV. A team of enzymes and other proteins functions in DNA replication

The general mechanism of DNA replication is conceptually simple, but the actual process:

A. Getting Started: Origins of Replication

DNA replication begins at special sites called origins of replication that have a specific sequence of nucleotides.

Specific proteins required to initiate replication bind to each origin.

The DNA double helix opens at the origin and replication forks spread in both directions away from the central initiation point creating a replication bubble.

Bacterial or viral DNA molecules have only one replication origin.

Eukaryotic chromosomes have hundreds or thousands of replication origins. The many replication bubbles formed by this process, eventually merge forming two continuous DNA molecules.

Replication forks = The Y-shaped regions of replicating DNA molecules where new strands are growing.

B. Elongating a New DNA Strand

1. Strand Separation

2. Synthesis of the New DNA Strands

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C. Hydrolysis of nucleoside triphosphates provides the energy necessary to synthesize the new DNA strands.

D. Continuous synthesis of both DNA strands at a replication fork is not possible, because:

 

The sugar phosphate backbones of the two complementary DNA strands run in opposite directions; that is, they are antiparallel.

Recall that each DNA strand has a distinct polarity. At one end (3 end), a hydroxyl group is attached to the 3 carbon of the terminal deoxyribose; at the other end (5 end), a phosphate group is attached to the 5 carbon of the terminal deoxyribose.

DNA polymerase can only elongate strands in the 5 to 3 direction.

The problem of antiparallel DNA strands is solved by the continuous synthesis of one strand (leading strand) and discontinuous synthesis of the complementary strand (lagging strand).

 

 

 

Leading strand = The DNA strand which is synthesized as a single polymer in the 5 3 direction towards the replication fork.

Lagging strand = The DNA strand that is discontinuously synthesized against the overall direction of replication.

Lagging strand is produced as a series of short segments called Okazaki fragments which are each synthesized in the 5 3 direction.

Okazaki fragments are 1000 – 2000 nucleotides long in bacteria and 100 to 200 nucleotides long in eukaryotes.

The many fragments are ligated by DNA ligase, a linking enzyme that catalyzes the formation of a covalent bond between the 3 end of each new Okazaki fragment to the 5 end of the growing chain.

Priming DNA Synthesis

Before new DNA strands can form, there must be small pre-existing primers to start the addition of new nucleotides.

Primer = Short RNA segment that is complementary to a DNA segment and that is necessary to begin DNA replication.

Primers are short segments of RNA polymerized by an enzyme called primase.

A portion of the parental DNA serves as a template for making the primer with a complementary base sequence that is about 10 nucleotides long in eukaryotes.

Primer formation must precede DNA replication, because DNA polymerase can only add nucleotides to a polynucleotide that is already correctly base-paired with a complementary strand.

Only one primer is necessary for replication of the leading strand, but many primers are required to replicate the lagging strand.

An RNA primer must initiate the synthesis of each Okazaki fragment.

The many Okazaki fragments are ligated in two steps to produce a continuous DNA strand:

DNA polymerase removes the RNA primer and replaces it with DNA.

DNA ligase catalyzes the linkage between the 3 end of each new Okazaki fragment to the 5 end of the growing chain.

E. Enzymes proofread DNA during its replication and repair damage to existing DNA

DNA replication is highly accurate, but this accuracy is not solely the result of base-pairing specificity.

Initial pairing errors occur at a frequency of about one in ten thousand, while errors in a complete DNA molecule are only about one in one billion.

DNA can be repaired as it is being synthesized (e.g. mismatch repair) or after accidental changes in existing DNA (e.g. excision repair).

1. Mismatch repair, corrects mistakes when DNA is synthesized.

2. Excision repair, corrects accidental changes that occur in existing DNA.


REFERENCES

Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts and J.D. Watson. Molecular Biology of the Cell. 2nd ed. New York: Garland, 1989.

Becker, W.M. and D.W. Deamer. The World of the Cell. 3rd ed. Redwood City, California: Benjamin/Cummings, 1996.

Campbell, N. Biology. 4th ed. Menlo Park, California: Benjamin/Cummings, 1996.

Culotta, E. and D.E. Koshland, Jr. "DNA Repair Works its Way to the Top." Science. December 23, 1994.

Watson, J.D., N.H. Hopkins, J.W. Roberts, J.A. Steitz and A.M. Weiner. Molecular Biology of the Gene. 4th ed. Menlo Park, California: Benjamin/Cummings, 1987.