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DNA as genetic material• Before we discuss the evidence gathered from

experiments to prove that the genetic material of most living organisms and many viruses is double-stranded DNA, let’s review what was known about genes and DNA at the time James Watson and Francis Crick elucidated the structure of DNA in 1953:

1. Genes—the hereditary “factors” described by Mendel—were known to be associated with specific character traits, but their physical nature was not understood.

2. The one-gene–one-enzyme theory postulated that genes control the structure of proteins.

3. Genes were known to be carried on chromosomes.4. The chromosomes were found to consist of DNA and

protein.

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The discovery of DNA as genetic material

• One of the first study that ultimately led to the identification of DNA as genetic material was done by Frederick Griffith involving the bacterium Streptococcus pneumoniae in 1928.

• This bacterium, which causes pneumonia in humans, is normally lethal in mice.

• Griffith used two strains that are distinguishable by the appearance of their colonies when grown in laboratory cultures. In one strain, a normal virulent type, the cells are enclosed in a polysaccharide capsule, giving colonies a smooth appearance; this strain is labelled S. In the other strain, a mutant nonvirulent type that is not lethal, the polysaccharide coat is absent, giving colonies a rough appearance; this strain is called R.

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• Griffith injected mice with living R type bacteria. The mice were not affected and after a while the bacteria disappeared from the animal’s blood stream.

• He also injected mice with living S type bacteria. The mice died, and S type bacteria could be isolated from their blood.

• Griffith killed some virulent cells by boiling them and injected the heat-killed cells into mice. The mice survived, showing that the carcasses of the cells do not cause death.

• However, mice injected with a mixture of heat-killed virulent cells and live nonvirulent cells did die. Live cells could be recovered from the dead mice; these cells gave smooth colonies and were virulent on subsequent injection. Somehow, the cell debris of the boiled S cells had converted the live R cells into live S cells. The process is called transformation.

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• In 1944, Oswald Avery, C. M. MacLeod, and M. McCarty separated the classes of molecules found in the debris of the dead S cells and tested them for transforming ability, one at a time. These tests showed that the polysaccharides themselves do not transform the rough cells.

• In screening the different groups, it was found that only one class of molecules, DNA, induced the transformation of R cells. DNA is the agent that determines the polysaccharide character and hence the pathogenic character. It seemed that providing R cells with S DNA was equivalent to providing these cells with S genes.

• The demonstration that DNA is the transforming principle was the first demonstration that genes are composed of DNA.

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Demonstration that DNA is the transforming agent. DNA is the only agent that produces

smooth (S) colonies when added to live rough

(R) cells.

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Hershey-Chase experiment

• In 1952 Alfred Hershey and Martha Chase used bacteriophage (virus) T2 to show that DNA is the genetic material. Most of the phage structure is protein, with DNA contained inside the protein sheath of its “head.”

• They reasoned that phage infection must entail the introduction (injection) into the bacterium of the specific information that dictates viral reproduction.

• Hershey and Chase incorporated the radioisotope of phosphorus (32P) into phage DNA and that of sulfur (35S) into the proteins of a separate phage culture. P is not found in proteins but is an integral part of DNA; S is present in proteins but never in DNA.

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Bacteriophage

DNA

Tail

Tail fiber

Head

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• When the 32P-labelled phages were used, most of the radioactivity ended up inside the bacterial cells, indicating that the phage DNA entered the cells. 32P can also be recovered from phage progeny.

• When the 35S-labelled phages were used, most of the radioactive material ended up in the phage ghosts, indicating that the phage protein never entered the bacterial cell.

• They concluded that DNA is the hereditary material; the phage proteins are mere structural packaging that is discarded after delivering the viral DNA to the bacterial cell.

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The Hershey-Chase experiment, which demonstrated that the

genetic material of phage is DNA, not protein.

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Support for the hypothesis that DNA is the genetic material

1. Experiments have shown that DNA is located almost exclusively in the nucleus of eukaryotic cells, only in cell locations where chromosomes, the carrier of genetic information, are present.

2. Cytochemical analysis demonstrated that the amount of DNA per diploid cell in a given organism is constant from one cell type to another. The amount of DNA in haploid germ cells (sperm and egg) was shown to be half the amount of DNA in diploid cells. This relates directly to the inheritance patterns of genes.

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3. Comparative studies indicated that different organisms have different amount of DNA. The amount of DNA per cell is in proportion to the complexity of the cell. The higher the organism in the evolutionary scale, the greater the content of DNA per cell. This is expected as different organisms differ in their complexity and therefore have different numbers of chromosomes and genes.

4. DNA is metabolically stable, meaning that it is not rapidly degraded like many other cellular molecules. Furthermore, the amount of DNA per cell in a given species of higher organism remains fairly constant, unaffected by changes in the environment or nutrition. This stability would be expected for the genetic material.

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5. There is a direct correlation between the absorption of ultraviolet light by DNA and the rate of mutation. The rate of mutation is highest at the wavelength of maximum absorption, suggesting that DNA is the hereditary material and when irradiated undergoes chemical changes which cause mutations.

6. DNA is capable of accurate replication. This mechanism ensures the faithful copying of the genetic information at each cell division, thus genetic information can be passed unchanged from one generation to the next.

7. DNA codes for all the instructions for making proteins, the macromolecules needed for the cell to perform different functions.

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The Structure and Function of Genes

• The complete set of instructions for making an organism is called its genome. It contains the master blueprint for all cellular structures and activities for the lifetime of the cell or organism.

• The genome consists of tightly coiled threads of deoxyribonucleic acid (DNA) and associated protein molecules, organized into structures called chromosomes.

• For each organism, the components of these slender threads encode all the information necessary for building and maintaining life, from simple bacteria to remarkably complex human beings.

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• An organism has some form of nucleic acid which is the chemical carrier of its genetic information.

• There are two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which code for all the information that determines the nature of the organism's cells.

• DNA codes for all the instructions needed for the cell to perform different functions. Human DNA contains enough information to produce about 100,000 proteins

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COMPOSITION OF NUCLEIC ACIDS

• Nucleic acids are one of several macromolecules in the body in addition to fats, proteins and carbohydrates.

• Nucleic acids are polymers made up of four nucleotides linked together in long chains known as polynucleotides.

• A nucleotide can itself be further broken down to yield three components:

- a pentose sugar, - a nitrogenous base, and - Phosphate group

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• When a sugar bonds together with a Nitrogenous base, it forms a structure known as a nucleoside.

• There are two types of nucleic acids: DNA and RNA. DNA stores genetic information, and RNA allows that information to be used in the cell.

• Both DNA and RNA contain nucleotides with similar components. In RNA, the sugar component is ribose, as indicated by the name "ribonucleic acid". In DNA, or deoxyribonucleic acid, the sugar component is deoxyribose. The prefix deoxy means that an oxygen atom is missing from one of the ribose Carbon atoms.

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• Nucleotides containing ribose are known as ribonucleotides, and those containing deoxyribose are known as deoxyribonucleotides.

• There are Four Nitrogen bases that are found in DNA: Adenine, Guanine, Thymine and Cytosine.

• The same bases are also found in RNA, except that there is Uracil instead of Thymine.

• These bases are divided into two categories (purines and pyrimidines) based on their molecular structure.

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• Purines - Adenine and Guanine

• Pyrimidines- Thymine (only in DNA), Cytosine, and Uracil (only in RNA).

• The four deoxyribonucleotides of DNA are:

- deoxyadenosine 5´-monophosphate (dAMP),

- deoxyguanosine 5´-monophosphate (dGMP),

- deoxycytidine 5´-monophosphate (dCMP)

- deoxythymidine 5´-monophosphate (dTMP).

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THE STRUCTURE OF NUCLEIC ACID CHAINS

• To form polynucleotides of either DNA or RNA , nucleotides are linked together by covalent bond between the phosphate groups. These phosphate linkage are called phosphodiester bonds.

• Nucleotides are joined together in DNA and RNA by phosphodiester bonds between the phosphate component of one nucleotide and the hydroxyl component in the sugar molecule of the next nucleotide.

• An ester bond is a bond which occurs between a Carbon atom and an Oxygen atom.

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• More and more nucleotides can be added on by the same process of forming ester bonds until an immense chain is formed.

• But no matter how long a polynucleotide chain is, one end of the nucleic acid molecule always has a free -OH group on the sugar at the Carbon known as C3' (called the 3' end) and the other end of the molecule always has a phosphate group at C5' (the 5' end).

• The Carbons get this name from a counting system illustrated in the next diagram.

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• This "counting system" allows the strand of nucleic acid to be oriented: the 5' end of the molecule always ends with a phosphate and the 3' end of the strand always ends with a sugar.

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• In 1953 James Watson and Francis Crick proposed a model for the physical and chemical structure of the DNA molecule.

• According to the Watson-Crick model, a DNA molecule consists of two polynucleotide strands coiled around each other in a helical manner - "twisted ladder" structure.

• The sugar-phosphate backbone is on the outside of the double helix, and the bases are on the inside, so that a base on one strand points directly toward a base on the second strand.

• The two strands of the DNA double helix run in opposite directions, one in the 5' to 3' direction, the other in the 3' to 5' direction. The term that describes how the two strands relate to each other is known as antiparallel.

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THE TWO STRANDS OF DNA ARE

ANTIPARALLEL

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• The two strands are held together by hydrogen bonds between the nitrogenous bases. In the double helix, adenine and thymine form two hydrogen bonds to each other but not to cytosine or guanine. Similarly, cytosine and guanine form three hydrogen bonds to each other in the double helix, but not to adenine or thymine.

• Hydrogen bonds occur only between a hydrogen atom on one base and either an oxygen or nitrogen atom on the other base. This explains why only two hydrogen bonds can form between A's and T's and three can form between G's and C's, because a hydrogen bond can only form where a H atom comes in close proximity to an Oxygen or Nitrogen atom of a base on the opposite strand.

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THERE ARE TWO HYDROGEN BONDS BETWEEN ADENINE AND THYMINE

THERE ARE THREE HYDROGEN BONDS BETWEEN GUANINE AND CYTOSINE

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THE GENERALIZED STRUCTURE

OF DNA

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• Every base pair contains one purine and one pyrimidine ALWAYS. Again, this is related to the structure of each base and how a proper "fit" (both in base size and chemical makeup) allows the DNA helix to exist in a physically and chemically stable structure. This type of base pairing is called complementary rather than identical.

• This complementary base pairing in the two strands explains why the amount of purines and pyrimidines always occur in equal amounts in a double-stranded DNA. Specifically the amount of adenine is equal to that of thymine and the amount of guanine is equal to that of cytosine.

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• The specific pairings observed are A with T and G with C.

• The specific A – T and G – C pairs are called complementary base pairs.

• The nucleotide sequence in one strand dictates the nucleotide sequence of the other strand, e.g. if one chain has the sequence

5'-TATTCCGA-3' the opposite chain must bear the sequence 3'-ATAAGGCT-5'