1 The Griffith Experiment
Genes Are Made of DNA
2 The Avery Experiments
3 The Hershey-Chase Experiment 4 The Fraenkel-Conrat Experiment
5 Discovering the Structure of DNA
6 How the DNA Molecule Replicates
From Gene to Protein
8 The Genetic Code
10 Architecture of the Gene
Regulating Gene Expression
11 Turning Genes Off and On
Altering the Genetic Message
13 Kindsof Mutation
14 Cancer and Mutation
When Mendel solved the puzzle of heredity, he with one stroke removed the mystery from the question of why we resemble our parents. As we learned, we resemble our parents because we are built from copies of their chromosomes. These chromosomes contain sets of instructions called genes that determine what they, and we, are like. However, Mendel's work leaves a key question unanswered: What is a gene? After almost a century of research, biologists are now able to answer this question fully. We have learned what genes are made of and how they work. We understand in considerable detail how the information in genes is converted into organisms with eyes, arms, and inquiring minds. The first step in this new understanding came with the discovery that the hereditary information of genes is stored in a long spiral molecule called DNA that forms the core of chromosomes.
Genes are made of DNA
1. The Griffith Experiment
When biologists began to examine chromosomes in their search for genes, they soon learned that chromosomes are made of two kinds of macromolecules, both of which you encountered in: proteins (long chains of amino acid subunits linked together in a string) and DNA (deoxyribonucleic acid) (long chains of nucleotide subunits linked together in a string). It was possible to imagine that either of the two was the stuff that genes are made of-information might be stored in a sequence of different amino acids, or of different nucleotides. But which one is the stuff of genes, protein or DNA? This question was answered clearly in a variety of different experiments, all of which shared the same basic design: If you separate the DNA in an individual's chromosomes from the protein, which of the two materials is able to change another individual's genes?
In 1928, British microbiologist Frederick Griffith made a series of unexpected observations while experimenting with pathogenic (disease-causing) bacteria. When he infected mice with a virulent strain of Streptococcus pneumoniae bacteria (then known as Pneumococcus), the mice died of blood poisoning. However, when he infected similar mice with a mutant strain of S. pneumoniae that lacked the virulent strain's polysaccharide coat, the mice showed no ill effects. The coat was apparently necessary for infection. The normal pathogenic form of this bacterium is referred to as the S form because it forms smooth colonies in a culture dish. The mutant form, which lacks an enzyme needed to manufacture the polysaccharide capsule, is called the R form because it forms rough colonies.
To determine whether the polysaccharide coat itself had a toxic effect, Griffith injected dead bacteria of the virulent S strain into mice~ the mice remained perfectly healthy. Finally, he injected mice with a mixture containing dead S bacteria of the virulent strain and live, coatless R bacteria, each of which by itself did not harm the mice. Unexpectedly, the mice developed disease symptoms and many of them died. The blood of the dead mice was found to contain high levels of live, virulent Streptococcus type S bacteria, which had surface proteins characteristic of the live (previously R) strain. Somehow, the information specifying the polysaccharide coat had passed from the dead, virulent S bacteria to the live, coatless R bacteria in the mixture, permanently transforming the coatless R bacteria into the virulent S variety.
Key concepts:Hereditary information can pass from dead cells to living ones and transform them.
2. The Avery Experiments
The agent responsible for transforming Streptococcus went undiscovered until 1944. In a classic series of experiments, Oswald Avery and his coworkers Colin MacLeod and Maclyn McCarty characterized what they referred to as the "transforming principle." They first prepared the mixture of dead S Streptococcus and live R Streptococcus that Griffith had used. Then Avery and his colleagues removed as much of the protein as they could from their preparation, eventually achieving 99.98% purity. Despite the removal of nearly all protein, the transforming activity was not reduced. Moreover, the properties of the transforming principle resembled those of DNA in several ways:
Same chemistry as DNA. When the purified principle was analyzed chemically, the array of elements agreed closely with DNA.
Same behavior as DNA. In an ultracentrifuge, the transforming principle migrated like DNA; in electrophoresis and other chemical and physical procedures, it also acted like DNA.
Not affected by lipid and protein extraction. Extracting the lipid and protein from the purified transforming principle did not reduce its activity.
Not destroyed by protein- or RNA-digesting enzymes. Protein-digesting enzymes did not affect the principle's activity; nor did RNA-digesting enzymes. Destroyed by DNA-digesting enzymes. The DNAdigesting enzyme destroyed all transforming activity.
The evidence was overwhelming. They concluded that "a nucleic acid of the deoxyribose type is the fundamental unit of the transforming principle of Pneumococcus Type III"-in essence, that DNA is the hereditary material.
Key concepts: Avery's experiments demonstrate conclusively that DNA is the hereditary material.
3.The Hershey-Chase Experiment
Avery's result was not widely appreciated at first, because most biologists still preferred to think that genes were made of proteins. In 1952, however, a simple experiment carried out by Alfred Hershey and Martha Chase was impossible to ignore. The team studied the genes of viruses that infect bacteria. These viruses attach themselves to the surface of bacterial cells and inject their genes into the interior; once inside, the genes take over the genetic machinery of the cell and order the manufacture of hundreds of new viruses. When mature, the progeny viruses burst out to infect other cells. These bacteria- infecting viruses have a very simple structure: a core of DNA surrounded by a coat of protein. In this experiment, Hershey and Chase used radioactive isotopes to "label" the DNA and protein of the viruses. In one preparation, the viruses were grown so that their DNA contained radioactive phosphorus (11P); in another preparation, the viruses were grown so that their protein coats contained radioactive sulfur ("S). The two preparations were then mixed together, and the mixture was allowed to infect bacteria. After a few minutes Hershey and Chase shook the suspension forcefully to dislodge attacking viruses from the surface of bacteria, used a rapidly spinning centrifuge to isolate the bacteria, and then asked a very simple question: What did the viruses inject into the bacterial cells, protein or DNA'? They found that the interiors of the bacterial cells contained the `P label but not the "S label. The conclusion is clear: the genes that viruses use to specify new generations of viruses are made of DNA and not protein.
Key concepts: The hereditary material of bacteriophages is DNA and not protein.
4. The Fraenkel-Conrat Experiment
Some viruses contain RNA instead of DNA, and yet they manage to reproduce quite satisfactorily. What genetic material do they use? In 1957, Heinz Fraenkel-Conrat and his coworkers answered this question for two RNA-containing viruses: tobacco mosaic virus JMV), which infects the leaves of tobacco leaves, and Holmes ribgrass virus (HRV), which infects grass. TMV, the better studied, consists of a single strand of RNA 6,390 nucleotides long, surrounded by a protein coat of 2,130 identical subunits. The protein can be separated from the RNA by a simple chemical treatment. When this is done, the isolated RNA is infective, while the protein is not, suggesting that RNA is the hereditary material of these viruses. If the dissociated RNA and protein subunits are mixed together in solution, they recombine to form fully active virus particles.
Fraenkel-Conrat and his coworkers further investigated this conclusion with a simple but compelling exchange experiment. First they chemically dissociated each virus, separating its protein coat from its RNA. They then manufactured hybrid viruses by combining the protein of one with the RNA of the other. When they infected healthy tobacco plants with a hybrid virus composed of HRV RNA and TMV protein, the tobacco leaves developed lesions characteristic of HRV. Clearly, the hereditary properties of the virus were determined by the nucleic acid in its core, not the protein in its coat.
Later studies have shown that many other viruses contain RNA rather than DNA. When DNA viruses infect a cell, their DNA is often inserted into the host cell's DNA as if they were the cell's own genes. Viruses containing RNA use a more indirect method. They first make an intermediate double-stranded form of DNA from the RNA, using a special kind of polymerase enzyme called reverse transcriptase. This DNA copy may then insen into the cell's DNA. Because the path of information flows from RNA to DNA rather than from DNA to RNA, these RNA viruses are called retroviruses. The HIV virus that causes acquired immunodeficiency syndrome (AIDS) is a retrovirus, as are many turnor-forming viruses. Transcription of the retrovirus RNA, necessary to produce new virus particles, takes place only after its DNA copy has been integrated into the host DNA. Thus, integration is an obligatory step in the life cycle of a retrovirus.
Key concepts: DNA is the genetic material for all cellular organisms and most viruses, although some viruses use RNA.
Fraenkel-Conrat's virus reconstitution experiment. Both TMV and HRV are plant RNA viruses that infect tobacco plants, causing lesions on the leaves. In this experiment, TMV and HRV were both dissociated into protein and RNA. Then hybrid virus particles were produced by mixing the HRV RNA and the TMV protein. When the reconstituted virus particles were painted onto tobacco leaves, HRV-type lesions developed. From these lesions, normal HRV virus particles could be isolated in great numbers; no TMV viruses could be isolated from the lesions. Thus the RNA (HRV) and not the protein (TMV) contains the information necessary to specify the production of the viruses.
5. Discovering the Structure of DNA
DNA is a long, chainlike molecule made up of subunits called nucleotides. Each nucleotide has three parts: a central sugar, a phosphate (PO) group, and an organic base. The sugar and the phosphate group are the same in every nucleotide of DNA, but there are four different kinds of bases, two large ones with double-ring structures and two small ones with single rings. The large bases, called purines, are A (adenine) and G (guanine). The small bases, called pyrimidines, are C (cytosine) and T (thymine). A key observation, made by Erwin Chargaff, was that DNA molecules always had equal amounts of purines and pyrimidines. In fact. with slight variations due to imprecision of measurement, the amount of A always equals the amount of T, and the amount of G always equals the amount of C. This observation (A = T, G = C), known as Chargaff's rule, suggested that DNA had a regular structure. The significance of Chargaff's rule became clear in 1953, when scientists began to study the structure of DNA using X-ray diffraction techniques. In these experiments, DNA molecules are bombarded with X-ray bearns, and when individual rays encounter atoms, their paths are bent or diffracted; each atomic encounter creates a pattern on photographic film like the ripples created by tossing a rock into a smooth lake. The first of these studies, carried out by Rosalind Franklin at Kings Colle-e, London, suggested that the DNA molecule had the shape of a coiled spring, a form called a helix.
Two workers at Cambridge University, Francis Crick and James Watson, learned informally of Franklin's results and, using Tinkertoy models of the bases, deduced the true structure of DNA: The DNA molecule is a double helix, a winding staircase of two strands whose bases face one another. Chargaff's rule is a direct reflection of this structure-every bulky purine on one strand is paired with a slender pyrimidine on the other strand. Specifically, A pairs with T, and G pairs with C. Because hydrogen bonds can form between the base pairs, the molecule keeps a constant thickness.
Key concepts: The DNA molecule has two strands of nucleotides that form hydrogen bonds with each other.
The DNA double helix. This X-ray diffraction photograph was made in 1953 by Rosalind Franklin in the laboratory of Maurice Wilkins. It suggested to Watson and Crick that the DNA molecule was a helix, like a winding staircase. The dimensions of the double helix were suggested by the X-ray diffraction studies. In a DNA duplex molecule, only two base pairs are possible: adenine (A) with thymine M and guanine (G) with cytosine (C). A G-C base pair has three hydrogen bonds; an A-T base pair has only two.
6. How the DNA Molecule Replicates
The attraction that holds the two DNA strands together is the formation of weak hydrogen bonds between the bases that face each other from the two strands. That is why A pairs with T and not C-it can only form hydrogen bonds with T. Similarly, G can form hydrogen bonds with C but not T. In the Watson-Crick model of DNA, the two strands of the double helix are said to be complementarY to each other. One chain of the helix can have any sequence of bases, of A, T, G, and C, but this sequence completely determines that of its partner in the helix. If the sequence of one chain is ATTGCAT, the sequence of its partner in the double helix must be TAACGTA. Each chain in the helix is a complementary mirror image of the other. This complementarity makes it possible for the DNA molecule to copy itself during cell division in a very direct manner. The double helix need only "unzip" and assemble a new complementary chain along each naked single strand by base pairing A with T and G with C. This form of DNA replication is called semiconservative, because while the sequence of the original duplex is conserved after one round of replication, the duplex itself is not. Instead, each strand of the duplex becomes part of another duplex.
The Meselson-Stahl Experiment
The hypothesis of sern icon servati ve replication was tested in 1958 by Matthew Meselson and Franklin Stahl of the California Institute of Technology. These two scientists grew bacteria in a medium containing the heavy isotope of nitrogen, 15N, which became incorporated into the bases of the bacterial DNA. After several generations, the DNA of these bacteria was denser than that of bacteria grown in a medium containing the lighter isotope of nitrogen, 14N. Meselson and Stahl then transferred the bacteria from the 15N medium to the 14N medium and collected the DNA at various intervals.
By dissolving the DNA they had collected in a heavy salt called cesium chloride and then spinning the solution at very high speeds in an ultracentrifuge, Meselson and Stahl were able to separate DNA strands of different densities. The centrifugal forces caused the cesium ions to migrate toward the bottom of the centrifuge tube, creating a gradient of cesium concentration, and thus of density. Each DNA strand floats or sinks in the gradient until it reaches the position where its density exactly matches the density of the cesium there. Because 15N strands are denser than 14N strands, they migrate farther down the tube to a denser region of cesium.
The DNA collected immediately after the transfer was all dense. However, after the bacteria completed their first round of DNA replication in the 14N medium, the density of their DNA had decreased to a value intermediate between 14N-DNA and 15N-DNA. After the second round of replication, two density classes of DNA were observed, one intermediate and one equal to that of 14N-DNA.
Meselson and Stahl interpreted their results as follows: after the first round of replication, each daughter DNA duplex was a hybrid possessing one of the heavy strands of the parent molecule and one light strand; when this hybrid duplex replicated, it contributed one heavy strand to form another hybrid duplex and one light strand to form a light duplex. Thus, this experiment clearly confirmed the prediction of the Watson-Crick model that DNA replicates in a semiconservative manner.
How DNA Copies Itself
The copying of DNA before cell division is called DNA replication and is overseen by an enzyme called DNA polymerase. After an enzyme called helicase unwinds the DNA double helix, DNA polymerase reads along each naked single strand and adds the correct complementary nucleotide (A with T, G with C) at each position as it moves, creating a complementary strand. DNA ligase joins the ends of newly synthesized segments of DNA. The place where the parent DNA molecule becomes unzipped is called a replication fork. At replication forks, the polymerase very actively shuttles up one strand and down the other. Chromosomes each contain a single, very long molecule of DNA, but it is too long to copy conveniently all the way from one end to the other with a single replication fork. Each chromosome is instead copied in segments; each zone of about 100,000 nucleotides has its own replication fork.
The enormous amount of DNA that resides within the cells of your body represents a long series of DNA replications, starting with the DNA of a single cell-the fertilized egg. Living cells have evolved many mechanisms to avoid errors during DNA replication and to preserve the DNA from damage. These mechanisms of DNA repair proofread the strands of each daughter cell against one another for accuracy and correct any mistakes. But the proofreading is not perfect. If it were, no mistakes would occur, no variation in gene sequence would result, and evolution would come to a halt.
Key concepts: The basis for the great accuracy of DNA replication is complementarity. DNA's two strands are complementary mirror images of each other, so either one can be used as a template to reconstruct the other.)
Enzymes unzip the DNA by breaking the hydrogen bonds between the base pairs. The unpaired bases are now free to bind with other nucleotides with the appropriate complementary bases. The enzyme Primase begins the process by synthesizing short primers of RNA nucleotides complementary to the unpaired DNA. DNA polymerase now attaches DNA nucleotides to one end of the growing complementary strand of nucleotides. Replication proceeds continuously along one strand, called the leading strand. The process occurs in separate short segments called Okazaki fragments next to the other, or lagging strand. This difference is due to the fact that DNA polymerase can only add new nucleotides to the 3 prime end of a nucleotide strand. A primer begins any new strand, including each Okazaki fragment. An enzyme replaces the RNA primer with DNA nucleotides. Then an enzyme called DNA ligase binds the fragments to one another. There are now 2 DNA molecules. Each consists of an original nucleotide strand next to a new complementary strand. The two molecules are identical to each other.