The recognition of the microscopic and ultrastructural features of cells and their fundamental components, such as the nucleus, the cytoplasm with its organelles, and the cell membrane, all described in Chapter 2, shed little light on the manner in which cells function. The key questions were: How does a cell reproduce itself in its own image? How are the genetic characteristics of cells inherited, transmitted, and modified? How does a cell function as a harmonious entity within the framework of a multicellular organism?

The facts available to the investigators during the 100 years after the initial observations on cell structure were few and difficult to reconcile. The developments in organic chemistry during the 19th century documented that the cells are made up of the same elements as other organic matter, namely, carbon, hydrogen, oxygen, nitrogen, phosphorous, calcium, sulfur, and very small amounts of some other inorganic elements. Perhaps the most critical discovery was the synthesis of urea by Wöhler in 1828. Soon, a number of other organic compounds, such as various proteins, fats and sugars, were identified in cells. Of special significance for molecular biology was the observation that all proteins are composed of the same 20 essential amino acids. A further important observation was that most enzymes, hence substances responsible for the execution of many chemical reactions, were also proteins. The cell ceased to be a chemical mystery, but it remained a functional puzzle.

The observations by the Czech monk, Gregor Mendel (or Mendl), who first set down the laws governing dominant and recessive genetic inheritance by simple observations on garden peas, opened yet another pathway to molecular biology. Was there any possible link between biochemistry and genetics? The phenomenon of mitosis, or cell division, and the presence of chromosomes were first observed about 1850, apparently by one of the founding fathers of contemporary pathology, Rudolf Virchow. Several other 19th-century observers described chromosomes in some detail and speculated on their possible role in genetic inheritance, but, again, there was no obvious way to reconcile the chromosomes with the genetic and biochemical data.

In 1869, a Swiss biochemist, Miescher, isolated a substance from the nuclei of cells from the thymus of calves, named thymonucleic acid, and since renamed deoxyribonucleic acid or DNA. The relationship between DNA and the principles of genetic inheritance, as defined by Mendel, was not apparent for almost a century. A hint linking the chromosomes with the “thymonucleic” acid was provided by Feulgen and Rossenbach, who, in 1924, devised a DNA-specific staining reaction, which is known today as the Feulgen stain. It could be shown that chromosomes stained intensely with this stain (see Fig. 2-28). Interestingly, in the 1930s, the Swedish pioneer of cytochemistry, Torbjörn Caspersson, suggested that thymonucleic acid could be the substance responsible for genetic events in the cell.

It was not, however, until 1944 that Avery, MacCarty, and MacLeod, working at the Rockefeller Institute in New York City, described a series of experiments documenting that DNA was the molecule responsible for morphologic changes in the bacterium, Diplococcus pneumoniae, thus providing firm underpinning to the principle that the genetic function was vested in this compound. The universal truth of this discovery was not apparent for several more years, particularly because bacterial DNA does not form chromosomes. The understanding of the mechanisms of the function of DNA had to await the discovery of the fundamental structure of this molecule by Watson and Crick in 1953. For a recent review of these events, see Pennisi (2003).

DNA was once described as a “fat, cigar-smoking molecule that orders other molecules around.” In fact, the molecule of DNA is central to all events occurring within the cell. In bacteria and other relatively simple organisms not provided with a nucleus (prokaryotes), the DNA is present in the cytoplasm. In higher organisms (eukaryotes), most of the DNA is located within the nucleus of the cell. In a nondividing cell, the DNA was thought to be diffusely distributed within the nucleus. Recent investigations, however, strongly suggest that even in the nondividing cells, the chromosomes retain their identity and occupy specific territories within the nucleus (Koss, 1998). For further details of the nuclear structure, see Chapter 2. During cell division, the DNA is condensed into visible chromosomes (see Chap. 4). Small amounts of DNA are also present in other cell organelles, mainly in the mitochondria; hence, the suggestion
that mitochondria represent previously independent bacterial organisms that found it advantageous to live in symbiosis with cells (see Chap. 2). To understand how DNA performs the many essential functions, it is important to describe its structure. DNA forms the well-known double helix, which can be best compared to an ascending spiral staircase or a twisted ladder (Fig. 3-2). The staircase has a supporting external structure, or backbone, composed of molecules of a pentose sugar, deoxyribose, bound to one another by a molecule of phosphate. This external support structure of the staircase is organized in a highly specific fashion: the organic rings of the sugar molecules are alternately attached to the phosphate by their 5′ and 3′ carbon molecules^* (Fig. 3-3). This construction is fundamental to the understanding of the synthesis of nucleic acids, which always proceeds from the 5′ to the 3′ end, by addition of sugar molecules in the 3′ position. The steps of the staircase (or rungs of the ladder) are formed by matching molecules of purine and pyrimidine bases, each attached to a molecule of the sugar, deoxyribose, in the backbone of the molecule (Fig. 3-4; see Fig. 3-2). The purines are adenine (A) and guanine (G); the pyrimidines are thymine (T) and cytosine (C). It has been known since the 1940s, thanks to the contributions of the chemist, Chargaff, that in all DNA molecules, regardless of species of origin, the proportions of adenine and thymine on the one hand, and of guanine and cytosine on the other hand, were constant. This information, combined with data from x-ray crystallography of purified molecules of DNA, allowed Watson and Crick to construct their model of the DNA molecule. In it, the purine, adenine, and the pyrimidine, thymine (the A-T bond), and cytosine and guanine (the C-G bond) are always bound to each other. The triple C-G bond is stronger than the double A-T bond (see Figs. 3-2 and 3-4). This relationship of purines and pyrimidines is immutable, except for the replacement of thymine by uracil (U) in RNA (see below), and is the basis of all subsequent technical developments in the identification of matching fragments of nucleic acids (see below). The term base pairs (bp) is frequently used to define one matching pair of nucleotides and to define the length of a segment of double-stranded DNA. Thus, a DNA molecule may be composed of many thousands of base pairs. It is of critical importance to realize that the sequence of the purine-pyrimidine base pairs varies significantly, in keeping with the encoding of the genetic message, as will be set forth below.

DNA is an enormous molecule. If fully unwrapped, it measures about 2 meters in length (but only 2 nm in diameter) in each single human nucleus. Each of the 46 individual human chromosomes contains from 40 to 500 million base pairs and their DNA is, therefore, of variable length, but still averages about 3 cm. It is evident, therefore, that to fit this gigantic molecule into a nucleus measuring from 7 to 10 μm in diameter, it must be folded many times. The DNA is wrapped around nucleosomes, which are cylindrical structures, composed of proteins known as histones (see Fig. 4-5). This reduces the length of the molecule significantly. Further reduction of the molecule is still required, and it is assumed that DNA forms multiple coils and folds to form a compact structure that fits into the space reserved for the nucleus. An apt comparison is with a wet towel that is twisted to rid it of water and then folded and refolded to form a compact ball. The interested reader is referred to a delightful book by Calladine and Drew (1997) that explains in a simple fashion what is known today about packaging of DNA. Be it as it may, individual chromosomes are
composed of multiple coils of DNA, as shown in Figure 4-5 and discussed at some length in Chapter 4.

The elegance and simplicity of the structure of the double helix resolved the secret of inheritance of genetic material. Because the double helix is constructed of two reciprocal, matching molecules, it was evident to Watson and Crick that DNA replication can proceed in its own image: the double helix can be compared to a zipper, composed of two corresponding half-zippers. Each half of the zipper, or one strand of DNA, serves as a template for the formation of a mirror image, complementary strand of DNA (Fig. 3-5). Hence, the first event in DNA replication must be the separation of the two strands forming the double helix. The precise mechanism of strand separation is still not fully understood, although the enzyme primase plays an important role. A further complication in the full understanding of the mechanisms of DNA replication is that the DNA molecule is wrapped around nucleosomes (see above). How the nucleosomal DNA is unwrapped and replicated, or for that matter transcribed (see below), is not fully understood as yet.

The synthesis of the new strand, governed by enzymes known as DNA polymerases, follows the fundamental principle of A-T and G-C pairing bonds and the principle of the 5′-to-3′ direction of synthesis, as described above. Because the two DNA strands are reciprocal, the synthesis on one strand is continuous and proceeds without interruption in the 5′-to-3′ direction. The synthesis on the other strand also follows the 5′-to-3′ rule but must proceed in the opposite direction; hence, it is discontinuous (Fig. 3-6). The segments of DNA created in the discontinuous manner are spliced together by an enzyme, ligase. When both strands of DNA (half-zippers) are duplicated, two identical molecules (fullzippers) of DNA are created. This fundamental basis of DNA replication permits the daughter cells to inherit all the characteristics of the mother cell that are vested in the DNA. Replication of DNA takes place during a well-defined period in a cell’s life, the synthesis phase or S-phase of the cell cycle, before the onset of cell division (mitosis) (see Chap. 4). By the time the cell enters the mitotic division, the DNA, in the form of chromosomes; is already duplicated. Each chromosome is composed of two identical mirror-image DNA segments (chromatids), bound together by a centromere (see Fig. 4-2). It is evident that the mechanism of DNA replication is activated before mitosis, when the chromosomal DNA is not visible under the light microscope, because the chromosomes are markedly elongated.

The exact sequence of events leading to the entry of the cell into the mitotic cycle is still under investigation and may be influenced by extracellular signals (see review by Cook, 1999). Whatever the mechanism, a family of proteins, cyclins, causes the resting cell to enter and progress through the phases of the cell division. For a review of cyclins, see the article by Darzynkiewicz et al (1996) and Chapter 4.

It is also known that the replication of the chromosomes
is not synchronous and that some of them replicate early and others replicate late. It has been proposed that those genes common to all cells that ensure the fundamental cell functions and “housekeeping” chores, replicate during the first, early part of the S-phase, whereas the tissue-specific genes replicate late. During other phases of the cell cycle, the mechanism of DNA replication is either inactive or markedly reduced.

If one considers that during the lifetime of the human organism, DNA replication occurs billions of times and that even single errors of replication affecting critical segments of DNA may result in serious genetic damage that may lead to clinical disorders (see below), it is evident that efficient mechanisms of replication control must exist that will eliminate or neutralize such mistakes. Work on bacteria suggests that there are at least three controlling steps in DNA replication: selection of the appropriate nucleotide by DNA polymerases; recognition of the faulty structure by another enzyme; and finally, the repair of the damage. In eukaryotic cells, the molecule p53, which has been named “the guardian of the genome,” appears to play a critical role in preventing replication errors prior to mitosis. As discussed in Chapter 6, cells that fail to achieve DNA repair will be eliminated by the complex mechanism of apoptosis. Regardless of the technical details, it is quite evident that these control mechanisms of DNA replication in multicellular organisms are very effective.

Once the fundamental structure of DNA became known, attention turned to the manner in which this molecule governed the events in the cell. There were two fundamental questions to be answered: How were the messages inscribed in the DNA molecule (i.e., how was the genetic code constructed?) and how were they executed? It became quite evident that the gigantic molecule of DNA could not be directly involved in cell function, particularly in the formation of the enzymes and other essential molecules. Furthermore, it had been known that protein synthesis takes place in the cytoplasm and not in the nucleus; hence, it became clear that an intermediate molecule or molecules had to exist to transmit the messages from the nucleus to the cytoplasm (see Fig. 3-8A). The best candidate for this function was RNA. RNAs, or the ribose nucleic acids, were analyzed at about the same time that the basic chemical makeup of DNA became known, in the 1940s. They were known to differ from DNA in three respects: the sugar in the molecule was ribose, instead of deoxyribose (hence the name); the molecule, instead of being double-stranded, was singlestranded (although there are some exceptions to this rule, notably in some viruses composed of RNA); and the thymine was replaced by a very similar base, uracil (Fig. 3-7). Several forms of RNA of different molecular weight (relative molecular mass) were known to exist in the cytoplasm and the nucleus. However, they appeared to be stable and, accordingly, not likely to fulfill the role of a messenger molecule that had to vary in length (and thus in molecular mass)
to reflect the complexity of the messages encoded in the DNA. The molecule that was finally identified as a messenger RNA, or mRNA, was difficult to discover because it constitutes only a small proportion of the total RNA (2% to 5%) and because of its relatively short life span. The DNA code is transcribed into mRNA with the help of specific enzymes, transcriptases (Fig. 3-8A). The transcription, which occurs in the nucleus on a single strand of DNA, follows the principles of nucleotide binding, as described for DNA replication, except that in RNA, thymine is replaced by a similar molecule, uracil (Fig. 3-8B). As will be set forth in the following section, each molecule of mRNA corresponds to one specific sequence of DNA nucleotides, encoding the formation of a single protein molecule, hence a gene. Because the size of the genes varies substantially, the mRNAs also vary in length, thus in molecular mass, corresponding to the length of the polypeptide chain to be produced in the cytoplasm. The identification of and, subsequently, the in vitro synthesis of mRNA proved to be critical in the further analysis of the genetic code and in subsequent work on analysis of the genetic activity of identifiable fragments of DNA. For a recent review of this topic, see articles by Cook (1999) and Klug (2001).

In experimental in vitro systems, the bonds between the two chains of DNA can be broken by treatment with alkali, acids, or heat. Still, the affinity of the two molecules is such that once the cause of the strand separation is removed, the two chains will again come together, an event known as reannealing. These properties of the double-stranded DNA became of major importance in gene analysis and molecular engineering.

The unraveling of the structure of DNA and its mechanism of replication was but a first step in understanding the mechanism
of cell function. The subsequent step required deciphering the message contained in the structure. Since neither the sugar molecule nor the phosphate molecule had any specificity, the message had to be contained in the sequence of the nucleotide bases (i.e., A,G,T, and C), as was suggested by Watson and Crick shortly after the fundamental discovery of the structure of the DNA. It was subsequently shown that the DNA code is limited to the formation of proteins from the 20 essential amino acids. The specific sequences of nucleotides that code for amino acids could be defined only after the pure form of the intermediate RNA molecules could be synthesized.

By a series of ingenious and deceptively simple experiments, it was shown that different clusters of three nucleotides coded for each of the 20 amino acids, the primary components of all proteins. A sequence of three nucleotides, encoding a single amino acid, is known as a codon (Fig 3-9B). A series of codons, corresponding to a single, defined polypeptide chain or protein, constitutes a gene. As discussed in the foregoing, the code inscribed in the DNA molecule is transcribed into mRNA, which carries the message into the cytoplasm of the cell wherein protein formation takes place (see Fig. 3-8A). The code, therefore, was initially defined, not as a sequence of nucleotides in the DNA, but as it was transcribed into RNA. Because there are four nucleotides in the RNA molecule (A,G,C, and U, substituting for T), and three are required to code for an amino acid, there are 4 X 4 X 4 or 64 possible combinations. These combinations could be established by using synthetic RNA. Thus, the identity of the triplets of nucleotides, each constituting a codon, could be precisely established (see Fig. 3-9). It may be noted that only one amino acid, methionine, is coded by a unique sequence, AUG (adenine, uracil, guanine). It was subsequently proven that the codon for methionine initiated the synthesis of a sequence of amino acids constituting a protein. In other words, every protein synthesis starts with a molecule of methionine, although this amino acid can be removed later from the final product. All other amino acids are encoded by two or more different codons. There are also three nucleotide sequences that are interpreted as termination or “stop” codons. The stop codons signal the end of the synthesis of a protein chain.

Once the RNA code was established, it became very simple to identify corresponding nucleotide sequences on the DNA by simply substituting U(racil) by T(hymine). This reciprocity between DNA and RNA base sequences was also subsequently utilized in further molecular biologic investigations (see Fig. 3-8B).