On March 7th, 1953, Francis Harry Crick, a 35-year old graduate student at the Cavendish laboratory of the University of Cambridge, England, walked into the Eagle pub and declared, “we have found the secret of life”. He was referring to his and James Dewey Watson’s discovery of the structure of DNA that explained transmission of genetic information from cell to cell and from parent to offspring, and helped understand how genetic mutations are produced. Theirs was a brilliant interpretation of another investigator’s published and, reportedly, still unpublished research data. Crick’s career had evolved from physics to chemistry and biology. On the other hand, the 23-year old Watson had received a B.S degree from the University of Chicago and a Ph.D. degree in zoology from Indiana University. However, as a research fellow at the Cavendish laboratory, he abandoned his chosen field for the pursuit of glory, as he recounts in his memoirs entitled The double Helix: A personal account of the discovery of the structure of DNA, first published in 1968 [132]. In that self-serving account of the momentous discovery, he described his obsession with the DNA molecule and his anticipation that unraveling its structure would bring the Nobel Prize. After attending a 1951 lecture where the gifted Rosalind Franklin presented X-ray crystallography data and her helical concept of the DNA molecule, Watson and Crick built a three-chain DNA molecule model with the backbone on the inside that drew sharp criticism. The head of the Cavendish laboratory, Sir Lawrence Bragg, ordered the unlikely pair to leave DNA to King’s College where Franklin and her rival Maurice Hugh Wilkins were assigned the task. However, when Linus Pauling, the world’s leading structural chemist who would be awarded Nobel Prizes for Chemistry in 1954 and for Peace in 1962, and the odds-on favorite to solve the structure of DNA published a wrong structure for DNA, it became evident to them that someone else might soonlay claim to “the most important of all scientific prizes” [133]. According to a special report published on August 17, 1998 [134], “When Watson came calling in January 1953, Wilkins [who Watson described as ‘a beginner’ in X-ray diffraction work, wanted some professional help and hoped that ‘Rosy’, a trained crystallographer, could speed up his research], revealed he had been quietly copying Franklin’s data. He showed one of her x-ray photos” (Fig. 3.1).

Watson was so impressed that he later wrote, “The instant I saw the picture my mouth fell open and my pulse began to race…. the black cross of reflections which dominated the picture could arise only from a helical structure… mere inspection of the X-ray picture gave several of the vital helical parameters” [135]. Watson’s own admission, the fact that neither he nor Crick conducted bench research on DNA, relying instead on other investigators’ data to draw diagrams and construct tri-dimensional models, and the short time between this episode and the publication of their report leads to the inescapable conclusion: Franklin’s work was pivotal in their inferring the correct molecular structure of DNA. Their highly acclaimed and universally accepted model included two helical chains made of sugar-phosphate backbones, as Franklin’s work revealed, held together by complementary pairs of four nitrogen bases interlocked between them. Thus, Watson’s and Crick’s failure to acknowledge Franklin’s crucial role in their formulation of the structure of DNA reported in the April 2, 1953 Nature article [136] where he and Crick disclosed their final model is a most regrettable episode in the annals of great discoveries. To add insult to injury, Watson ridiculed Franklin in his book, stating, “So it was quite easy to imagine her the product of an unsatisfied mother who unduly stressed the desirability of professional careers that could save bright girls from marriages to dull men”. Franklin’s biographer adds “‘Rosy’ was depicted as an aggressive, perhaps belligerent, female subordinate with no respect for her superiors” who “refused to think of herself as an assistant to Wilkins” [137]. Crick, Watson, and Wilkins received the 1962 Nobel Prize for Physiology or Medicine “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material” [138]. Watson went on to receive the most honors and recognition, including honorary degrees from 22 universities. Franklin died of ovarian cancer in 1958, age 37.

From an anatomical standpoint, the genome is contained in tightly coiled strands of DNA organized in chromosomes, which are housed in the cell nucleus. To illustrate the minuscule size of the DNA, suffice it to say that, if unwound, the DNA of a single cell (one million cells fit on the head of a pin) would stretch 5 ft but would be only 50 trillionth of an inch wide. Stretching all the DNA of a human being would reach the sun and back. A human DNA molecule (Fig. 3.2) consists of two strands that wrap around each other like a twisted ladder or a spiral staircase, the so-called double helix, whose sugar and phosphate sides connect to each other by rungs of nitrogen-containing chemicals called bases. Each strand is a linearly repeated sequence called nucleotides, made of one sugar, one phosphate, and one nitrogenous base. There are four different bases: adenine (A), thymine (T), cytosine (C), and guanine (G). The order of the bases along the sugar-phosphate backbone, called the DNA sequence, is like a barcode that encrypts the genetic instructions necessary for the structural and functional integrity of an organism with its array of unique traits. Weak bonds between bases forming base pairs, of which there are approximately three billion in the human genome, hold the two DNA strands together. Each time a cell divides, its genome is duplicated by DNA replication, a complex process initiated by DNA polymerase, an enzyme that breaks the weak bonds between base pairs, unwinding the helix to allow separation of the two DNA strands. Once separated, each strand directs the synthesis of a complementary DNA strand, including matching bases following strict base pairing: adenine with thymine (A-T pair) and cytosine with guanine (C-G pair). Each daughter cell receives one parental and one new DNA strand, thus minimizing chances of errors (mutations) in gene transfer.

At the functional cellular level, genetic information encoded in nuclear DNA ultimately leads to production of regulatory proteins in the cell cytoplasm. This process requires an intermediary molecule, called ribonucleic acid (RNA). RNA polymerase first unzips a section of nuclear DNA and transcribes (copies), base-by-base, a given sequence of exposed bases, and moves into the cell cytoplasm as messenger RNA (mRNA) where it translates the genetic code (or message) into synthesis of the particular protein encoded in the exposed DNA.

Johann Mendel (Fig. 3.3), born in Hynčice, Moravia (in the Austro-Hungarian Empire) in 1822, entered religious life at the seminary of the Abbey of St. Augustine in Brünn, Moravia’s capital (Brno in today’s Czech Republic) in 1843, taking the name Gregor. He worked on the side as a substitute teacher in a secondary school in Znaim, near Brünn, and tried to upgrade to regular teacher but failed the certification examination. Paradoxically, his lowest mark was in biology. Sponsored by his Abbot, František Cyril Napp (1824–1867), himself an accomplished scholar, Mendel enrolled at the University of Vienna in 1851 where he studied physics, chemistry, mathematics, zoology, and botany. He returned to the abbey in 1853 and became its Abbot in 1867, a demanding responsibility that effectively ended his research career. Although he worked alone, Mendel did not operate in a vacuum, for he drew inspiration from his former teachers Friedrich Franz and Karl Nestler, and his scientific interests and pursuits were supported by a good library at the monastery, by other monks, most notably Aurelius Thaler, a botanical expert who in 1830 had established an experimental garden at the monastery, and by colleagues at the Brünn’s Natural Science Society. Mendel’s now famous paper entitled Versuche über pflanzenhybriden (Experiments with plant hybridization), describing his work on heredity, was presented orally before the Society in February 1865, and published in the Society’s transactions in 1866 [140]. From his modest monastery garden, Mendel had unraveled the secrets of heredity that would earn him the title of father of modern genetics. Yet, his contemporaries failed to understand the importance of Mendel’s work on genetics and his observations fell into oblivion to the point that, for several decades, they were unknown to the public and to scientists, including Charles Darwin [141].

Mendel’s success in choosing seemingly rudimentary techniques applied to the study of ordinary garden peas (Pisum sativum) leading to deciphering the secrets of genetics that rest on carefully designed experiments, painstakingly collecting large amounts of data, analyzing results in light of a starting hypothesis, and testing results at each step with a new set of experiments. Choosing garden peas was a carefully considered decision that demonstrated his genius. Indeed, he selected inexpensive and easily available peas of distinct shapes and colors with a short generation time that produce many offspring, enabling him accurately and systematically to sort sequential generations of cross-pollination results. Moreover, in addition to an anatomy designed for self-pollination that prevents cross-pollination, garden peas can be cross-pollinated at will by the experimenter by placing pollen from one plant on the female flowers of another [142]. Mendel selected seven traits or characteristics for study involving various colors and shapes, but first grew the plants for 2 years to ensure a pure line. That is, all offspring produced by 2 years of self-pollination would be identical with regards to each trait under study. His first major observation contradicted the then popular notion known as blending inheritance that assumed that all traits were inherited by the first generation offspring (F₁) as a blend or average of the parents’ (P). Thus, crossing a tall plant with a short one, for example, was expected to produce a medium-sized offspring plant. When Mendel pollinated tall or short plants within themselves, offspring plants remained tall or short, as expected. Curiously, when he cross-pollinated plants with green pea pods with plants that had yellow pods, he noticed that all offspring hybrid plants (F₁) exhibited green pea pods, as if the yellow pea pod trait had vanished. Yet, when he pollinated two (F₁) hybrid plants between themselves, some of their offspring (F₂) exhibited yellow pods and others had green pods. This phenomenon reappeared regardless of the trait studied and did so at a constant ratio of approximately 3:1 (Table 3.1). Mendel correctly concluded that hereditary traits are discrete packets or particles that pass unchanged from one generation to the next, although each trait might not be expressed in each generation. He called these packets elemente (elements), and called dominant those elements that appeared in the first offspring (F₁) and recessive those that were hidden in the first generation but re-surfaced in the second (F₂). He further concluded, also correctly, that paired traits pass from one generation to the next as separate and independent elements, each inherited from one parent. While genetics, particularly human genetics, is more complex, with each trait, physical and otherwise, generally being influenced by more than one gene, Mendel’s concept of elements, which we now call alleles, and his notion that elements are paired and inherited as separate and independent entities from one another in a dominant or recessive fashion remain largely accurate. Yet, despite being pivotal to understanding genetics, Mendel’s momentous work fell into oblivion until 1900 when his article Experiments with plant hybridization was simultaneously re-discovered by Hugo De Vries of the University of Amsterdam, Carl Correns of the Kaiser Wilhelm Institute for Biology in Berlin, Erik von Tschermak of the University of Vienna, and British botanist William Bateson. The latter became a fervent advocate of Mendel’s, published a book in defense of his work [144] extending his conclusions to animals and demonstrating that, contrary to Mendel’s findings, certain traits are inherited together by genes located in close proximity on the same chromosome, a phenomenon now known as linkage. Mendel died at the monastery in 1884, where his tombstone reads, “Scientist and biologist in charge of the Augustinian monastery in Old Brno. He discovered the laws of heredity in plants and animals. His knowledge provided a permanent scientific basis for recent progress in genetics.”

Genes are the fundamental physical and functional units of heredity that are passed from parent to offspring. They are made of specific sequences of DNA bases located on a particular chromosome that encode (contain information for) the production of specific proteins that serve as cellular signals. The size of genes varies widely, from approximately 10,000–150,000 base pairs. However, only a fraction (10 %) of the three billion base pairs that constitute the genome represents protein-encoding sequences (exons) of genes, the rest being intercalated sequences (introns) with no known coding function. Additionally, only a small fraction of the approximately 25,000 human genes are expressed in any particular cell. For example, hemoglobin genes are expressed in red blood cell precursors, not in muscle or brain cells. Yet, the very presence of all genes in every cell makes each of them a potential source for cloning virtually any cell lineage, under the right conditions. Gene expression begins with the synthesis of an RNA copy (transcription) of the DNA gene sequence, in the nucleus, followed by its transport mRNA to cytoplasmic ribosomes, where the encoded genetic information is translated into protein synthesis. However, before moving to the cytoplasm, non-functional introns are snipped out and exons are spliced (linked) together, thus giving rise to the proper protein-encoding sequences. Once in the cell cytoplasm, the mRNA serves as a template to translate the encoded information (codons) into a string of individual amino acids that constitute the building blocks of protein synthesis. Codons are sequences of three DNA bases within exons that direct cells to produce a specific amino acid. For example, the sequence ATG codes for the amino acid methionine. There are 64 possible codons encoding 20 amino acids, thus allowing for code redundancy for all but 2 amino acids: methionine (AUG) and tryptophan (UGG). The other 18 amino acids are encoded by 2–6 codons. For example, AAA and AAG encode lysine and UCU, UCC, UCA, UCG, AGU, and AGC encode serine. In addition, there is an initiation codon, usually AUG, that initiates translation of mRNA, and a termination codon, usually UAA, UGA or UAG, that ends it. Thus, when the RNA reads a gene sequence it is prompted where to start and where to end the transcription process. Hence, from a logistic point of view, the genetic code is a series of codons, contained in genes in turn housed in chromosomes located in the cell nucleus, that specify which amino acids will be synthesized and in what order. The 20 amino acids, assembled in a variety of different combinations and lengths, give rise to approximately 100,000 proteins encoded in the human genome that are necessary to maintain the structural and functional integrity of human beings. Errors in DNA or RNA transcription, and in exon splicing, can result in mutations, which in turn can lead to a faulty translation of the gene code, including failure to synthesize the gene-encoded protein or production of an aberrant protein. The outcome of either failure will be a functional disruption of the protein-targeted cell.

Chromosomes are microscopic units that house all genes. Thus, it might be expected that the number of chromosomes would increase with increasing complexity of the organism according to an evolutionary scheme. However, this is not the case. While a humble bacterium might function with a single chromosome and mosquitoes need 6, humans 46, dogs 78, and goldfish 94, the tropical plant Ophioglossum (snake tongue) has over 1,200. The 46 human chromosomes are organized in two sets of 23 pairs: 22 pairs of autosomes ⁴ (numbered 1 through 22) plus 1 pair of allosomes ⁵; XX for female and XY for male. Except for sex chromosomes that determine gender and are thus distinct and different, each set of autosomal chromosomes bears identical copies of the entire human genome, and is inherited as a result of sexual reproduction; one copy from the father, the other from the mother. Indeed, germ cells or gonads (spermatozoid or sperm for short in males, ovum or egg in females) contain only 23 chromosomes: 22 autosomes plus allosome X in a female ovum, and 22 autosomes plus allosome Y or X, in a male sperm. During reproduction the male sperm delivers its entire genetic load, either 22X or 22Y, into the female egg (22X) so that the fertilized egg will contain two identical and complementary pairs of 22 autosomes plus 1 pair of allosomes, either XX or XY. Women’s cells contain 44XX where one X allosome is inherited from each parent, whereas in men’s (44XY), the X allosome derives from the mother and the Y allosome comes from the father, who therefore is the parent that determines the gender of the offspring, whether male or female. Genetic alterations or mutations are associated with over 4,000 human diseases, including cancer, and have been mapped to specific chromosomes, as illustrated in Fig. 3.4 [145].

Alterations of any of the 22 autosomal chromosomes are associated with autosomal diseases, such as sickle cell anemia. Aberrations in sex chromosomes (X or Y) lead to sex-linked diseases such as hemophilia A. Genetic alterations involving major structural chromosomal abnormalities, such as multiple copies of a chromosome (as seen in Down syndrome), translocation of part of a chromosome to another (as occurs in Burkitt’s lymphoma), or deletions of a chromosome or parts thereof (exemplified by the DiGeorge syndrome), are visually detectable under the microscope. This is because appropriately stained chromosomes acquire light and dark transverse bands (reflecting variations in amounts of A-T or G-C base pairs) that enable cytogeneticists to identify each individual chromosome (Fig. 3.5), and recognize structural abnormalities [147]. This test, called chromosome banding, is used routinely in the clinical setting. More subtle defects can now be detected via more sophisticated approaches, including molecular techniques.

Chromosomal analysis is of great clinical interest because many chromosomal abnormalities are linked to a number of disorders, including mental retardation, infertility, and cancer. Cancer management is impacted by chromosomal analysis because some cancers, especially hematologic malignancies, harbor structural chromosomal abnormalities that have diagnostic or prognostic significance. In fact, a small number of chromosomal abnormalities are virtually diagnostic by themselves. They include t(9;22), the so-called Philadelphia chromosome (Fig. 3.6), the hallmark of chronic myelocytic leukemia (CML) also shared by a small subset of acute lymphocytic leukemia (ALL), and t(15;17), an abnormality that is specific for acute promyelocytic leukemia. Beyond its diagnostic value, the t(9;22) translocation confers a growth advantage to CML cells that led to the development of Imatinib mesylate (Gleevec®), the first successful post-genomic molecularly targeted agent to control rather than kill malignant cells. However, most cancers exhibit either no chromosomal abnormalities detectable by current methodology, as is the case with most solid tumors, or exhibit non-specific but diagnostically and prognostically helpful abnormalities, as is the case with most hematologic malignancies. Examples of these include gene translocations, such as t(14;18) in follicular-type lymphoma, t(8;14) in Burkitt’s lymphoma, trisomy 12 (three copies of chromosome 12) in CLL, and del(16)(q22) in a subset of acute myelocytic leukemia (AML). Additionally, many genetic aberrations are sub-microscopic, precluding their visual detection by chromosomal banding. Such cases can be unmasked by more powerful techniques that use DNA probes such as FISH analyses [148], comparative genomic hybridization [149], spectral karyotyping [150], recombinant DNA techniques [151], and others [152]. One example of sub-microscopic chromosomal abnormalities is point mutations that characterize certain hemoglobinopathies, where single amino acid substitutions occur on one of the four hemoglobin chains. To illustrate, sickle cell disease and hemoglobin C, two hemoglobinopathies with different symptoms, clinical profiles, and prognoses, result when glutamic acid on position 6 of the β chain is replaced by valine or lysine, respectively.

Cells undergo two fundamentally different but complementary processes: cell cycle and cell differentiation. Cell division, which occurs via the cell cycle, ensures self-renewal of undifferentiated precursor cells, also called stem cells. In contrast, cell differentiation is designed to generate highly specialized non-dividing mature cells with distinct and varied functions. Together, these genetically controlled cellular processes sustain the structural and functional integrity of the entire organism and ensure genetic transfer to the next generation. For example, bone marrow stem cells possess the ability to divide, thus ensuring a constant pool of self-renewing precursor cells. However, stem cells also give rise to a diversity of progenitor cells that, while losing self-renewing potential, undergo differentiation into various types of cells capable of carrying out highly specialized functions. These include red cells to ensure oxygen delivery to all tissues, white cells to seek, engulf, and kill invading microorganisms, and platelets to instantly plug any vascular leak as our first line of defense against accidental blood loss. Both cell division and cell differentiation are highly regulated processes necessary to fulfill their respective functions. For instance, many more bone marrow stem cells must enter into differentiation to produce platelets, with a T1/2⁶ of 7 days, than to produce granulocytes or red blood cells with a T1/2 of 36–48 h and 120 days, respectively. Such stem cells are pre-committed to differentiating into each cell type in contrast to pluripotent stem cells, which are the source of all cell lines in some tissues or organs. Some pre-committed cell lines do not cycle or differentiate until the proper conditions are met. An illustrative case is that of memory T-lymphocytes that remain in G₀ (see below) until they are awoken by re-exposure to the original antigen trigger, when they re-enter G₁ in order to replicate in an explosive manner so as to confer swift protection from the antigen’s pathogenic effects.

The cell cycle is divided into two major phases visible under the microscope: M-phase (mitosis or cell division) and Interphase [153]. The M-phase comprises prophase, prometaphase, metaphase, anaphase, and telophase, whereas the Interphase includes the S-phase (DNA synthesis), G₁, and G₂ stages or gaps between M and S, and between S and M, respectively, where cells remain metabolically active in preparation for the next phase. For rapidly proliferating human somatic⁷ cells that typically exhibit a total cycling time of 24 h, the M-phase lasts approximately 1 h, with most of the cell-cycling time being spent in Interphase, and G₁, S, and G₂ phases lasting approximately 11, 8, and 4 h, respectively [154]. Cells out of cycle are said to be quiescent or in G₀ and require external stimuli to move out of G₀ and into G₁ [155]. In animal cells, this step is triggered by growth factors that might include epidermal growth factor (EGF), fibroblastic growth factor (FGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF) or by exposure to antigens each binding to receptive cells’ specific surface receptors, but also hormones entering such cells. Once in G₁, cells must pass through a restriction point to enter the S-phase, without which they will remain dormant, though metabolically active, until the proper growth factor becomes available. Alternatively, many cells remain in G₀ for extended periods of time until the need arises to replace injured or dead cells as occurs in kidney, liver, and other internal organs. Other cells remain permanently in G₀, as is the case of neurons. The different events that occur in each phase of the cell cycle must be exquisitely coordinated with one another to ensure their completion in a proper sequence to prevent an aberrant cell division from transferring incomplete or defective copies of genetic material to daughter cells. Such coordination is ensured by checkpoints and feedback controls (Fig. 3.7). An important checkpoint occurs in G₂ that, upon detecting unreplicated DNA sequences, blocks cells in G₂ and prevents their progress through the M-phase, allowing both repair of DNA damage to take place and orderly completion of the S-phase. Cell cycle checkpoints are under the control of numerous genes that promote or inhibit the cell cycle depending on whether or not defective DNA-carrying cells must be repaired or eliminated. In broad terms, genes that ensure genomic integrity during the cell cycle are called tumor suppressor genes and include caretakers that prevent genomic instability and mutations from occurring, and gatekeepers that regulate cell cycle progression and maintain genomic stability by inducing apoptosis⁸ or senescence⁹ on genomically aberrant cells before they become cancer cells [156]. Of these, RB1 and TP53 are considered the main cell cycle gatekeepers through the activity of their encoded proteins, pRB and p53. Their role is exerted through the E2F1, a protein that acts as a transcription factor that promotes cell-cycle progression from G1 to S. In normal cells, E2F is inhibited by pRB, which in turn can be temporarily inactivated by cyclin-dependent kinases of the m2m gene product. In cancer cells, the gene encoding TP53 is often mutated, which prevents G₁ arrest in response to DNA damage and allows damaged DNA to both replicate and be delivered to daughter cells. pRB is inactivated by several mechanisms, including loss of function, mutations, and by viral oncogenes, enabling E2F-driven excessive cancer cell proliferation. Mutated RB1 are a cause of childhood retinoblastoma, bladder cancer, and osteogenic sarcoma. p53, a protein encoded by tumor suppressor gene TP53 (located at 17p13.1), is believed to have a far-reaching role, and is sometimes called the guardian of the genome. It includes activation of genes that control the cell cycle (WAF1 and CIP1/p21), DNA damage repair (GADD45), G1 to S and G2 to M progression (14-3-σ), and apoptosis (BAX). Loss of the latter function is generally viewed as a common pathway in carcinogenesis. TP53 is the most frequently mutated gene in human somatic cancers, and is responsible for the Li-Fraumeni syndrome, a rare inherited condition associated with a high risk for developing sarcomas, brain tumors, breast cancer, and leukemias [157]. Standardized nomenclature is being assigned to human genes in order to bring uniformity in scientific communications and data retrieval. This will prevent past cacophony in this field. For instance, synonyms for the cyclin-dependent kinase inhibitor 2A (CDKN2A) include ARF, CDK4I, CMM2, INK4, INK4a, MTS1, p14, p16, p16INK4a, p19, and p19Arf [158].

Like organisms, cells are born, live, and die. Also like organisms, cells can die of accidental or natural causes. Accidental cell death is caused by exposure to injurious stimuli, such as excessive heat, acid, radiation, or hypoxia against which cells play an entirely passive role. In contrast, natural cell death is the result of a highly complex but controlled process called programmed cell death that includes apoptosis, which is genetically induced. Cell survival is also controlled by a stretch of DNA located at the end of each chromosome, called telomeres. These two major pathways to cell death control cells’ life span through distinct mechanisms. Apoptosis occurs when a cell commits ‘suicide’ in response to external signals that challenge and ultimately defeat its self-preservation mechanisms. In contrast, telomere-triggered cell death originates from within the cell as a mechanism that inherently limits its life span and by extension controls aging of the entire organism.