9CHAPTER 2

Basic Concepts in Molecular Biology

Wendy L. Kimber

This chapter introduces the fundamental concepts in molecular biology that underlie the principles of genetics and inheritance. It begins with a discussion of the way that genetic material is organized into genes and chromosomes and the mechanisms by which these are transmitted to the next generation. This is followed by an explanation of the molecular nature of genes and the processes of DNA replication and gene expression. The flow of information from DNA to RNA to protein is described along with the consequences of mutations on this system. Finally, a brief survey of current genetic technologies is presented, including the burgeoning field of genomics. This chapter provides the foundational concepts on which subsequent chapters are built.

CHROMOSOMES

The term chromosome is derived from the Greek words for color (chroma) and body (soma) as chromosomes were first observed as colored threads inside the nucleus of stained cells by scientists in the 1800s. These thread-like structures are present in the nucleus of all cells and are the basic units of heredity that are passed from parents to their offspring.

Chromosomes are composed of a single molecule (double strand) of DNA, which is wrapped around histone proteins (Figure 2.1). The association of DNA with histone proteins is known as chromatin. Chromosomes exist in the cell in one of two forms, condensed (closed) or relaxed (open). For most of the time, the DNA in chromosomes is only loosely wound around histone proteins so that the genes on the chromosomes are accessible to the transcriptional machinery of the cell. In this form, chromosomes exist as long slender threads that are not visible under a light microscope. Only when a cell is getting ready to divide does the DNA become compacted to take on the characteristic shape and form of a chromosome.

Before a cell divides it makes a duplicate copy of each chromosome; both chromosome copies remain temporarily stuck together, with each individual chromosome referred to as a chromatid (Figure 2.2). During cell division the DNA of both chromatids are wound tightly around histone proteins so that it forms a short tight bundle. This makes it easier for the cell to move the chromosomes around the cell, and is analogous to taking multiple lengths of yarn and winding them up into individual balls for ease of handling. In this condensed form, chromosomes are visible under the light microscope, and each individual chromosome can be identified on the basis of its size and the pattern of bands created when the chromosomes are stained with Giemsa (G banding). During the time that chromosomes are in this condensed form, the DNA is so tightly wound around the histones that transcription cannot take place. For this reason, as soon as nuclear division is complete, the chromosomes rapidly decondense so that the DNA is accessible once more and can be used by the cell to direct the production of proteins.

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FIGURE 2.1. The association of DNA and histone proteins to form chromatin.

Source: http://www.genome.gov/dmd/img.cfm?node=Photos/Graphics&id=85280

In the condensed state, certain features of a chromosome become visible. A constriction in the chromosome identifies the chromosome centromere, which holds the two chromatids together. During nuclear division (mitosis or meiosis), spindle fibers attach to specialized regions of the centromere known as kinetochores to move the chromosomes around the cell. The ends of each chromosome are called telomeres, which are made up of short DNA sequences that are repeated over many times and do not code for proteins. Telomeres have a protective function for the chromosome, and shortening of telomeres has been linked to aging and cancer.

Humans have 23 pairs of different chromosomes in a somatic (nonsex) cell, with one of each pair being inherited from each parent. Twenty-two of these chromosome pairs do not play a role in sex determination and are referred to as autosomes. The remaining pair are called the sex chromosomes or X and Y chromosomes, because these determine the sex of an individual. In humans, sex is determined by the presence or absence of the Y sex chromosome, with females having two X chromosomes and males having one X and one Y chromosome. In the absence of a Y chromosome, embryonic development proceeds along the default female pathway. In the presence of a Y chromosome, development is switched to that of male by a transcription factor that is encoded by the sex-determining region Y (SRY) gene, which is found only on the Y chromosome. Because somatic cells contain two copies of each chromosome type, they are referred to being diploid, and their chromosome number is denoted as 2n. Gametes, which are cells specialized for fertilization (sperm and oocytes), have only one of each chromosome and are said to be haploid and are given the n designation. The fusion of haploid male and female gametes during fertilization restores the diploid number of chromosomes (46) to the zygote, with one maternally derived chromosome and one paternally derived chromosome for each pair.

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FIGURE 2.2. The structure of a duplicated chromosome.

Source: http://www.genome.gov/dmd/img.cfm?node=Photos/Graphics&id=85281

CELL DIVISION

Cell Cycle

Cells preparing to divide progress through a series of phases, which collectively are known as the cell cycle (Figure 2.3). The cell cycle could be considered the “life cycle” of the cell; however, only cells that have been given “permission” to divide complete the cell cycle, and nondividing cells remain in the first phase of the cell cycle indefinitely, which is referred to as the resting phase or G₀.

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FIGURE 2.3. The four phases of the cell cycle.

Source: http://www.genome.gov/dmd/img.cfm?node=Photos/Graphics&id=85276

The first phase of the cell cycle, called the gap 1 (G₁) phase, occurs immediately following cell division. When the parental cell divides in half to create two daughter cells, these are initially half the size of the parental cell with only half the number of organelles. The first task for this newly created cell is to increase its size and synthesize additional organelles. Therefore, the G₁ phase is considered to be a period of growth. Once a cell has reached its full size and capacity it does not automatically proceed to cell division. There are strict controls on which cells are allowed to divide, and division is only permitted if there is a need for more cells for the purposes of growth, repair, or regeneration. Therefore, there are a host of cell cycle controls that prevent a cell from leaving G₁ and proceeding with division. These are enforced by proteins, such as cyclins, that enforce cell cycle checkpoints, the maintenance of G₁ state, and prevent cells from leaving G₁ and progressing through the cell cycle. Mutation of one or more of these genes results in loss of cell cycle control, and cells progress through the cell cycle and divide in an uncontrolled way, leading to cancer. The mitotic index is a measurement of cell proliferation that measures the ratio of mitotic (dividing) cells to nondividing cells within a population. An elevated mitotic index can be indicative of the presence of cancerous cells that have lost cell cycle control.

When cell division is required, a chemical signal will be received by the cell, which leads to the removal of G₁ checkpoint controls, and the cell then enters the second phase of the cell cycle, the S phase. During this phase of the cell cycle, DNA synthesis occurs, and all of the chromosomes are replicated, leading to a doubling of the DNA content of a cell. There is no checkpoint at the end of the S phase; rather, cells proceed directly into the gap 2 (G₂) phase, which is another period of growth. During the G₂ phase, the cell prepares for nuclear and cell division, by synthesizing the proteins that will be required to drive this process. The gap phases of the 13cell cycle (G₁ and G₂) were named by early researchers studying the cell cycle by observing visible changes under the microscope. In contrast to S and M phases, the processes taking place during G₁ and G₂ do not create any visible changes in cellular morphology, leading scientists to name them “gap” phases to reflect what they incorrectly thought of as periods of inactivity in the cell.

At the end of the G₂ phase there is another cell cycle checkpoint, and cells can only progress to the next phase of the cell cycle when their DNA has been checked and found to be undamaged. If any DNA damage is found, it must be repaired before cells can proceed any further. Cells that pass the G₂ checkpoint proceed into the M phase, which is where nuclear division and eventually cytokinesis (cell division) takes place. The G₁, S, and G₂ phases are known collectively as interphase, to reflect that these are the phases leading up to nuclear and cell division.

The ploidy status, which is the number of sets of chromosomes of a cell, is determined by the method of nuclear division that is used to create the two nuclei during the M phase of the cell cycle, as a precursor to cytokinesis. Nuclear division occurs during the M phase of the cell cycle by one of two methods, mitosis or meiosis, depending on the function of the cell.

Mitosis

Somatic cells do not participate in reproduction and are therefore diploid, having two copies of each chromosome, referred to as homologous chromosomes. During the S phase of the cell cycle, a somatic cell makes a copy of all of its chromosomes; this double complement of chromosomes must become organized into two separate nuclei, each containing a complete set of chromosomes, before cell division can occur. Somatic cells achieve this through the process of mitosis, a method of nuclear division which, in humans, separates a set of 46 chromosomes into each of two nuclei. Mitosis is followed by cell division to generate two diploid daughter cells that have maintained the chromosome number of 46. This method of nuclear division is used by all dividing cells except gametes, for the purpose of growth, regeneration, and repair, and generates daughter cells that are genetically identical to the original parental cell. Organisms that reproduce asexually also divide by mitosis, leading to offspring that are identical, genetic clones of the parent.

Mitosis begins with all of the chromosomes being in their duplicated state, having been replicated during the S phase. Both of the chromosome duplicates, known as sister chromatids, remain fastened together at the centromere. The goal of mitosis is to pull apart these chromosome copies and move them into separate nuclei. Mitosis is divided into five stages, the first of which is prophase (Figure 2.4). During this phase, the chromosomes condense to become shorter and thicker in preparation for being moved around the cell. At the beginning of prophase, stained chromosomes are barely visible as thin threads; however, by the end of prophase the chromosomes have condensed to such an extent that they are then visible as the shapes that we commonly associate with chromosomes. The end of prophase is marked by the disappearance of the nuclear membrane to allow access of the spindle fibers to each chromosome. Metaphase is the most characteristic stage of mitosis, as this is the phase where spindle fibers move the chromosomes so that they are lined up along the center of the cell (metaphase plate). This stage of the cell cycle is used for preparing karyotypes (images of individual chromosomes for the purpose of identifying abnormalities in chromosome number or structure). When preparing cells for karyotyping, they are treated with a chemical inhibitor, which prevents the cells from leaving metaphase. This ensures that the chromosomes are in their most condensed state and therefore most highly visible under a light microscope. Before the cell proceeds to the next phase of mitosis, a cell cycle checkpoint is encountered, which ensures that all spindle fibers are attached to the chromosome centromeres. This is very important because errors at this point could lead to incorrect separation of duplicated chromosomes (nondisjunction), leading to conditions of aneuploidy (abnormal chromosome number). (For a more detailed description of aneuploidy, see Chapter 4.) Once this cell checkpoint has been verified, cells enter anaphase, which is when the spindle fibers abruptly shorten, pulling duplicated chromosomes apart and toward opposite poles of the cell. When adequate separation of the chromosome pairs has been achieved, the nuclear membranes quickly re-form around each complete set of chromosomes in the last phase of mitosis known as telophase. At this stage the chromosomes rapidly decondense, allowing access to the DNA for the resumption of transcription. For a very brief period, the cell has two nuclei; however, as soon as telophase is complete, the cell quickly divides its cell contents in half to form two cells. At this point, the cell cycle is complete and each newly created daughter cell enters the G₁ phase.

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FIGURE 2.4. The four phases of mitosis, which achieves nuclear division in somatic cells.

Source: http://www.genome.gov/dmd/img.cfm?node=Photos/Graphics&id=85204

15Meiosis

Gametes, which are cells specialized for reproduction (sperm and eggs), do not use mitosis for nuclear division because a haploid nucleus must be generated during cell division. In order to generate two haploid nuclei with only one of each chromosome type, the nucleus must divide using meiosis. Meiosis is a form of nuclear division that includes two rounds, resulting in the formation of four haploid cells. The first nuclear division, meiosis I, separates each pair of homologous chromosomes to generate two haploid nuclei, and for this reason is referred to as a reductive division. Although the first round of meiosis has generated two haploid nuclei, each chromosome is still in the duplicated state with the two sister chromatids connected at the centromere. The second round of meiotic nuclear division, meiosis II, is similar to mitosis in that it functions to separate duplicated sister chromatids, creating haploid nuclei, each with a set of unduplicated chromosomes; this is an equational division. Both meiosis “I” and “II” are divided into the same four stages of prophase, metaphase, anaphase, and telophase as mitosis (Figure 2.5). A phase is identified as being from meiosis I or II by the use of the Roman numeral I or II. For example, metaphase I indicates that this is metaphase from meiosis I and metaphase II indicates metaphase of meiosis II; the absence of a number indicates that this is a phase of mitosis (e.g., metaphase). A comparison of mitosis and meiosis is shown in Table 2.1.

FIGURE 2.5. The four phases of meiosis, which create haploid nuclei during nuclear division of germ cells.

Source: http://www.genome.gov/dmd/img.cfm?node=Photos/Graphics&id=85196

Meiosis begins, like mitosis, when the chromosomes are in their duplicated state. In prophase I, the chromosomes begin to condense and become visible as thin threads, as in prophase of mitosis. However, during prophase I, a process unique to meiosis occurs in which homologous chromosomes find each other and pair up in a zippering-like process known as synapsis. The synapsed chromosome pair is known as a bivalent, although it actually represents four chromosome copies and is sometimes referred to as a tetrad. Once the chromosomes are paired up, the chromosomes continue to condense, becoming visibly shorter and thicker, as well as more closely associated with each other due to the formation of the synaptonemal complex between the chromosomes; this protein complex is thought to mediate synapsis. During this period when the chromosomes are so intimately associated, chromatids on opposite chromosomes (referred to as nonsister chromatids) swap segments in the process of crossing over (Figure 2.6). In humans, crossovers occur on average at two points on each chromosome during meiosis, and the frequency and location of crossing over have been used to map genes (Box 2.1). A physical connection occurs between chromosomes during crossing over and is called a chiasma (plural chiasmata) because it resembles the Greek letter chi (χ). Crossing over is a significant genetic event as it is an important source of genetic variation during sexual reproduction, leading to new combinations of alleles on chromosomes. Prophase I, like prophase of mitosis, ends with the breakdown of the nuclear membrane and the attachment of the spindle fibers to the centromeres of each tetrad.

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FIGURE 2.6. Crossing over during prophase I of meiosis creates new combinations of alleles on chromosomes.

Source: http://www.genome.gov/Glossary/resources/crossing_over.pdf

18Metaphase I of meiosis is characterized by the unique way that the chromosomes are lined up along the metaphase plate. In metaphase of mitosis and meiosis II, chromosomes are lined up along the middle of the cell in a single file; however, in metaphase I of meiosis I, the chromosomes remain paired up in a tetrad, and are lined up in two rows of chromosomes along the metaphase plate. The way that the tetrads are aligned on the metaphase plate is random with respect to the maternal chromosome and paternal chromosomes. This introduces another source of genetic variation, as a random mix of maternal and paternal homologous chromosomes will segregate to different nuclei with only one chromosome of each pair existing in a given gamete. It is a matter of chance as to whether, for example, a chromosome number 1, which was originally from the mother, and a chromosome number 2, which was originally from the father, end up in the same gamete or not, or whether, by chance, all maternally derived chromosomes end up in the same gamete, a concept that is described by Mendel’s Law of Independent Assortment.

The phases of anaphase I and telophase I are very similar to mitosis; however, in this case anaphase is identified by the shortening of spindle fibers to pull apart the tetrads and move duplicated chromosomes consisting of two sister chromatids to opposite poles, in contrast to anaphase of mitosis where single chromatids are separated. In telophase I, the nuclear membrane quickly re-forms around each duplicated chromosome and the cell immediately begins to divide. The end result is two haploid cells with only one of each chromosome type.

Cells completing meiosis I go immediately into meiosis II as there is no need for DNA replication to occur. Although the cells are now haploid, each chromosome is still in the duplicated state and the sister chromatids need to be separated from each other. The stages of meiosis II follow more closely those of mitosis, and differs only in that the cell is now haploid. During prophase II, the chromosomes shorten and thicken, the nuclear membrane disappears, and the spindle fibers attach to the centromeres. Metaphase II is characterized by the alignment of a haploid number of chromosomes along the metaphase plate. In human cells, this means that there will only be 23 chromosomes aligned on the metaphase plate. The sister chromatids are separated during anaphase II and the nuclear membrane re-forms around each set of chromosomes during telophase II. Cytokinesis completes the process, with the end result being the generation of four haploid cells.

Gametogenesis

Diploid germ cells divide using meiosis to produce haploid gametes. In males, this process is called spermatogenesis and begins with the division of diploid spermatogonia in the seminiferous tubules of the testes. In humans, spermatogenesis begins at puberty and continues throughout the lifetime of the male, with cells moving continuously through all of the stages of meiosis. In contrast, the process of female gamete formation, oogenesis, is discontinuous, with cells arrested at two points in this process. Oogenesis takes place in the oogonial cells in the ovary during the first 6 months of embryonic development. Meiosis is not completed at this time and instead is arrested at the prophase stage of 19meiosis I in primary oocytes, which remain in this state indefinitely. At the onset of puberty in human females, one oocyte per month is released from the ovary during ovulation. Ovulation triggers the completion of meiosis I in an oocyte, which then goes on to complete meiosis I and enter meiosis II. This cell becomes arrested for the second time at metaphase II of meiosis II. The oocyte remains arrested at this stage unless it encounters sperm and fertilization occurs. Sperm entry during fertilization stimulates the oocyte to complete meiosis II, prior to fusion of the pronuclei containing the maternal and paternal chromosomes.

Cell division during spermatogenesis is symmetrical, with meiosis I generating two equally sized cells, which each divide by meiosis II to generate a total of four equally sized spermatids. In contrast, cell division during oogenesis is asymmetric, with each cell division generating one cell that contains the majority of the cell contents and a much smaller cell, called a polar body, containing a nucleus and very little cytoplasm. The end result of oogenesis is the creation of one large ovum containing a large amount of cytoplasm and cellular contents, as well as three small polar bodies that do not participate in reproduction. During the process of sperm maturation, spermiogenesis, spermatids actively eliminate most of the cytoplasm from the cell. Therefore, at fertilization, the ovum contributes most of the cellular contents including mitochondria, and sperm contributes the haploid male pronuclei. The inheritance of mitochondria from the mother is an interesting genetic phenomenon because mitochondria also contain DNA. Mitochondria are the sites of energy production in the cell, and have a genome of 16,500 base pairs of DNA containing 37 genes. It is now known that some genetic disorders result from mutations in mitochondrial DNA, which are discussed further in Chapter 4.

CHROMOSOMES AND INHERITANCE

Genes are arranged in a linear fashion along a chromosome, and are always found at the same position on the same chromosome, with this position being referred to as its locus (plural: loci). There is one copy of each gene at a given locus; however, somatic cells are diploid, having two of each chromosome: one maternal and one paternal in origin. Therefore, there are two copies of each gene per cell: one gene copy inherited from the mother and one from the father. The exceptions are the X and Y chromosomes of the male. The Y chromosome has a much lower number of genes than the X chromosome, with around 50 genes compared to the approximately 800 genes on the X chromosome. In addition, some genes are unique to the Y chromosome, such as SRY, and function in male development. For this reason, a gene on the X chromosome often does not have a corresponding copy on the Y chromosome.

A single gene contains the information for making a specific polypeptide and is the hereditary determinant of a trait. Different versions of the same gene often exist, usually differing by one or a few base pairs, which produce the same protein but with slightly differing activities. Different versions of the same gene are called alleles, and lead to variations in a given trait. For any given gene, if both alleles are identical, an individual is said to be homozygous. If an individual has two different alleles (versions) of the same gene, he or she is said to be heterozygous for that gene. The term genotype is used to describe the genetic makeup of a person when discussing combinations of specific gene alleles, and phenotype refers to an observable trait that is a result of a genotype. A trait or characteristic is considered dominant if the trait 20encoded by the allele is apparent when one copy of the allele is paired with a different allele of the same gene. A trait is considered to be recessive when the phenotype for that allele is seen only in individuals with two copies (homozygous) of that allele. In males, only one recessive allele is needed to express a recessive phenotype if the gene is located on the X chromosome with no corresponding allele on the Y chromosome. These traits are called X-linked recessive traits. Genes on the X chromosome of the male are often referred to as hemizygous, when a second allele on the Y chromosome is absent. In order to avoid a double dosage of X-linked gene products in females, a process known as X-inactivation silences gene expression from one of the X chromosomes in each somatic cell. A situation of codominance occurs when both alleles are expressed, as in the case of the AB blood group. A summary of these genetic terms is presented in Box 2.2.

To explain patterns of inheritance more simply, geneticists often use capital letters to represent alleles for dominant traits and lowercase letters to represent recessive ones. Thus, a person who is heterozygous for a given allele pair can be represented as Aa, one who is homozygous for two dominant alleles as AA, and one who is homozygous for two recessive alleles as aa. For autosomal recessive traits, the homozygote (AA) and the heterozygote (Aa) may be indistinguishable on the basis of phenotypic appearance, but they may be distinguishable biochemically because they may make different amounts or types of a gene product. This information can often be used in carrier screening for recessive disorders to determine genetic risk and for genetic counseling. When geneticists discuss a particular gene pair or disorder, normality is usually assumed for the rest of the person’s genome, and the term normal is often used unless stated otherwise.

 

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