Summary: Essential Cell Biology (Alberts et al) - First part

This summary is based on the 3rd edition of Essential Cell Biology from Alberts et al. The remaining chapters can be accessed when logged in and can be found here: Second part of the summary


1. Introduction to cells

Unity and diversity of cells

Cells are the fundamental units of life; all living things are made of cells. The present-day cells are believed to have evolved from an ancestral cell that excited more than 3 billion years age. Cells vary enormous in appearance and function, however all living cells have a similar basic chemistry.

With the invention of the microscope, it became clear that plants and animals are assemblies of cells, that cells can also exist as independent organisms, and that cells individually are living in the sense that they can grow, reproduce, convert energy from one form into another, respond to their environment, and so on. Although cells are varied when viewed from the outside, all living things are fundamentally similar inside. And in all living things, genetic instructions, called genes, are stored in DNA molecules. In every cell, the instructions in the DNA are read out, or transcribed, into a chemically related set of molecules made of RNA. The messages carried by the RNA molecules are in turn translated into yet another chemical form: they are used to direct the synthesis of a huge variety of large protein molecules that dominate the behaviour of the cell. In sum, the reproduction process exists of replication (DNA synthesis), transcription (RNA synthesis) and translation (protein synthesis). Unfortunately, the copying of DNA is not always perfect, and the instructions are occasionally corrupted. Later is this summary we will discuss this further.

Cells are enclosed by a plasma membrane that separates the inside of the cell from the environment. And all cells contain DNA as a store of genetic information and use it to guide the synthesis of proteins. Cells in a multicellular organism, though the all contain the same DNA, can be very different. They use their genetic information to direct their biochemical activities according to cues they receive from their environment.

Cells under the microscope

Cells of animal and plant tissues are typically 5-20 micrometer in diameter and can be seen with a light microscope, which also reveals some of their internal components (organelles). The electron microscope permits the smaller organelles and even individual molecules to be seen, but specimens require elaborate preparation and cannot be viewed alive. So, the invention of the light microscope led to the discovery of cells

The presence or absence of a nucleus is used as the basis for a simple but fundamental classification of all living things. Organisms whose cells have a nucleus are called eukaryotes. Organisms whose cells do not have a nucleus are called prokaryotes. Bacteria, the simplest of present-day living cells, are prokaryotes. Different species of prokaryotes are diverse in their chemical capabilities and inhabit an amazingly wide range of habitats. Prokaryotes are divided into two groups: eubacteria and archaea. As mentioned above eukaryotic cells possess a nucleus. They probably evolved in a series of stages from cells more similar to bacteria. An important step appears to have been the acquisition of mitochondria, origination as engulfed bacteria living in symbiosis with larger anaerobic cells.

There are a lot of organelles found in eukaryotic cells: the nucleus is the most prominent organelle in most plant and animal cells. It contains the genetic information of the organism, stored in DNA molecules. The rest of the cell’s contents, apart from the nucleus, constitute the cytoplasm. Chloroplasts are green organelles found only in the cells of plants and algae, not in the cells of animals or fungi. They perform photosynthesis and in the process they release oxygen as a molecular by-product. Other organelles are the mitochondria, which are generators of chemical energy for the cell. Mitochondria contain their own DNA and reproduce by dividing in two. Furthermore, they take the energy from the oxidation of food molecules to produce adenosine triphosphate (ATP). The endoplasmatic reticulum (ER) is the site at which most cell membrane components, as well as materials destined for export from the cell, are made.  The Golgi apparatus often modifies chemically the molecules made in the ER and directs them to various locations of the cell. Lysosomes are organelles in which intracellular digestion occurs and peroxisomes generate a dangerously reactive chemical, hydrogen peroxide. Finally, the cytoskeleton is responsible for directed cell movements.

Model organisms

Free-living single-celled eucaryotic micro organisms include some of the most complex eucaryotic cells known, and they are able to swim, mate, hunt and devour food. Other types of eukaryotic cells, derived from a fertilized egg, cooperate to form large, complex multicellular organisms composed of thousands of billions of cells.

Biologists have chosen a small number of organisms as a focus for intense investigation. These include the bacterium E. coli, brewer’s yeast, a nematode worm, a fly, a small plant, a mouse and the human species itself.
Although the minimum number of genes needed for a viable cell is probably less than 400, most cells contain significantly more. Yet even such a complex organism as a human has only about 30.000 genes – twice as many as a fly, seven times many as E. coli.

2. Chemical components of cells

Chemical bonds

The cell is the structural and functional unit of all known living organisms, but the smallest particle of an element that still retains its distinctive chemical properties is an atom. Each atom has as center a positively charged nucleus, which is surrounded by a cloud of negatively charged electrons. The nucleus consists of two kinds of particles:

  • positively charged protons

  • neutrons, which are electrically neutral

The number of protons present in an atomic nucleus determines its atomic number. Because the whole atom is electrically neutral, the number of negatively charged electron surrounding the nucleus is equal to the number of positively charged protons that the nucleus contains. Isotopes of an element have nuclei with the same number of protons (the same atomic number) but different numbers of neutrons.

The atomic weight of an atom, or the molecular weight of a molecule, is its mass relative to that of a hydrogen atom. The mass of an atom or a molecule is often specified in daltons. If a substance has a molecular weight of M, a mass of M grams of the substance will contain 6 x 10^23 molecules. This quantity is called one mole of the substance. The concept of mole is used widely in chemistry as a way to represent the number of molecules that are available to participate in chemical reactions. There are 92 naturally occurring elements, each differing from the others in the number of protons and electrons in its atoms. Living organisms are made of only a small selection of these elements.

The outermost electrons determine how atoms interact. The number and arrangement of its electrons determine the chemical properties of an atom. An atom is most stable when all of its electrons are at their lowest possible energy level and when each electron shell is completely filled. The number of electrons an atom must acquire of lose to attain a filled outer shell is known as its valence. Chemical bonds form between atoms as electrons move to reach a more stable arrangement. Clusters of two or more atoms held together by covalent bonds are known as molecules. There are two ways to create chemical bonds:

  • An ionic bond is formed when electrons are donated by one atom to another.
  • A covalent bond is formed when two atoms share a pair of electrons. If two pairs of electrons are shared, a double bond is formed. Double bonds are shorter and stronger than single bonds.

Also covalent and noncovalent chemical bonds have different strengths and lengths. Noncovalent bonds as a rule are much weaker.

Another noncovalent bond is the hydrogen bond, by which water is held together. These bonds are much weaker than covalent bonds. Molecules carrying positive or negative charges (ions) dissolve readily in water and are called hydrophilic, meaning that the are ‘water-loving’. Hydrophobic (water fearing) molecules on the other hand, are uncharged and form few or no hydrogen bonds, and so do not dissolve in water.

Substances that release protons when they dissolve in water and thus forming H3O+, are termed acids. The higher the concentration of H3O+, the more acidic the solution. The opposite of an acid is a base; any molecule capable of accepting a proton is called a base or alkaline. The concentration of H3O+ is expressed using the pH scale.

Molecules in Cells

Living organisms contain a distinctive and restricted set of small carbon-based molecules that are essentially the same for every living species. The main categories are:

  • Sugars: a primary source of chemical energy for cells and can be incorporated intro polysaccharides for energy storage

  • Fatty acids: also important for energy storage, but their most essential functions is in the formation of cell membranes. There are two kinds of fatty acids saturated and non-saturated. The first has no double bounds between its carbon atoms and contains the maximum possible numbers of hydrogens. The non-saturated fatty acids have tails with one or more double bounds. These double bounds create kinks in the molecules, interfering with their ability to pack together in a solid mass. How tightly the fatty acids, found in cell membranes, pack affects the fluidity of the membrane.

  • Amino acids: the subunits of proteins. The covalent linkage between two adjacent amino acids in a protein chain is called a peptide bound, the chain of amino acids is also known as a polypeptide.

  • Nucleotides: the subunits of DNA and RNA

These four families of small organic molecules, together with the macromolecules made by linking them into long chains, account for a large fraction of a cell’s mass.

Macromolecules in Cells

The vast majority of the dry mass of al cell consists of macromolecules, formed as polymers of sugars, amino acids, or nucleotides. Macromolecules are intermediated both in size and complexity between small molecules and cell organelles. They have many remarkable properties that are not easily deduced from the subunits from which they are made. Their remarkable diversity arises from the fact that each macromolecule has a unique sequence of subunits.

Noncovalent bounds specify the precise shape of a macromolecule: weak noncovalent bonds form between different regions of a macromolecule. Two types of noncovalent bounds are discussed earlier: ionic bounds and hydrogen bounds, but there is a third type of weak bound that result from ‘van der Waals attractions’. These attractions are a form of electrical attraction caused by fluctuating electric charges that whenever two atoms come within a very short distance of each other. These weak noncovalent bounds can cause the macromolecule to fold into a unique three-dimensional shape with a special chemistry, as seen in proteins.

3. Energy, catalysis and biosynthesis

Living organisms are able to exist because of a continual input of energy. Part of this energy is used to carry out essential functions, like reactions that support cellular metabolism, growth and reproduction, and the remainder is lost in the form of heat.

Catalysis and the use of energy

All animals live on energy stored in the chemical bonds of organic molecules made by other organisms, which they take as in food. Animals obtain food by eating plants or by eating animals that feed on plants. But ultimately, the primary source of energy for most living organisms is the sun.

Plants and photosynthetic bacteria use solar energy to produce organic molecules from carbon dioxide. They use the energy they derive from sunlight to form chemical bonds between atoms, linking them into small chemical building blocks such as sugar, amino acids, nucleotides and fatty acids. These small molecules in turn are converted into the macromolecules that form the plant.
The reactions of photosynthesis take place in two stages:

  1. In the light-dependent stage energy for the sunlight is captured and transiently stored as chemical bond energy in specialized small molecules that carry energy in their reactive chemical groups. Oxygen is released as a by-product of the first stage.

  2. In the second stage the molecules that serve as energy carriers are used to help drive a carbon-fixation process in which sugars are manufactured from carbon dioxide gas and water. By producing sugars, these light-independent reactions generate a critical source of stored chemical bond energy and materials.

The net result of the entire process of photosynthesis is:

Light energy + CO2 + H20 ® sugars + O2 + energy

To use to energy to live, grow and reproduce, organisms must extract it in a usable form. In both plants and animals, energy is extracted from food molecules by a process of oxidation, or controlled burning. Next to oxidation there is a process called cellular respiration.  Photosynthesis and cellular respiration are complementary processes; cellular respiration uses the O2 to form CO2 from the same carbon atoms that had been taken up as CO2 and converted into sugars by photosynthesis. In this process, the organisms obtain the chemical bond energy that they need to survive.

Oxidation refers to the removal of electrons and reduction (the converse of oxidation) refers to the addition of electrons. Because the number of electrons is conserved in a chemical reaction – there is no net loss or gain – oxidation and reduction always occur simultaneously.

Cells use enzymes to catalyze the oxidation of organic molecules in small steps, through a sequence of reactions that allows useful energy to be harvested.

Enzymes lower the barriers that block chemical reactions

Each of the many hundreds of chemical reactions that occur in a cell is specifically catalyzed by an enzyme. Large numbers of different enzymes work in sequence to form chains of reactions, called metabolic pathways, each performing a particular set of functions in the cell.

Catabolic reactions break down food molecules through oxidative pathways and release energy. Anabolic reactions generate the many complex molecules needed by the cell, and they require an energy input. In animal cells, both the building blocks and the energy required for the anabolic reactions are obtained by catabolism.

Enzymes catalyze reactions by binding to particular substrate molecules in a way that lowers the activation energy required for making and breaking specific covalent bonds. The rate at which an enzyme catalyzes a reaction depends on how rapidly it finds its substrate and how quickly the product forms and then diffuses away. These rates vary widely from one enzyme to another, and they can be measured after mixing purified enzymes and substrates together under a set of defined conditions. In general, the stronger the binding of the enzyme and substrate, the slower their rate of dissociation.

If a reactions leads to a release of free-energy, this energy can be harnessed to do work or drive chemical reactions. Chemical reactions proceed only in the direction that leads to a loss of free energy; in other words, the spontaneous direction for any reaction is the direction that goes ‘downhill’. This kind of reaction is often said to be energetically favorable. But even energetically favorable reactions require activation energy to get them started!

As mentioned above, the push over the energy barrier is greatly aided by enzymes. A substance that can lower the energy barrier, and hence the activation energy of a reaction is termed a catalysts. Like all other catalysts, enzyme molecules themselves remain unchanged after participating in a reaction and therefore can function over and over again.

If the concentration of the substrate is increased progressively from a very low value, the concentration of the enzyme-substrate complex, and therefore the rate at which product is formed, initially increases in a linear fashion in direct proportion to substrate concentration. But at a very high concentration of substrate it reaches a maximum value, termed Vmax. At this point, the active sites of all enzyme molecules in the sample are fully occupied with substrate, and the rate of product formation depends only on how rapidly the substrate molecule can be processed; also called the turnover number.

The concentration of substrate needed to make the enzyme work efficiently is often measured by a different parameter, the Michaelis’ constant (Km). An enzyme’s Km is the concentration of substrate at which the enzyme works at half its maximum speed (0.5 Vmax). A low value of Km indicated that a substrate binds very tightly to the enzyme, and a large value corresponds to weak binding. But enzymes cannot change the equilibrium point for reactions!

The free-energy change for a reaction determines whether it can occur

Although enzymes speed up reactions, they cannot by themselves force energetically unfavorable reactions to occur. But this can be done through enzymes that directly couple energetically favorable reactions, which release energy and produce heat, to energetically unfavorable reactions, which use this energy. But (according to the second law of thermodynamics) a chemical reaction can proceed only if it results in a net increase of the disorder in the universe. The criterion for an increase of disorder can be expressed most conveniently in term of free energy (G) of a system. The free-energy change for a reaction, ∆G, measures the disorder, and it must be less than zero for a reaction to proceed.

Energetically favorable reactions are those that create disorder by decreasing the free energy of a system to which the belong; in other words, they have a negative ∆G. Conversely, energetically unfavorable reactions, with a positive ∆G, create order in the universe. Because energetically unfavorable reactions require energy, they can take place only if they are coupled to a second reaction with a negative ∆G so large that the net ∆G of the entire process is negative.

In concluding, by creating a reaction pathway that couples an energetically favorable reaction to an energetically unfavorable one, enzymes cause otherwise impossible chemical transformations to occur.

The free-energy change for a chemical reaction, ∆G, depends on the concentration of the reacting molecules, and it may be calculated from these concentrations if the equilibrium constant (K) of the reaction (of the standard free-energy change ∆Gº for the reactants) is known. Because the equilibrium constant of a reaction is related directly to the standard free energy change (∆Gº), it is often employed as a measure of the binding strength between molecules. This value is very useful as it indicates the specificity of the interactions between molecules.

Equilibrium constants govern all of the associations (and dissociations) that occur between macromolecules and small molecules in the cell. The equilibrium constant becomes larger as the binding energy between the two molecules increases, and the more likely that these molecules will be paired.

Activated carrier molecules and biosynthesis

In living systems energy capture is achieved by means of a couple reactions, in which an energetically favorable reaction is used to drive and energetically unfavorable one that produces an activated carrier molecule. Coupling mechanisms require enzymes, and they are fundamental to all of the energy transactions in the cell. The most important of the activated carrier molecules are ATP, NADH and NADPH. ATP carries high-energy phosphate groups, whereas NADH an NADPH carry high-energy electrons.

1) ATP

The most important of the activated carrier molecules in cells is ATP (adenosine 5’-triphosphate). ATP is synthesized in an energetically unfavorable phosphorylation reaction in which a phosphate group is added to ADP (adenosine 5’-diphosphate). When required, ATP gives up this energy packet in an energetically favorable hydrolysis to ADP and inorganic phosphate. The regenerated ADP is then available to be used for another round of the phosphorylation reaction that forms ATP, creating an ATP cycle in the cell. Therefore, an energetically unfavorable biosynthetic reaction can be driven by ATP hydrolysis. For example, the synthesis of a polynucleotide likes RNA and DNA.

2) NADH an NADPH

NAD+ and NADP+ each pick up a ‘packet of energy’ in the form of two high-energy electrons plus a proton (H+), becoming NADH and NADPH, respectively. Like ATP, NADPH is an activated carrier that participates in many important biosynthetic reactions that would otherwise be energetically unfavorable. NADH, by contrast, has a special role as an intermediate in the catabolic system of reactions that generate ATP through the oxidation of food molecules.

Food molecules provide the carbon skeletons for the formation of larger molecules. The covalent bonds of these larger molecules are typically produced in reactions that are coupled to energetically favorable bond changes in activated carrier molecules such as ATP and NADPH.

4. Protein structure and function

Proteins are by far the most structurally complex and functionally sophisticated molecules known. Proteins are assembled from a set of 20 different amino acids, each with different chemical properties. A protein molecule is made from a long chain of these amino acids, each linked to its neighbor through a covalent peptide bond. Proteins, therefore, are also called polypeptides. Each polypeptide chain consists of a backbone that supports the different amino acid side chain. The polypeptide backbone if formed from the repeating sequence of atoms along the polypeptide chain. Attached tot this repetitive chain are any of the 20 different amino acid side chains. These side chains give each amino acid its unique properties, for example hydrophobic, nonpolar or positively charged.

Shape and structure of proteins

Each type of protein has a unique amino acid sequence that determines both its three-dimensional shape and its biological activity. Long peptides are very flexible and therefore proteins can fold in enormous number of ways. The folded structure of a protein is stabilized by noncovalent interactions between different parts of the polypeptide chain.

The final folded structure, of conformation, is the one in which the free energy (G) is minimized. A protein can be unfolded, or denatured, by treatment with certain solvents that disrupt the noncovalent interactions holding the folded chain together. When the denaturing solvent is removed, the protein often refolds spontaneously, or renatures, into its original conformation.

When proteins fold improperly, the can form aggregates that can damage cells and even whole tissue. Aggregated proteins underlie a number of neurodegenerative disorders, including Alzheimer’s disease and Huntington’s disease. Although a protein chain can fold into its correct conformation without outside help, protein folding in a living cell in generally assisted by special proteins called molecular chaperones. These proteins bind to partly folded chains and help them to fold along the most energetically favorable pathway.  However, the final three-dimensional shape of the protein is still specified by its amino acid sequence: chaperones merely make the folding process more efficient and reliable.

Although the overall conformation of each protein is unique, two regular folding patterns are often found in parts of them. Hydrogen bonds between neighboring regions of the polypeptide backbone can give rise to regular folding patterns, known as a helices and b sheets. In an a helix the N-H of every peptide bond is hydrogen-bonded to the C=O of a neighboring peptide bond located four peptide bonds away in the same chain. In the case of the b sheet the individual polypeptide chains in the sheet are held together by hydrogen-bonding between peptide bonds in different strands, and the amino acid side chains in each strand project alternately above and below the plane of the sheet.

A a helix is generated when a single polypeptide chain turns around itself to form a structurally rigid cylinder. Sometimes a pair of a helices will wrap around one another to form a particularly stable structure, known as coiled-coil. This structure forms when the two a helices have most of their nonpolar (hydrophobic) side chains on one side, so that they can twist around each other with these side chains facing inwards. b Sheets are made when hydrogen bonds form between segments of polypeptide chains lying side by side. When the structure consists of neighboring polypeptide chains that run in the same orientation, it is considered a parallel b sheet; when it forms from a polypeptide chain that fold back and forth upon itself the structure is an antiparallel b sheet. b Sheets provide an ideal ice-binding surface in an antifreeze protein.

Levels of organization

The structure of many proteins can be subdivided into smaller globular regions of compact three-dimensional structure, known as protein domain. So, a protein’s structure begins with its amino acid sequence, which is thus considered its primary structure. The next level of organization includes the a helices and b sheets that form within certain segments of polypeptide chain; these folds are elements of the protein’s secondary structure. The full, three-dimensional conformation formed by an entire polypeptide chain is referred as the tertiary structure. Finally, if a particular protein molecule is formed as a complex of more than one polypeptide chain, than the complete structure is designed its quaternary structure.

The same weak noncovalent bonds that enable a polypeptide chain to fold into a specific conformation also allows proteins to bind to each other to produce larger structures in the cell. Any region on a protein’s surface that interacts with another molecule through sets of noncovalent bonds is termed a binding site. If a binding site recognizes the surface of a second protein, the tight binding of two folded polypeptide chains at this site will create a larger protein molecule with a precisely defined geometry. Each polypeptide chain in such a protein is called a subunit. Each of these protein subunits may contain more than one domain.

There are different types of proteins:

  1. Globular proteins: in which the polypeptide chain folds up into a compact shape like a ball with an irregular surface. Enzymes tend to be globular proteins.

  2. Fibrous proteins: these have a relatively simple, elongated three-dimensional structure. These proteins are especially abundant outside the cell, where they form the gel-like extracellular matrix that helps cells bind together to form tissues. These proteins are secreted by cells into their surface roundings, where they often assemble into sheets of long fibrils. Collagen is the most abundant of these fibrous proteins in animal tissues, and another example is elastin.

To help maintain their structures, the polypeptide chains in such proteins are often stabilizes by covalent cross-linkages. The most common cross-links in proteins are covalent sulfur-sulfur bonds. These disulfide bonds (also called S-S bonds) form as proteins are being exported from cells. Their formation is catalyzed in the endoplasmatic reticulum by a special enzyme that links together two –SH groups from cysteine side chains that are adjacent in the folded protein.

How proteins work

The biological function of a protein depends on the detailed chemical properties of its surface and how it binds to other molecules, called ligands. The ability of a protein to bind selectively and with high affinity to a ligand is due to the formation of a set of weak, noncovalent bonds and favorable hydrophobic interactions. Each individual bond is weak, so that an effective interaction requires that many weak bonds be formed simultaneously. The region of a protein that associates with a ligand, known as its binding sites, usually consists of a cavity in the protein surface formed by a particular arrangement of amino acids. These amino acids belong to widely separated regions of the polypeptide chain that are brought together when the proteins fold.

All proteins must bind to particular ligands to carry out their various functions. But this binding capacity seems to have been most highly developed for proteins in the antibody family. Antibodies, or immunoglobulins, are proteins produced by the immune system in response to foreign molecules. Each antibody binds to a particular target molecule, either inactivating that target directly or marking it for destruction. An antibody is Y-shaped and has two identical binding sites for its antigen, one on each arm of the Y.

For many proteins, binding to another molecule is their only function, but there are some proteins for which ligand binding is simply a necessary first step in their functions. This class of proteins is called enzymes. Enzymes are proteins that first bind tightly to specific molecules, called substrates, and then catalyze the formation or breakage of covalent bonds in these molecules. At the active site of an enzyme, the amino acid side chains of the folded protein are precisely positioned so that they favor the formation of the high-energy transition states that the substrates must pass through to be converted to product.
The three-dimensional structure of many proteins has evolved so that the binding of a small ligand can induce a significant change in protein shape.

Although the order of amino acids in proteins gives molecules their shape and the versatility to perform different functions, sometimes the amino acids by themselves are not enough. So proteins often employ small nonprotein molecules to perform functions that would be difficult or impossible using amino acids alone. Examples of these proteins are retinal (the light-sensitive molecule attached to rhodopsin in our eyes) and heme (gives hemoglobin and blood its red color, and enables hemoglobin to pick up oxygen in the lungs and release it in the tissues).

How proteins are controlled

Inside the cell most proteins and enzymes do not work continuously or at full speed. Instead, their activity is regulated so that the cell can maintain itself in a state of equilibrium, generating only those molecules it requires to thrive under the current conditions. To achieve this balans, the activities of cellular proteins are controlled in an integrated fashion, with consideration of what reactions are occurring in other parts of the cell. By coordinating when and how proteins function, the cell ensures that it does not deplete its energy reserves by accumulating molecules it does not require.

Regulation of enzyme activity occurs at many levels. At one level, the cell controls how many molecules of each enzyme it makes by regulating the expression of the gene that encodes that protein. At another level, the cell controls enzymatic activities by confining sets of enzymes to particular subcellular compartments, enclosed by distinct membranes (both mechanisms are discussed later in this summary). But the most rapid and general process used to adjust reactions rates operated at the level of the enzyme itself. In this case, an enzyme’s activity changes in response to other specific molecules that it encounters.

  • Feedback inhibition (negative feedback): it prevents an enzyme from acting
  • Positive regulation (positive feedback): the enzyme’s activity is stimulated

The interaction between sites that are located on separate regions of a protein molecule is known to depend on a conformational change in the protein: binding at one of the sites causes a shift in the protein’s structure from one folded shape to a slightly different folded shape. Feedback inhibition, for example, triggers a conformational change. Many, if not most, protein molecules are allosteric: they can adopt two or more slightly different conformations that differ in catalytic activity, and by a shift from one to another, their activity can be regulated. This is true not only for enzymes but for many other proteins like receptors, structural proteins, and motor proteins. The enzyme can be turned on or off by ligands that bind to a distinct regulatory site to stabilize either the active or the inactive conformation.

Phosphorylation can control protein activity by triggering a conformational change

Enzymes are not only regulated by the binding of small molecules. A second method commonly used by eukaryotic cells to regulate protein activity involves attaching a phosphate group covalenty to one of its amino acids side chains. Removal of the phosphate group by a second enzyme returns the protein to its original conformation and restores its initial activity. This reversible protein phosphorylation controls the activity of many different types of proteins in eukaryotic cells.

Protein phosphorylation involves the enzyme-catalyzed transfer of the terminal phosphate group of ATP to the hydroxyl group on a serine, threonine, of tyrosine said chain of the protein. This reaction is catalyzed by a protein kinase. The reverse reaction – removal of the phosphate group, or dephosphorylation – is catalyzed by a protein phosphatase. Cells contain hundreds of different protein kinases, each responsible for phosphorylating a different protein or set of proteins. Cells also contain many different protein phosphatases.

For many proteins, a phosphate group is added to a particular side chain and then removed in a continuous cycle. Phosphorylation cycles of this kind allow proteins to switch rapidly from one state to another. The energy required to drive this cycle is derived from the free energy of hydrolysis of ATP.

GTP-Binding proteins are also regulated by cyclic gain and loss of a phosphate group

Eucaryotic cells have another way to regulate protein activity by phosphate addition and removal. Instead of being enzymatically transferred from ATP to the protein, the phosphate is part of a guanine nucleotide (either GTP or GDP) that is bound tightly to the protein. Such GTP-binding proteins are in their active conformations with GTP bound; the protein itself then hydrolyses this GTP to GDP, by releasing a phosphate, and flips to an inactive conformation. As with protein phosphorylation, this process is reversible.

The GTP-binding proteins often bind to other proteins to control enzyme activities, and their crucial role in intracellular pathways will be discussed later in this summary.

In concluding, many thousands of proteins in a typical eucaryotic cell are regulated either by cycles of phosphorylation and dephosphorylation, or by the binding and hydrolysis of GTP by a GTP-binding protein.

Nucleotide hydrolysis

As mentioned above, conformational changes in proteins play a central part in enzyme regulation and cell signaling. But conformational changes also play another important role in the operation of the cell: they enable proteins whose major function is to move other molecules, the motor proteins, to generate the forces responsible for muscle contraction and the movements of the cell. The hydrolysis of ATP to ADP by motor proteins produces directed movements in the cell.

Proteins often form large complexes that function as protein machines

Highly efficient proteins machines are formed by assemblies of allosteric proteins. In most protein machines the hydrolysis of bound nucleoside triphosphates (ATP or GTP) drives an ordered series of conformational changes in some of the individual protein subunits, enabling the ensemble of proteins to move coordinately. In this way, the appropriate enzymes can be moved directly into the positions where they are needed to carry out successive reactions in a series as, for example, protein synthesis or DNA replication.

5. DNA and chromosomes

The structure and function of DNA

Life depends on stable and compact storage of genetic information. Genetic information is carried by very long deoxyribonucleic acid (DNA) molecules and encoded in the linear sequence of nucleotides A, T, G and C.
A DNA molecule consists of two long polynucleotide chains known as DNA chains, or DNA strands. Each of these chains is composed of four types of nucleotide subunits, and the two chains are held together by hydrogen bonds between the base portions of the nucleotides, nucleotides are composed of a five-carbon sugar to which are attached one or more phosphate groups and a nitrogen-containing base. In the case of the nucleotides in DNA, the sugar is deoxyribose attached to a single phosphate group, and the base may by adenine (A), cytosine (C), guanine (G), or thymine (T). The nucleotides are covalently linked together in a chain through the sugars and phosphates, which thus form a ‘backbone’ of alternating sugar-phosphate-sugar-phosphate.

The two polynucleotide chains in the DNA double helix are held together by hydrogen-bonding between the bases on the different strands; G-C and A-T. All the bases are therefore on the inside of the helix, with the sugar-phosphate backbones on the outside.
Each strand of DNA has a chemical polarity due to the linkage of alternating sugars and phosphates in its backbone The members of each base pair can fit together within the double helix only if the two strands of the helix are antiparallel, that is, only if the polarity of one strand is oriented opposite to that of the other strand. A consequence of these base-pairing requirements is that each strand of a DNA molecule contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. This is crucial importance for the copying of DNA, as we will see later in the summary.

The structure of eukaryotic chromosomes

Large amounts of DNA are required to encode all the information needed to make just a single-celled bacterium, and far more DNA is needed to encode the instructions for the development of multicellular organisms like ourselves.

In eukaryotic cells, enormously long double-stranded DNA molecules are package into chromosomes can be easily apportioned between the two daughter cells at each cell division. In eucaryotes the DNA in the nucleus is distributed among a set of different chromosomes. Each chromosome consists of a single, enormously long, linear DNA molecule associated with proteins that fold and pack the fine thread of DNA into a more compact structure. The complex of DNA and protein is called chromatin.
With the exception of the germ cells (sperm and eggs) and highly specialized cells that lack DNA entirely, human cells each contain two copies of each chromosome, one inherited from the mother and one from the father; the maternal and paternal chromosomes of a pair are called homologous chromosomes (homologs). The only nonhomologous chromosome pairs are the sex chromosomes in males, where a Y chromosome is inherited from the father and an X chromosome from the mother.

A display of the full set of 46 chromosomes is called the human karyotype. Cytogeneticists use alterations in banding patterns to detect chromosomal abnormalities that are associated with some inherited defects and with certain types of cancer.

The genetic material of a eukaryotic cell is contained within one or more chromosomes, each formed from a single, enormously long DNA molecule that contains many genes. In general, the more complex an organism is, the larger its genome. Furthermore, how the DNA is apportioned over chromosomes also differ from one species to another. Humans have 46 chromosomes, but a species of small deer has only 6 chromosomes. Thus, although gene number is roughly correlated with species complexity, there is no simple relationship between gene number, chromosome number and total genome size.

‘Life-cycle of chromosomes’

To form a functional chromosome, a DNA molecule must be able to replicate, and the replicated copies must be separated and partitioned reliably into daughter cells at each cell division. These processes occur through an ordered series of stages, known collectively as the cell cycle.

Two stages are important:

  1. the interphase, when chromosomes are duplicated;
  2. and mitosis, when they are distributed to the two daughter nuclei.

During interphase, the cell is actively expressing its genes, and during this stage the chromosomes are extended as long, thin, tangled threads of DNA in the nucleus. Still during the interphase and before cell division, the DNA is replicated and the chromosomes are duplicated. Once DNA replication is complete, the cell can enter M phase, when mitosis occurs. Mitosis is the division of the nucleus. During this stage, the chromosomes condense, gene expression largely ceases, the nuclear envelope breaks down, and the mitotic spindle forms from microtubules and other proteins. The condensed chromosomes are captured by the mitotic spindle, and one complete set of chromosomes is pulled to each end of the cell. A nuclear envelope form around each chromosome set, and in the final step of M phase, the cell divides to produces two daughter cells.

Three DNA sequence elements are needed to produce a eukaryotic chromosome that can be replicated and then segregated at mitosis. These sequences ensure that the chromosome can be replicated efficiently and passed on to daughter cells.

  1. Telomere: contain repeated nucleotide sequences that enable the ends of chromosomes to be replicated. They also protect the end of the chromosome from being mistaken by the cell as a broken DNA molecule in need of repair. But the function of telomeres is discussed later in this summary.

  2. Replication origin

  3. Centromere: this allows one copy of each duplicated chromosome to be apportioned to each daughter cell.

The nucleus is delimited by a nuclear envelope formed by two concentric membranes. The nuclear envelope is supported by two networks of protein filaments: one, the nuclear lamina, forms a thin layer underlying and supporting the inner nuclear membrane; while the other, less regularly organized, surrounds the outer nuclear membrane. The two membranes are punctured at intervals by nuclear pores, which actively transport selected molecules to and from the cytosol.

The most obvious example of chromosome organization in the interphase nucleus is the nucleolus. This is a region where the parts of different chromosomes carrying genes for ribosomal RNA cluster together. Here, ribosomal RNAs are synthesized and combined with proteins to form ribosomes, the cell’s protein-synthesizing machines.

Chromosomes and DNA

Chromosomes in eukaryotic cells consist of DNA tightly bound to a roughly equal mass of specialized proteins. These proteins fold the DNA into a more compact form so that it can fit into a cell nucleus.

The proteins that bind to the DNA to form eukaryotic chromosomes are traditionally divided into two general classes: the histones and the nonhistone chromosomal proteins. The complex of DNA and both classes of protein in chromosomes is called chromatin. Histones are responsible for the first and most fundamental level of chromatin packing: they pack DNA into a repeating array of DNA-protein particles called nucleosomes.

An individual nucleosome core particle consists of a complex of eight histone proteins (two molecules each of the histone H2A, H2B, H3 and H4) and a double stranded DNA of about 146 nucleotide pairs that winds around this histone octamer. Each nucleosome core particle is separated from the next by a region of linker DNA.

All four of the histones that make up the nucleosome core are relatively small proteins with a high proportion of positively charged amino acids. The positive charges help the histones bind tightly to the negatively charged sugar-phosphate backbone of DNA. Each of the core histones also has a long N-terminal amino acid ‘tail’, which extends out from the DNA histone core. These histone tails are subject to several types of covalent modification that control many aspects of chromatin structure.

Nucleosomes are further packed upon one another to generate a more compact structure, the 30-nm fiber. This happens with the aid of histone H1 molecules, which is thought to pull the nucleosomes together into a regular repeating array. This fiber can be further coiled and folded, producing more compact chromatin structures. But, some forms of chromatin are so highly compacted that the packaged genes cannot be expressed into protein.

As daughter cells complete their separation following mitosis, the mitotic chromosomes unfold into a more extended form: the interphase chromosomes. However, the chromatin in an interphase chromosome is not in the same packing state throughout the chromosome. In general, regions of the chromosome that contain genes that are being expressed are more extended, while those that contain quiescent genes are more compact. The most highly condensed form of interphase chromatin is called heterochromatin. Heterochromatin typically makes up about 10% of an interphase chromosome, and in mammalian chromosomes, it is typically concentrated around the centromere region and in the telomeres at the ends of the chromosomes. Most DNA that is folded into heterochromatin does not contain genes. However, genes that do become packaged into heterochromatin usually become resistant to being expressed because heterochromatin is unusually compact. The rest of the interphase chromatin, which is in a variety of more extended states, is called euchromatin.

Changes in nucleosome structure allow access to DNA

Eucaryotic cells have several ways to rapidly adjust the local structure of their chromatin. One approach takes advantage of chromatin remodeling complexes, protein machines that use the energy of ATP hydrolysis to change the structure of nucleosomes.

In concluding, chromatin structure is dynamic: by temporarily altering its structure (using chromatin remodeling complexes and enzymes that modify histone tails) the cell can ensure that proteins involved in gene expression, replication, and repair have rapid, localized access to the necessary DNA sequences.

6. DNA replication, repair and recombination

The ability of a cell to maintain order in a chaotic environment depends on the accurate duplication of the vast quantity of genetic information carried in its DNA. This duplication process, called DNA replication, must occur before a cell can produce two genetically identical daughter cells.

Despite systems for protecting the genetic instructions form copying errors and accidental damage, permanent changes, or mutations, sometimes do occur. Mutations in the DNA often affect the information it encodes. Occasionally, this can benefit the organism in which a mutation occurs. However, mutations are often detrimental: they are responsible for thousands of inherited diseases and many types of cancer. Without the cellular systems that are continually monitoring and repairing damage to DNA, it is questionable whether life could exist at all.

DNA replication

As mentioned earlier, each strand of the DNA double helix contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. Each strand can therefore act as a template for the synthesis of a new complementary strand.
The ability of each strand of a DNA molecule to act as a template for producing a complementary strand enables a cell to copy, or replicate, its genes before passing them on to its descendants. This replication is performed by a cluster of proteins that together form a ‘replication machine’. In each round of replication, each of the two strands of DNA is used as a template for the formation of a complementary DNA strand. The original strands, therefore, remain intact through many cell generations. DNA replication is called ‘semiconservative’ because each daughter DNA double helix is composed of one conserved strand and one newly synthesized strand.

DNA synthesis

1) DNA synthesis begins at replication origins

The DNA double helix is normally very stable, because the two DNA strands are locked together by the large numbers of hydrogen bonds between the bases on both strands. The process of DNA replication begins by initiator proteins that bind to the DNA and pulled the two strands apart, by breaking the hydrogen bonds between the bases. Although the hydrogen bonds collectively make the DNA helix very stable, individually each hydrogen bond is weak. Separating a short length of DNA does not therefore require a large energy input. The positions at which the DNA is first opened are called replication origins, and they are usually marked by a particular sequence of nucleotides. An A-T base pair is held together by fewer hydrogen bonds than is a G-C base pair, therefore DNA rich in A-T base pairs is relatively easy to pull apart, and A-T-rich stretches of DNA are typically found at replication origins.
Once an initiator protein binds to DNA at the replication origin and locally opens up the double helix, it attracts a group a proteins that carry out DNA replication. This group operates as a protein machine, with each member carrying out a specific function.

2) New DNA synthesis occurs at replication forks

As a DNA molecule replicates, its two strands are pulled apart to form one or more Y-shaped replication forks. At these forks, the replication machine is moving along the DNA, opening up the two strands of the double helix and using each strand as a template to make a new daughter strand. The enzyme DNA polymerase, situated in the fork, catalyzes the addition of nucleotides to the 3’ end of a growing DNA strand by forming a phosphodiester bond between this end and the 5’-phosphate group of the incoming nucleotide.

In concluding, DNA polymerase can catalyze the growth of the DNA chain in only one direction: the 5’-to-3’ direction. This problem is solved by the use of a ‘backstitching’ maneuver. The DNA strand whose 5’ end must grow is made discontinuously, in successive separate small pieces, with the DNA polymerase working backward from the replication fork in the 5’-to-3’ direction for each new piece. These pieces, called Okazaki fragments, are later ‘stitched’ together (by the enzyme ligase) to form a continuous new strand. The DNA strand that is synthesized discontinuously in this way is called the lagging strand; the strand that is synthesized continuously is called the leading strand. Because both of the new strands are synthesized in the 5’-to-3’ direction, the DNA replication forks are asymmetrical.

DNA polymerase

DNA polymerase replicated a DNA template with remarkable fidelity, making less tan one error in every 10^7 bases read. This is possible because the enzyme removes its own polymerization errors as it moves along the DNA (proofreading). Before the enzyme adds a nucleotide to a growing DNA chain, it checks whether the previous nucleotide added is correctly base-paired to the template strand. If so, the polymerase adds the next nucleotide; if not, the polymerase removes the mispaired nucleotide by cutting the phosphodiester bond it has just made, releases the nucleotide, and tries it again. Thus, DNA polymerase possess both a 5’-to-3’ polymerization activity and a 3’-to-5’ exonuclease (nucleic acid-degrading) activity.

This proofreading mechanism explains why DNA polymerase synthesize DNA only in the 5’-to-3’ direction. If DNA is synthesized in the 3’-to-5’ direction is would be unable to proofread: because if it removed an incorrectly paired nucleotide, the polymerase would create a chain end that is dead and unable to elongate.

Because the polymerase can join a nucleotide only to a base-paired nucleotide in a DNA double helix, it cannot start a completely new DNA strand. A different enzyme is needed and it is called primase. This enzyme makes short lengths of RNA, called primers, which are subsequently erased and replaced with DNA.

For the leading strand, an RNA primer is needed only to start replication at a replication origin; once a replication fork has been established, the DNA polymerase is continuously present with a base-paired 3’ end as it tracks along the template strand. But on the lagging strand, where DNA synthesis is discontinuous, new primers are needed continually.

To produce a continuous new DNA strand from the many separate pieces of RNA and DNA made on the lagging strand, three additional enzymes are needed. These act quickly to remove the RNA primer (nuclease), replace it with DNA (repair polymerase), and join the DNA fragments together (ligase).

Proteins at a replication fork

DNA replication requires the cooperation of many proteins: at the head of the replication machine is a helicase, a protein that uses the energy of ATP hydrolysis to speed along DNA, unzipping the double helix as it moves. Another component of the replication machine, the single-strand binding proteins clings to the single-stranded DNA exposed by the helicase and transiently prevents is from re-forming base pairs. Yet another protein, called a sliding clamp, keeps the DNA polymerase firmly attached to the DNA template; on the lagging strand, the sliding clamp releases the polymerase from the DNA each time an Okazaki fragment is completed. This clamp proteins forms a ring around the DNA helix and binds polymerase, allowing it to slide along a template strand as it synthesis new DNA.

Most of the proteins involved in DNA replication are thought to be held together in a large multienzyme complex that moves as a unit along the DNA, enabling DNA to be synthesized on both strands in a coordinated manner.  

Genetic information can be stored stably in DNA sequences only because a variety of DNA repair enzymes continuously scan the DNA and correct replication mistakes and replace damaged nucleotides. DNA can be repaired easily because one strand can be corrected using the other strand as a template.

Telomerase replicates the ends of eukaryotic chromosomes

In eucaryotes, a special enzyme called telomerase replicated the DNA at the ends of the chromosomes. Telomerase adds multiple copies of the same telomere DNA sequence to the ends of the chromosomes, thereby producing a template that allows replication of the lagging strand to be completed.

DNA repair

To survive and reproduce, individuals must be genetically stable. This stability is achieved not only through the extremely accurate mechanism for replicating DNA, but also through mechanisms for correcting the rare copying mistakes made by the replication machinery and for repairing the accidental damage that continually occurs to the DNA. Most of these changes in DNA are only temporary because they are immediately corrected by processes collectively called DNA repair.

Only rarely do the cell’s DNA replication and repair processes fail and allow a permanent change in the DNA. Such a permanent change is called a mutation. For example, a single nucleotide change causes the inherited disease sickle-cell anemia. It is very important to protect reproductive cells (germ cells) against mutation, because a mutation in one of these cells will be passed on to all the cells in the body of the multicellular organism that develops from it, including the germ cells for production of the next generation. However, the other cells (somatic cells) must also be protected from genetic change to safeguard the health and well-being of the individual. Therefore, cells have acquired an elegant set of mechanisms to reduce the number of mutations that occur in their DNA.

DNA mismatch repair system

The rare copying mistakes that slip through the DNA replication machinery are dealt with by the mismatch repair proteins, which monitor newly replicated DNA and repair copying mistakes. The overall accuracy of DNA replication, including mismatch repair, is one mistake per 10^9 nucleotides copied.

A complex of mismatch repair proteins recognizes the DNA mismatches, removes (excises) one of the two strands of DNA involved in the mismatch, and resynthesizes the missing strand. To be effective in correcting replication mistakes, this mismatch repair system must always excise only the newly synthesized DNA, and thus, eliminating the mutation by using the original (old) template strand as the template. The importance of mismatch repair in humans was recognized when it was discovered that an inherited predisposition to certain cancers (especially some types of colon cancer) is caused by a mutation in the gene responsible for producing one of the mismatch repair proteins.

DNA is continually suffering damage in cells. There are many ways in which the DNA can be damaged, and these require other mechanism, than mismatch repair proteins, for their repair. Depurination and deamination are the most frequent chemical reactions known to create serious DNA damage in the cell. Furthermore, the ultraviolet radiation in sunlight causes also DNA damage.
The basic mechanism of DNA repair involved three steps:

  • Excision
  • Resynthesis
  • Ligation

In step one (excision), the damage is recognized and removed by one of a variety of different nuclease, which cleave the covalent bond that join the damaged nucleotides to the rest of the DNA molecule, leaving a small gap on one of the DNA double helix in this region. In step two (resynthesis), a repair DNA polymerase binds to the 3’-hydroxyl ends of the cut DNA strand. In then fills the gap by making a complementary copy of the information stored in the undamaged strand. In step three (ligation), DNA ligase seals the nick left in the sugar-phosphate backbone of the repaired strand. Nick sealing requires energy from ATP hydrolysis, remakes the broken phosphodiester bond between the adjacent nucleotides.

teps two and three are nearly the same for most types of DNA repair, including mismatch repair. However, step one uses a series of different enzymes, each specialized for removing different types of DNA damage.

DNA recombination

Homologous recombination is the process by which two double-stranded DNA molecules of similar nucleotide sequence can cross over to create DNA molecules of novel sequence. Homologous recombination begins with a double-strand break in a chromosome, creating a complete break in the DNA molecule. The 5’ ends at the break are then chewed back by a DNA-digesting enzyme, creating protruding single-stranded 3’ ends. Each of these single strands then searches for a homologous, complementary DNA helix with which to pair, leading to the formation of a ‘joint-molecule’ between the two chromosomes. The nicks in the DNA strands are then sealed and this is known as a cross-strand exchange or ‘Holliday junction’. To regenerate two separate DNA molecules, the two crossing strands must be cut. The structure undergoes a series of rotational movements so that the two original noncrossing strands become crossing and vice versa.

Homologous recombination provides many advantages to cells and organisms:

  • The process allows an organism to repair DNA that is damaged on both strands of the double helix
  • It can fix other genetic accidents that occur during nearly every round of DNA replication
  • It is essential for the accurate chromosome segregation that occurs during meiosis in fungi, plants and animals.

In homologous recombination, DNA rearrangements occur between DNA segments that are very similar in sequence. A second type of recombination, called site-specific recombination, allows DNA exchange to occur between DNA double helices that are not similar in nucleotide sequences. Mobile genetic elements are thought to play a crucial role in this process. Mobile genetic elements are DNA sequences that can move from place to place in the genomes of their host. This movement creates change in the host genome provides a source of genetic variation.

More than 50% of the human genome consists of DNA that is repeated many times in the genome. Approximately two-thirds of this repeated DNA (about 34% of the total genome) consists of two classes of transposons that have multiplied to especially high copy numbers in the genome. Retrotransposons move via an RNA intermediate, instead of a DNA intermediate. One type of retrotransposon, the L1 element (or LINE-1), is a highly repeated sequence that constitutes about 15% of the total mass of the human genome.

Viruses are mobile genetic elements that can escape from cells
Viruses are little more than genes packaged in protective protein coats. They require host cells in order to reproduces themselves. Viral genomes can be made of DNA or RNA and can be single-stranded or double-stranded. One group of RNA viruses – the retrovirus – must copy their RNA genomes into DNA in order to replicate. The human immunodeficiency virus (HIV), which is the cause of AIDS, is a retrovirus.

7. From DNA to protein

When a particular protein is needed by the cell, the nucleotide sequences of the appropriate portion of the immensely long DNA molecule in a chromosome is first copies into another type of nucleic acid – RNA (ribonucleic acid). It is these RNA copies of short segments of the DNA that are used as templates to direct the synthesis of the protein.

The flow of genetic information in all living cells is therefore from DNA ® RNA ® protein. The conversation of the genetic instructions in DNA into RNAs and proteins is termed gene expression.

From DNA to RNA

To express the genetic information carried in DNA, the nucleotide sequence of a gene is first transcribed into RNA. Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds. It differs from DNA chemically in two ways:

  1. The nucleotides in RNA are ribonucleotides; they contain the sugar ribose rather than deoxyribose

  2. Although RNA and DNA both contain the bases adenine (A), guanine (G), and cytosine (C), RNA contains uracil (U) instead of thymine (T) found in DNA.

Next to the chemical differences, there are also important differences in overall structure. Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded. This difference has important functional consequences; because RNA is single-stranded it can fold up into a variety of three-dimensional shapes. As we will see later in this summary, this ability allows RNA to carry out different functions, like for example catalytic functions.

All of the RNA in a cell is made by transcription. The enzymes that carry out transcription are called RNA polymerase. RNA polymerases catalyze the formation of phosphodiester bonds that link the nucleotides together and form the sugar-phosphate backbone of the RNA chain. The RNA polymerase moves stepwise along the DNA, unwinding the DNA helix to expose a new region of the template strand for complementary base-pairing. Nucleotide sequences in the DNA molecule indicate to the RNA polymerase where to start and stop transcribing. A promotor contains a sequence of nucleotides indicating the starting point for RNA synthesis. A subunit of bacterial polymerase, called sigma (s) factor, is primarily responsible for recognizing the promotor sequence on DNA.

RNA polymerase make about one mistake for every 10^4 nucleotides copied into RNA, compared with an error rate for DNA polymerase of about one in 10^7 nucleotides. Although RNA polymerase catalyzes essentially the same chemical reactions as DNA polymerase, there are some important differences between the two enzymes:

  • RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides
  • Unlike the DNA polymerase involved in DNA replication, RNA polymerase can start an RNA chain without a primer

Several types of RNA are produces in cells

Cells make several different functional types of RNA, including messenger RNA (mRNA), which carries the instructions for making proteins, ribosomal RNA (rRNA), which is a component of ribosome’s; and transfer RNA (tRNA), which acts as an adaptor molecule in proteins synthesis.

Although the templating principle by which DNA is transcribed into RNA is the same in all organisms, the way in which the RNA transcripts are handled before the can be used by the cell differs a great deal between bacteria and eucaryotes. Bacterial DNA lies directly exposed to the cytoplasm, which contains the ribosomes on which protein synthesis takes places. In eukaryotic cells, by contrast, DNA is enclosed within the nucleus. Transcription takes place in the nucleus, but protein synthesis takes place on ribosomes in cytoplasm. So, before mRNA can be translated, it must be transported out of the nucleus. In addition, before RNA exists in the nucleus, it must go through several different RNA processing steps. Two processing steps that occur only on transcripts destines to become mRNA molecules are:

  • RNA capping
  • Polyadenylation

These two modifications are thought to increase the stability of the eukaryotic mRNA molecule, to aid its export from the nucleus to the cytoplasm, and to generally identify the RNA molecule as an mRNA. They are also used by the protein-synthesis machinery as an indication that both ends of the mRNA are present and that the message is therefore completed.

Genes are interrupted by noncoding sequences

In eukaryotic DNA most genes are composed of a number of smaller coding regions (exons) interspersed with noncoding regions (introns). When a eukaryotic gene is transcribed from DNA into RNA, both exons and introns are copied. After capping, as the RNA polymerase continues to transcribe the gene, the process of RNA splicing begins, in which the intron sequences are removed from the newly synthesized RNA and the exons are stitched together. RNA splicing is performed by RNA molecules that recognize intron-exon boundaries and participate in the chemistry of splicing. These RNA molecules, called small nuclear RNAs (snRNAs), bind with additional proteins to form small nuclear ribonucleoprotein particles (snRNPs). These snRNPs form the core of the spliceosome, the largely assembly of RNA and protein molecules that performs RNA splicing in the cell. To splice an RNA, a group of snRNPs assemble at an intron-exon boundary, cut out the intron, and rejoin the RNA chain.

In concluding, eucaryotic mRNAs go through several additional RNA processing steps before the leave the nucleus, including RNA capping and polyadenylation. These reactions, along with splicing, are tightly coupled to transcription and take place as the RNA is being transcribed. The mature mRNA then moves to the cytoplasm. Furthermore, RNA splicing enables eucaryotes to increase the already enormous coding potential of their genomes.

From RNA to protein

Translation is the ‘transfer of the information’ in RNA into proteins. Because there are only 4 different nucleotides in mRNA and 20 different types of amino acids in a protein, this translation cannot be accounted for by a direct one-to-one correspondence between a nucleotide in RNA and an amino acid in protein. The rules by which the nucleotide sequence of a gene, through the medium of mRNA, is translated into the amino acid sequence of a protein are known as the genetic code.

Translation of the nucleotide sequence of mRNA into a protein takes place in the cytoplasm on large ribonucleoprotein assemblies called ribosomes. These attach to the mRNA and move stepwise along the mRNA chain, translating the message into protein. The nucleotide sequence in mRNA is read in sets of three nucleotides (codons), each codon corresponding to one amino acid. The correspondence between amino acids and codons is specified by the genetic code. The possible combinations of the 4 different nucleotides in RNA give 64 (4x4x4) different codons in the genetic code. Most amino acids are specified by more than one codon.

Transfer RNAs

The codons in an mRNA do not directly recognize the amino acids they specify. Rather, the translation of mRNA into proteins depends on adaptor molecules that can recognize and bind both to the codon and to the amino acid. These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs).

tRNA acts as an adaptor molecule in protein synthesis and enzymes called aminoacyl-tRNA synthetases link amino acids to their appropriate tRNAs. Each tRNA contains a sequence of three nucleotides, the anticodon, which matches a codon in mRNA by complementary base-pairing between codon and anticodon.

Recognition and attachment of the correct amino acid depends on enzymes called aminoacyl-tRNA synthetases, which covalently couple each amino acid to its appropriate set of tRNA molecules. There is a different synthetase enzyme for each amino acid. The synthetase-catalyzed reaction that attaches the amino acid to the 3’ end of the tRNA is one of the many cellular reactions coupled to the energy-releasing hydrolysis of ATP. And it produces a high-energy bond between the charged tRNA and the amino acid. The energy of this bond is used at a later stage in protein synthesis to covalently link the amino acid to the growing polypeptide chain.

Decoding of the RNA message

Each ribosome has a binding site for mRNA and three binding sites for tRNA. The tRNA sites are designated the A-, P-, and E-sites. Protein synthesis begins when a ribosome assembles at an initiation condon (AUG) in mRNA, a process that is regulated by proteins called translation initiation factors. Of all the charged tRNAs in the cell, only the charged initiator tRNA is capable of tightly binding to the P-site of the small ribosome subunit. The completed protein chain is released from the ribosome when a stop codon (UUA, UAG, of UGA) is reached. Proteins known as release factors bind to any stop codon that reaches the A-site on the ribosome, and this binding alters the activity of the peptidyl transferase in the ribosome, finally causing the release of the protein into the cytoplasm.
The stepwise linking of amino acids into a polypeptide chain is catalyzes by an rRNA molecule in the large ribosomal subunit. Thus the ribosome is an example of a ribozyme, an RNA molecule that can catalyze a chemical reaction.

Protein breakdown

The degradation of proteins in the cell is carefully controlled. Cells possess specializes pathways to enzymatically break proteins down into their constituent amino acids – a process called proteolysis. The enzymes that degrade proteins are known collectively as proteases. Proteases act by hydrolyzing the peptide bonds between amino acids. One function of the proteolytic pathways is to rapidly degrade those proteins whose lifetimes must be short. Another is to recognize and eliminate proteins that are damaged or misfolded. Eliminating improperly folded proteins is critical for an organism, because neurodegenerative disorders such as Huntington’s and Alzheimer’s are caused by the aggregation of misfolded proteins. Some proteins are degraded in the cytosol by large protein complexes called proteasomes. Proteasomes act primarily on proteins that have been marked for destruction by the covalent attachment of a small protein called ubiquitin.

RNA and the origins of life

From our knowledge of present-day organisms and their molecules, it seem likely that living systems began with the evolution of RNA molecules that could catalyze their own replication. We have seen that a protein is able to catalyze a biochemical reaction because is has a special surface on which a given substrate can react. In the same way, RNA molecules, with their unique folded three-dimensional shapes, can serve as enzymes. Although the fact that they are constructed of only four different subunits limits their catalytic efficiency and the range of chemical reactions they can catalyze compared with proteins. So, most catalytic functions in present-day cells have been taken over by proteins.

It has been proposed that, as cells evolved, the DNA double helix replaced RNA as a more stable molecule for storing increased amounts of genetic information, and proteins replaced RNAs as major catalytic and structural components.

The flow of information in present-day living cells is DNA ® RNA ® protein, with RNA serving primarily as a go-between. Some important reactions, however, are still catalyzed by RNA; these are thought to provide a glimpse into the ancient, RNA-based world.

8. Control of gene expression

An overview of gene expression

A typical eukaryotic cell expresses only a fraction of it genes, and the distinct types of cells in multicellular organisms arise because different sets of genes are expressed as a cell differentiates. So, the different cell types of a multicellular organism contain the same DNA and, therefore all the genetic instructions necessary to direct the formation of a complete organism. Hence, the cells of an organism differ not because they contain different genes, but because they express them differently.

Although all of the steps involved in expressing a gene can in principle be regulated, for most genes the initiation of transcription is the most important point of control. Thus a cell can control the proteins it makes by:

  • Controlling when and how often a given gene is transcribed
  • Controlling how the primary RNA transcript is spliced or otherwise processed
  • Selecting which mRNAs are translated by ribosomes
  • Selectively activating or inactivating proteins after they have been made

How transcription switches work

We saw earlier that the promoter region of a gene attracts the enzyme RNA polymerase and correctly orients the enzyme to begin its task of making an RNA copy of the gene. The promoters of both bacterial and eukaryotic genes include an initiation site, where transcription begins. In addition to the promoter, nearly all genes have regulatory DNA sequences that are used to switch the gene on or off. Regulatory DNA sequences do not work by themselves; to have any effect these sequences must be recognized by proteins called gene regulatory proteins that bind to the DNA. It is the combination of a DNA sequence and its associated protein molecules that acts as the switch to control transcription.

Although each gene regulatory protein has unique features, most bind to DNA using one of a small number of protein structure motifs. The precise amino acid sequence that is folded into the DNA-binding motif determines the particular DNA sequence that is recognized. The DNA-binding motifs are the homeodomain that consists of three linked α helices; the zinc finger that is built from an α helix and a β sheet held together by a molecule of zinc; and the leucine zipper that is formed by two α helices.

Repressors turn genes off, activators turn them on

RNA polymerase binds to the DNA and initiates transcription at a site called promoter. However, within the promoter is a short DNA sequence that is recognized by a gene regulatory protein. When the regulatory protein binds to this nucleotide sequence, termed the operator, it blocks access of RNA polymerase to the promoter. This prevents transcription of the operon and production of the tryptophan-producing enzymes; it switches genes off. The gene regulatory protein is known as the tryptophan repressor, and it is regulated in a clever way: the repressor can bind only to DNA if it has also bound several molecules of the amino acid tryptophan. Therefore, the tryptophan repressor in an allosteric protein: the binding of tryptophan causes subtle change in its three-dimensional structure so that it can now bind to the operator DNA.

Other bacterial gene regulatory  proteins operate in the opposite manner by switching genes on, or activating them. These activator proteins bind to a regulatory sequence on the DNA and then interacts with the RNA polymerase to help it initiate transcription. Without the activator, the promoter fails to initiate transcription efficiently.

Differences in regulation of transcription

Regulation of transcription in eucaryotes differs in four important ways from that in bacteria:

  1. While bacteria contain a single type of RNA polymerase, eukaryotic cells have three: RNA polymerase I / II / III. These polymerases are responsible for transcribing different types of genes. RNA polymerases III and I transcribe the genes encoding tRNA, rRNA and small RNAs. RNA polymerase II transcribes the vast majority of eukaryotic genes.

  2. Bacterial RNA polymerase is able to initiate transcription without the help of additional proteins. However, eucaryotic RNA polymerases require the assembly of proteins called general transcription factors. The general are thought to position the RNA polymerase correctly at the promoter, to aid in pulling apart the two strands of DNA to allow transcription to begin, and to allow RNA polymerase to leave the promoter as transcription begins.

  3. In bacteria, regulatory proteins usually bind to regulatory DNA sequences close to where RNA polymerase binds and then either activate or repress transcription of the gene. In eucaryotes, these regulatory DNA sequences are often separated from the promoter by many thousands of nucleotide pairs. So, in eucaryotes gene activation occurs at a distance.

  4. Initiation of transcription in eukaryotic cells must also take into account the packing of DNA into nucleosomes and more compact forms of chromatin structure.

Eucaryotic gene regulatory proteins act in two fundamental ways:

  1. They can directly affect the assembly process of RNA polymerase and the general transcription factors at the promoter
  2. They can locally modify the chromatin structure of promoter regions

The molecular mechanisms that create specialized cell types

In eucaryotes, the expression of a gene is generally controlled by a combination of gene regulatory proteins. The regulatory proteins do not each function individually, but they work together as a ‘committee’ to control gene expression.

In multicellular plants and animals, the production of different gene regulatory proteins in different cell types ensures the expression of only those genes appropriate to the particular type of cell. Although all cells must be able to switch genes on and off, multicellular organisms require special gene switching mechanisms for generating and maintaining their different types of cells. Once a cell in a multicellular organism has become differentiated into a particular cell type, it will generally remain differentiated, and if it is able to divide, all it progeny cells will be of that same cell type. This means that the changes in gene expression that give rise to a differentiated cell must be remembered and passed on to its daughter cells through all subsequent cell divisions. Cells have several ways of ensuring that daughter cells remember what kind of cells they are supposed to be:

  1. Trough a positive feedback loop, where a key gene regulatory protein activates transcription of its own gene in addition to that of other cell-type-specific genes

  2. Trough the propagation of a condensed chromatin structure from parent to daughter cell even though DNA replication intervenes

A single gene regulatory protein, if expressed in the appropriate precursor cell, can trigger the formation of a specialized cell type or even an entire organ.

9. How genes and genomes evolve

The vast diversity of life we see around us has arisen through changes in DNA sequences that have accumulated since the first cells on earth arose some 3.5 billion years ago.

Generating genetic variation

Genetic changes that offer an organism a selective advantage or those that are selectively neutral are the most likely to be perpetuated. Changes that seriously compromise an organism’s fitness are eliminated through natural selection.

Genetic variation (the raw material for evolutionary change) occurs by a variety of mechanisms and each of these forms of genetic variation has played an important part in the evolution of modern organisms:

  1. Mutations within a gene: an existing gene can be modified by mutations that change a single nucleotide or that delete or duplicate on or more nucleotides in its DNA sequence. These so called point mutations typically arise from small errors in DNA replication or repair.

  2. Gene duplication: an existing gene, a larger segment of DNA, or even a whole genome can be duplicated, creating a set of closely related genes within a single cell. Gene duplication is one of the most important sources of genetic diversity. Once a gene has been duplicated, one of the two gene copies is free to mutate and become specialized to perform a different function. Repeated rounds of this process of duplication and divergence can allow one gene to give rise to a whole family of genes within a single genome.

  3. Gene deletion: individual genes, or whole blocks of genes can be lost through chromosome breakage and failures of repair.  

  4. Exon shuffling: the evolution of new proteins is thought to have been greatly facilitated by the organization of eukaryotic genes as relatively short exons separated by long, noncoding introns. The presence of introns greatly increases the probability that a chance recombination event generate a functional hybrid gene by joining together two initially separate exons coding for quite different protein domains; this process called exon shuffling.

  5. Horizontal (intracellular) gene transfer: a piece of DNA can be transferred from the genome of one cell to that of another, even to that of another species. This process is rare among eucaryotes, but common among procaryotes.

Reconstructing life’s family tree

By comparing the nucleotide or amino acid sequences of contemporary organisms, we are beginning to able to reconstruct how genomes have evolved in the billions of years that elapsed since the appearance of the first cells.

Examining the human genome

The human genome contains 3.2 x 10^9 nucleotide pairs divided among 22 autosomes and 2 sex chromosomes. The human genome sequence refers to the complete nucleotide sequence of the DNA contained in these 24 chromosomes.
Individual human differ from one another by an average of 1 nucleotide pair in every 1000; this variation underlies our individuality and provides the basis for identifying individuals by DNA analysis.

The first characteristic feature of the human genome is how little of it (only a few percent) codes for proteins or for structural or catalytic RNAs. Much of the remaining DNA is made up of transposable elements that have gradually colonized our genome over evolutionary time. A second feature of the human genome is the very large average size of 27.000 nucleotide pairs. Only about 1300 nucleotide pairs are required to encode a protein of average size, and most of the remaining DNA is a gene consists of long stretches of noncoding DNA that interrupt the relatively short protein-coding exons. Finally, the nucleotide sequence of the human genome has revealed that the critical information it carries seems to be in an alarming state of disarray.

A major obstacle in interpreting the nucleotide sequences of human chromosomes is the fact that much of the sequence appears unimportant. Comparative genome analyses provide a valuable tool for indentifying genes as well as functionally important regulatory sequences. Knowing the location, and possibly the function, of a gene in one genome consequently makes it easier to identify and predict the function of the corresponding gene in the other genome. Such comparisons have revealed that mice and humans share most of the same genes, and that large blocks of the mouse and human genomes contain these genes in the same order.

Even with the human genome in hand, many questions will continue to challenge cell biologists throughout the next century. Perhaps most puzzling is to determine how organisms built from essentially the same set of proteins can be so different. This will require understanding how genes are regulated and alternatively spliced to define each organism’s developmental programs.

10. Manipulating genes and cells

Isolating cells and growing them in culture

Several approaches can be used to separate a particular type of cell from the cells that surround it in the body. If the cells are part of a compact tissue, they must first be dissociated from each other. This is often accomplished using proteolytic enzymes and other agent that disrupt the adhesive bonds between cells. Next, the different types of cells in the tissue must be isolated from each other. A fluorescence-activated cell sorter allows the isolation of specific types of cells. The isolated cells can be used for biochemical analysis or for establishing cell cultures.

Many animal and plant cells survive and proliferate in culture provided they have suitable medium containing nutrients and the necessary growth factor proteins. Experiments performed using cultured cells are said to be carried out in vitro (‘in glass’) to contrast them with experiments on intact organisms, which are said to be carried out in vivo (‘in the living’).

Most vertebrate cells cease to proliferate after a finite number of cell divisions. Like most human somatic cells, these cells do not express the enzyme telomerase, whose renew the ends of chromosomes at each cell division. As a result the chromosomes of human somatic cells progressively shrink at each cell division, and cell division stops when critical information is lost from the ends of chromosomes. This feature ensures that somatic cells do not divide indiscriminately and develop into cancerous cells. Cells that can divide indefinitely as the result of a genetic change are said to be immortalized and can be propagated in culture as a cell line. Immortalized cell lines can be regenerated by providing the cells with the gene that encodes the catalytic subunit of telomerase. The cell lines provide a convenient source of homogeneous cells.

Among the most promising cell lines to be developed are the human embryonic stem (ES) cell lines. The critical importance of these cell lines is the fact that the cells are undifferentiated; and given the appropriate treatment, they can give rise to any tissue in the body.

How DNA molecules are analyzed

Recombinant DNA technology has revolutionized the study of the cell, making it possible for researchers to pick any gene at will from the thousands of genes in a cell, and after an amplification step, to determine the exact molecular structure of the gene. A crucial element in this technology is the ability to cut a large DNA molecule into a specific and reproducible set of DNA fragments using restriction nucleases, each of which cuts the DNA double helix only at a particular nucleotide sequence. In general, a nuclease catalyzes the hydrolysis of a phosphodiester bond in a nuclei acid. The restriction nucleases used in DNA technology come mainly from bacteria.

After a large DNA molecule is cleaved into smaller pieces using restriction nuclease, it is often desirable to separate the DNA fragments from one another. This is usually accomplished using gel electrophoresis, which separates the fragments on the basis of their length. When a voltage is applied across the gel slab, the DNA fragments migrate toward the positive electrode (DNA is negatively charged); the larger fragments migrate more slowly and after several hours the DNA fragments become spread out across the gel according to size. Isolating a particular DNA fragment is simple: a small section of the gel can now be cut out. Techniques are now available for rapidly determining the nucleotide sequence of any isolated DNA fragment.

In 1970 researchers developed methods that allows the nucleotide sequence of any purified DNA fragment to be determined simply and quickly. These techniques have made it possible to determine the complete nucleotide sequences of the genomes of dozens of single-celled organisms (including bacteria, archaea, and yeasts), as well as several more complex organisms.

Several schemes for sequencing DNA have been developed, but the enzymatic or dideoxy method is the most commonly used technique. The process of interpreting a genome sequence by locating its genes and assigning functions to them is called annotation. Identifying genes is easiest when the DNA sequence is from a simple genome that lacks introns and other nonessential DNA.

Nucleic acid hybridization

We have seen that the two strands of a double helix DNA are held together by weak hydrogen bonds that can be broken by heating the DNA to around 90°C or by subjecting it to extremes of pH. These treatments release the two strands from each other but do not break the covalent bonds between the nucleotides. If this process is slowly reversed (slowly lowering the temperature to normal body temperature of by bringing the pH back to neutral), the complementary strands will readily re-form double helices. This process is called hybridization or renaturation, and its results from a restoration of the complementary hydrogen bonds.

Nucleic acid hybridization can detect any given DNA or RNA sequence in a mixture of nucleic acid fragments. This technique relies on the fact that a single strand of DNA or RNA will form a double helix only with another nucleic acid strand of the complementary nucleotide sequence. Single-stranded DNAs of known sequences and labeled with fluorescent dyes or radioisotopes are used as probes in hybridization reactions. Nucleic acid hybridization can be used to detect the precise location of genes in chromosomes, or RNAs in cells and tissues.

DNA hybridization facilitates the diagnosis of genetic diseases

To search for a nucleotide sequence by hybridization, a piece of nucleic acid is needed to search with. This DNA probe is a single-stranded DNA molecule that is used in hybridization reactions to detect nucleic acid molecules containing a complementary sequence. In the past, scientists were limited to using probed that could be obtained from natural sources. Today, short DNA strands of any sequence can be made by chemical (nonenzymatic) synthesis in the laboratory. Of the many uses of DNA probes, one of the most important is in identifying carriers of genetic diseases. More than 3000 different human genetic diseases are caused by mutations in single genes, including sickle-cell anemia. For some of these diseases, it is now possible to identify early in a pregnancy fetuses that carry two copies of a defective gene; this information may be the factor in decisions relating to possible termination of the pregnancy. A common laboratory procedure used to visualize the hybridization is called Soutern blotting.

The same techniques can also be used to ascertain an individual’s susceptibility to future diseases. For example, they can identify individuals who have inherited abnormal copies of a DNA mismatch repair gene.

Hybridization and microarrays

Another important use of nucleic acid hybridization is to determine, for a population of cells, exactly which genes are being transcribed into mRNA and which genes are transcriptionally silent. DNA microarrays have revolutioned they way of analyzing genes by allowing the RNA products of thousands of genes to be monitored at the same time. By examining the expression of so many genes simultaneously, it is possible to identify and study the complex gene expression patterns that underlie cellular physiology, like responses to hormones.

DNA cloning

DNA cloning techniques enable a DNA sequence to be selected from millions of other sequences and produced in unlimited amounts in pure form. DNA ligase reseals the nicks in the DNA backbone that arise during DNA replication and DNA repair. So, DNA fragments can be joined together in vitro using DNA ligase to form recombinant DNA molecules not found in nature.

The first step in a typical cloning procedure is to insert the DNA fragments to be cloned into a DNA molecule capable of replication, such as a plasmid or a viral genome. This recombinant DNA molecule is then introduced into a rapidly dividing host cell, usually a bacterium, so that the DNA is replicated at each cell division. The bacteria are then lysed, and the plasmid DNA is purified from the rest of the cell contents. The purified preparation of plasmid DNA will contain millions of copies of the original DNA fragment.

Human genes are isolated by DNA cloning

A collection of cloned fragments of chromosomal DNA representing the complete genome of an organism is known as a genomic library. The library is often maintained as clones of bacteria, each clone carrying a different DNA fragment.  Complementary DNA (cDNA) libraries contain cloned DNA copies of the total mRNA of a particular cell type or tissue. cDNA is synthesized from mRNA and unlike genomic DNA clones, cloned cDNAs contain only protein-coding sequences; they lack introns, gene regulatory sequences and promoters. They are thus most suitable for use when the cloned gene is to be expressed to make a protein.

The polymerase chain reaction

Cloning via DNA libraries was once the only route the gene isolation. However, a method known as the polymerase chain reaction (PCR) provides a quicker alternative for many cloning applications, particularly for those organisms whose complete genome sequence is known. PCR is based on the use of DNA polymerase to copy a DNA template in repeated rounds of replication. But because the oligonucleotide primers have to be chemically synthesized, PCR can be used only to clone DNA whose beginning and end sequences are known. Guided by these primers, DNA polymerase is then used to make many copies of the sequences required.
There are several useful applications of PCR:

  1. PCR is now the method of choice for cloning relatively short DNA fragments from a cell.

  2. PCR is able to detect infections by pathogens at very early stages. Short sequences complementary to the pathogen’s genome are used as primers, following many cycles of amplification, the presence or absence of even a few copies of an invading genome in a sample of blood can be ascertained. In this way PCR can be used to detect the presence of a viral genome in a sample of blood.

  3. PCR is used in forensic science. Its extreme sensitivity makes it possible to work with a very small sample and still obtain a DNA fingerprint of the person from whom it came. The genome of each human differs in DNA sequence from the genome of every other human; the DNA amplified by PCR using a particular primer pair is therefore quite likely to differ in sequence from one individual to another.

DNA engineering

Genetic engineering has far-reaching consequences. Bacteria, yeasts, and mammalian cells can be engineered to synthesize a particular protein from any organism in large quantities, thus making it possible to study proteins that are otherwise rare or difficult to isolate.

There are more than 10.000 human genes whose functions are unknown. Clues to a protein’s function can be obtained by examining when and where its gene is expressed in the cell or in the organism. Determining the pattern and timing of a gene’s expression can be accomplished by joining the regulatory region of the gene under study to a reporter gene, one whose activity can be easily monitored. One of the most popular reporter proteins used today is green fluorescent protein (GFP), whose allows the tracking of its movements inside the cell. In the case of GFP, the protein can be monitored over time in living organisms.

Animals can be genetically altered

The ultimate test of the function of a mutated gene is to insert it into the genome of an organism and see what effect it has. Organisms into which a new gene has been introduced, or those whose genomes have been altered in other ways using recombinant DNA techniques, are known as transgenic organisms. Several types of gene alterations can be made in genetically engineered organisms:

  1. Gene replacement: the normal gene is completely replaced by a mutant copy of the gene. This will provide information on the activity of the mutant gene, without interference from the normal gene, and thus the effect of small and subtle mutations can be determined

  2. Gene knockout: the normal gene is completely inactivated. This is used to obtain information on the possible function of the normal gene in the whole animal

  3. Gene addition: a mutant gene is added to the genome. Even this alteration can still provide useful information when the introduced mutant gene overrides the function of the normal gene

There is another way discovered to inactivate genes, known as RNA interference (RNAi). This technique relies on introducing intro a cell or organism a double stranded RNA molecule whose nucleotide sequence matches that of the gene to be inactivated. The RNA molecule hybridizes with the mRNA produced by the target gene en direct its degradation. Small fragments of this degraded RNA are subsequently used by the cell to produce more double-stranded RNA which directs the continued elimination of the target mRNA. Because these short RNA fragments can be passed on to progency cells, RNAi can cause heritable changes in gene expression.

In concluding, cloned genes can be permanently inserted into the genome of a cell or an organism by the techniques of genetic engineering. Cloned DNA can be altered in vitro to create mutant genes that can then be reinserted into a cell or an organism to study gene function.

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