Biotechnology Demystified

Biotechnology Demystified

Sharon Walker, Ph.D.

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DOI: 10.1036/0071448128

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This book is dedicated to Diane Nicholson and to Red

Cloud—they shaped my view of life and love.

ABOUT THE AUTHOR

Sharon Walker, Ph.D, is a Diplomat of the American Board of Toxicology (DABT) and has done extensive research in various areas of biomedicine. Dr. Walker has many years' experience as a safety analyst for high hazard operations, and has taught graduate courses in immunology, epidemiology, cell biology, and statistics.

ACKNOWLEDGMENTS

I gratefully acknowledge the substantial contribution of Bret Wing, who provided a thorough and thoughtful review of this book. I would also like to thank David McMahon who gave invaluable guidance during the development of this book. Finally, many thanks to Lee, Brad, and Lindsey for their encouragement, and to my Uncle Ed and Aunt Sarah for their support.

CONTENTS

Introduction

CHAPTER 1 Biomolecules and Energy

Review of Molecular Forces

Formation of Hydrogen Bonds

Building Blocks and Reagents: Lipids, Carbohydrates, Proteins, and Nucleic Acids

Energy

Summary

Quiz

CHAPTER 2 Cell Structures and Cell Division

The Barrier: Cell Membranes

The Genetic Material: The Nucleus

The Energy System: Mitochondria

Control of Cell Chemistry

Ribosomes

Endoplasmic Reticulum

Golgi Apparatus

Transporting Material and Waste Elimination

Cell Duplication—Mitosis

Sexual Reproduction—Meiosis

Summary

Quiz

CHAPTER 3 Information Methods of a Cell

History

Forming the Code

Coding for Proteins

Transcription

Mutations

Translation

The Rest of the DNA…Is It Junk?

One Gene, One Protein…NOT!

Control of Protein Production

Summary

Quiz

CHAPTER 4 Genetics

Mendelian Genetics

Sex-Linked Characteristics

Inheritance of Characteristics Controlled by More Than One Gene or Pair of Alleles

Mutations

Genetic Diseases

Influence of Environment

Gene Therapy

Summary

Quiz

CHAPTER 5 Immunology

Antigens

Antibodies

Structure of Antibodies

Cell-mediated Immunity

Immune Response to Proteins

Other Components of Immunity

Summary

Quiz

CHAPTER 6 Immunotherapy and Other Bioengineering Applications

Nonspecific Immune Stimulation

Antisera

Monoclonal Antibodies

Nanobodies

A Library of Antibodies

ELISA

Other Assays Using Monoclonal Antibodies

Autoimmune Disease

Cancer

Addictive Disorders

Allograft Rejection

Summary

Quiz

CHAPTER 7 Recombinant Techniques and Deciphering DNA

Recombinant Techniques

Deciphering DNA

Summary

Quiz

CHAPTER 8 Proteomics

One Gene, More Than One Protein

Structure of Proteins

Protein Folding and Misfolding

Proteolysis—Protein Breakdown

Reducing Protein Misfolding in Bioengineered Systems

Famous Misfolded Proteins

Research into Protein Type and Function

Summary

Quiz

Reference

CHAPTER 9 Stem Cells

Somatic Cells, Germ Cells, Stem Cells, Adult Stem Cells, and Embryonic Stem Cells

Adult Stem Cells

Embryonic Stem Cells

Somatic Cell Nuclear Transfer—Cloning

Controversy and Legal Constraints

Summary

Quiz

CHAPTER 10 Medical Applications

Recombinant DNA

Vaccines

Production of rDNA Proteins Using Bacteria, Yeast Cells, and Mammalian Cells

Products from Transgenic Animals

Transgenic Plants/Biofarms

The rDNA Protein Market

Alternatives for Protein Therapeutics—Upcoming Technology

Protein Therapeutics/Protein Misfolding

Antisense Technology and Prevention of Viral Infections

Use of Ribozymes in Viral Infections and Cancer Therapy

Forensics

DNA Sequencing

Detection of Bacteria, Viruses, and Fungi

Detection of Mutations

Summary

Quiz

References

CHAPTER 11 Agricultural Applications

Current GM Crops

Herbicide Tolerance

Insect Resistance

Pathogen Resistance

Nutritional Enhancements

Hardiness

Control of Seeds

Other Controversies Involving GM Crops

Future GM Crops

Bioengineering of Livestock

Feeding the World

Summary

Quiz

References

CHAPTER 12 Industrial and Environmental Applications

Use of Bioreagents in Industry

Finding Suitable Bioreagents

Modifying Enzymes and Molecule Sex

Inside or Outside of the Cell

Bioreactors

Environmental Applications—Hazardous Waste

Environmental Applications—Air Emissions

Summary

Quiz

References

CHAPTER 13 The Future

The Legal Dilemmas

Public Confidence

The Issue of Labeling

Genetic Pollution

Bioethics

Developing Nations

Worst Case Scenarios

Summary

Quiz

References

Final Exam

Answers to Quiz and Exam Questions

Index

INTRODUCTION

Dear Bioengineer to be—I wrote this book for you (and Pam and Raul illustrated this book for you). Personally, as I contemplate these subjects, I experience a sensation similar to the one I had when I watched Neil Armstrong step onto the lunar surface. I would describe it as a new vantage point; not of another point in space, but of our own world. It's another perspective—just like looking back toward Earth from a lunar orbit.

As you move into bioengineering, you may be overcome by a feeling that you have trespassed into forbidden territory just because of the enormous potential for change. Characteristics can and have jumped surrealistically among unrelated species. As you will learn, oil from palm trees can now be produced by rapeseed plants, and pig fetuses can glow like fireflies. We have our hands in the very clay of life. Life is no longer immutable, not only in the characteristics of a species but potentially real time, in the genetics of individuals.

Behind this subject is intense human drama. Some of the drama has reached the popular press, including the emotional debates over embryonic stem cells and the spectacular collapse of the Korean project that claimed to have developed cloned human cell lines. Some of the drama was behind the scenes, such as the race to decipher the human genome—a race between a huge, long-standing government project and an upstart private enterprise that had the audacity to claim the project could be compressed into much shorter time. Some of the drama is yet to come, as we face the potential of designer babies and production of super-athletes. Certainly a basic understanding of this technology and its potential should be a part of the education of all of us, because it is changing our world.

The subject of bioengineering is easier to follow if you have a basic background in science. However, the early chapters summarize the essential information on biological systems such that you should be able follow the descriptions of the technology. The intent was to give you enough of the basic terminology to enable you to understand the literature on this subject, without overwhelming you, dear reader, with foreign words.

This book is about enormous promise and unsettling risks. It involves unique ethical issues and questions about appropriate boundaries for commercial enterprises, if there are any such boundaries. It is about your world now and to come.

CHAPTER 1

Biomolecules and Energy

As a bioengineer, you will be using the systems, subsystems, and molecular units devised by nature towards your own ends. Of course, the more you understand about the natural way of things, the more effective you will be in using and modifying nature. One of the huge benefits reaped from the recent mushrooming of biotechnology initiatives is an unprecedented leap in the knowledge of cellular systems. Before we discuss the recent advances in our knowledge, let's review the basics.

Before you can begin, you must have some knowledge of what you have to work with. First, we need to do a quick review of molecular forces and then we'll describe the building blocks for constructing the most complex of all systems—a living organism.

Review of Molecular Forces

You will need a basic knowledge of how molecules interact to understand how cells function. We don't need to delve into chemistry very far, but you should understand how atoms stick together to form molecules. Biomolecules (molecules in biological systems) abide by the same physical laws as any other physical structure.

There are two ways that atoms can associate with one another to form molecules, through ionic or covalent bonds. To understand this review, you need to know that positively-charged protons exist in the nucleus of an atom, together with neutral neutrons. The negatively-charged electrons travel in shells around the nucleus. Atoms have the same number of electrons as protons, so the atom is neutrally charged. Simple enough.

For the discussion of the electron shells, envision the shells as a series of shelves that must be filled in a certain order. Each shell can accommodate a specific number of electrons. Hydrogen, for example, has one electron in the first shell that exists—the shell with the lowest possible energy state. (Nature seeks the lowest energy state available.) There is one electron because hydrogen has only one proton and the number of electrons in an atom equals the number of protons. The first shell needs to have two electrons before it is full; yes, it wants one more electron than it has protons. So, hydrogen will react easily with other molecules that want to give up an electron. Contrast the behavior of hydrogen with that of helium. Helium has two electrons. The electron shell is perfectly happy, and helium will not react with anything.

When the first shell is full, the next shell, with a higher energy level, receives the additional electrons. The next element is lithium, which has three protons and three electrons. The first shell has two electrons and is stable. However, that third electron in the outer shell wants a buddy badly, so lithium is highly reactive and will give up the third electron at the drop of a hat.

Let's use hydrogen and lithium to explain the difference between ionic and covalent bonds. Ionic bonds, shown in Figure 1-1, are formed when one atom gives up an electron to another atom. Lithium will donate its extra third electron to another atom that is short a full house, such as chlorine. When this reaction occurs, lithium assumes a positive charge because the atom has lost the negatively-charged electron, and chlorine receives a negative charge by picking up one more electron that it has positively-charged protons. If this reaction occurs while these atoms are in solution, the positively-charged and the negatively-charge chlorine will float around independently of one another. If you keep adding lithium and chlorine, eventually there are so many atoms that the water cannot keep them apart. The opposite charges will cause these atoms to associate with one another and form lithium chloride salt. The lithium chloride salt is a molecule; however, the forces keeping these two atoms together are not strong and can be easily disrupted by adding more water. And an atom or molecule with a stronger charge can definitely compete for one of the

Figure 1-1 Ionic bonds

partners. Table salt (sodium chloride) is a familiar example of a molecule that forms ionic bonds in this fashion.

Covalent bonds, shown in Figure 1-2, are formed when atoms share an electron; the electron actually belongs to both molecules. Covalent bonds are much stronger than ionic bonds. Let's return to our example of hydrogen. Hydrogen, as you recall, is one electron short of a full house. Two hydrogen atoms will share their electrons, forming the hydrogen gas, or H2. There are two protons, one per hydrogen atom, and two electrons, shared equally by these atoms.

Often, one atom has a stronger pull than the other, resulting in polar regions on the molecule. Consider water. Water is formed when oxygen picks up two electrons, one from each of two hydrogen atoms, and shares these electrons with the hydrogen atoms. However, the sharing is not equal, as shown in Figure 1-3.

Formation of Hydrogen Bonds

Hydrogen bonds are formed between the hydrogen in one molecule and an electronegative molecule in another molecule. Hydrogen tends to form molecules with elements that are strongly electronegative, meaning that the hydrogen bears a partial

Figure 1-2 Covalent bonds

Figure 1-3 Formation of poles in a covalently bonded molecule

positive charge. This positive charge causes the formation of hydrogen bonds. Water is the perfect example of hydrogen bonds.

Consider two water molecules coming close together, as in Figure 1-4.

The hydrogen is strongly attracted to the electronegative poles of the oxygen. Notice that oxygen has two poles, corresponding to the two shared electrons. Hydrogen bonds have about a tenth of the strength of an average covalent bond. They are easily broken and reformed. However, they are significantly stronger than an ordinary dipole-dipole (ionic) interaction. Each water molecule can potentially form four hydrogen bonds with surrounding water molecules. Water

Figure 1-4 Water and the formation of hydrogen bonds

forms a matrix as the dipoles form between the molecules. This is why water molecules appear as though they are trying to cluster together and push out anything in the way.

Biomolecules are formed through covalent bonds, but ionic bonds are important in their interactions and in the relationship between different sections of a given molecule. Hydrogen bonds are very important in the interactions of biomolecules. Many biomolecules literally bristle with hydrogen. Among other things, the formation of hydrogen bonds is a primary force in the way proteins fold in on themselves.

Building Blocks and Reagents: Lipids, Carbohydrates, Proteins, and Nucleic Acids

In general, you have four atoms to work with in forming biomolecules: carbon, oxygen, nitrogen, and hydrogen. (Note: some cellular molecules contain metals, and sulfur is frequently present in disulfide bonds.) These elements form only four types of materials to use in your design of this organism: lipids, carbohydrates, proteins, and nucleic acids. All of these materials build off of a backbone of carbon. Carbon is wonderful because it so readily picks up the other three molecules by forming covalent bonds. Each carbon atom can attach up to four of the other atoms or attach other carbon atoms as well. Carbons also will form a ring where the carbons each share one or two electrons. In the diagrams shown in Figure 1-5, the sharing of two electrons is depicted by a double line, whereas the sharing of only one electron is depicted by a single line.

A biomolecule typically consists of a number of similar units hooked together like a structure made of LEGOs®. The individual building unit is known as a monomer and the entire structure is a polymer. As we go through this discussion, remember that modular assembly is the norm. Polymers may consist of repeating units of the

Figure 1-5 The wonderful carbon atom

same material or the units may be unique. Sometimes a polymer will have a given function and knocking off one monomer unit will give it a different function.

There are four classes of biomolecules based on their structure and function—lipids, carbohydrates, proteins, and nucleic acids.

LIPIDS

Individual cells owe their form to the physical behavior of lipids, which are the only type of molecule identified only by its physical behavior. Lipids are hydrophobic, meaning that they do not dissolve in water (whereas hydrophilic molecules are water loving and can dissolve in water). This characteristic makes lipids ideal for the formation of membranes. Lipids form large polymers that provide a mechanism for high caloric-content storage, in the form of fats (Figure 1-6 shows an example fat molecule). Some lipids influence biochemical reactions in the cell; in other words, they are bioactive. An example is cholesterol.

Figure 1-6 Example fat molecule: phosphatidylcholine (Based on Figure 2-7 of Schaum's Outlines Molecular and Cell Biology by William D. Stansfield, Jaime S. Colome, and Raul J. Cano, McGraw-Hill, 1996)

Lipids are the primary constituents of the cell membrane. See Chapter 2 for a discussion on how the hydrophobic nature of lipids results in the cell membrane structure.

CARBOHYDRATES

Carbohydrates are the most important source of energy for living organisms and have a formula that is some multiple of CH2O. For all carbohydrates, if you remove water, only a carbon remains. For multicellular organisms, such as us, the fundamental energy source is a five-carbon sugar known as glucose, a small carbohydrate monomer. If the small monomers are stacked up into large polymers, forming complex carbohydrates (see Figure 1-7 for an example), the resulting molecules can provide structural units. Examples are cellulose and collagen. Other complex carbohydrates (composed of large polymers containing many similar monomers hooked together) are used for energy storage. The carbohydrate energy storage molecules are starch in plants and glycogen in animals. This energy storage form has a lower caloric content than fat but is easier to access.

PROTEINS

Lipids and carbohydrates tend to form large, monotonous polymers. Proteins become much more interesting. In fact, proteins are so interesting and so diverse that it was difficult to convince researchers in the mid-1900s that proteins were not the information storage molecules for the organism. (We now know that nucleic acids are the information storage molecules.) Proteins form some structures; however, the most important function of proteins is to orchestrate the biochemical reactions that occur in the cell.

Figure 1-7 Example complex carbohydrate (Based on Figure 2-6 of Schaum's Outlines Molecular and Cell Biology by William D. Stansfield, Jaime S. Colome, and Raul J. Cano, McGraw-Hill, 1996)

Figure 1-8 Examples of amino acids

Proteins are made of amino acids, examples of which are shown in Figure 1-8. These are distinct from carbohydrates and fats because they contain nitrogen. All proteins contain a backbone structure with two carbon atoms and a nitrogen atom. The nitrogen has two hydrogen atoms attached, and the carbon on the end has an oxygen molecule and a hydroxyl group (OH) attached. The middle carbon provides the diversity among proteins because, theoretically, almost anything could be attached. In practice, there are only 20 amino acids, 12 of which the human body can manufacture. The other eight we must consume and are called essential amino acids.

Amino acids link together by forming a peptide bond, as shown in Figure 1-9. Note that the formation of the bond involves the atoms at the end but leaves that middle carbon atom to retain the characteristic side chains. Protein molecules are large, with the average one containing about 300 amino acids. With 20 amino acids available per position, there are thousands of different proteins that could be formed from 300 amino acids.

The protein is formed as a long chain with numerous side chains, which may be hydrophobic or hydrophilic. The side chains may attract one another by electrical charge or may react chemically. Hydrogen bonds form between side chains. Depending on which side chains are present and in what order, the molecule folds in on itself to form a particular shape. Proteins have a primary structure (linear), and as the molecule begins to fold in on itself, a secondary, tertiary, and a final shape form. This shape is important in allowing the protein to carry out its particular function. Chapter 8 will discuss protein metabolism (the production, use, and destruction of

Figure 1-9 Peptide bond (Based on Figure 2-9 of Schaum's Outlines Molecular and Cell Biology by William D. Stansfield, Jaime S. Colome, and Raul J. Cano, McGraw-Hill, 1996)

proteins) in detail because of proteins' importance in cell function and the potential for a bioengineer to intervene in the way proteins are handled by the organism.

NUCLEIC ACIDS

Nucleic acids were discovered in 1868 by Friederich Miescher. The name nucleic acid is of historic derivation; nucleic acids are simply acidic molecules that early scientists found in cell nuclei. The discussion of the structure of nucleic acids tends to be confusing because the most important part of the structure is composed of organic bases. There are five of these bases, four found in deoxyribose nucleic acid (DNA) and a fifth found in ribose nucleic acid (RNA). The four bases in DNA are adenine, guanine, thymine, cytosine, and the fifth base found in RNA is uracil. Although these names mean little, it is best to familiarize yourself with them because four of them (excluding uracil) are the all-important code of life. Using information encoded in these four compounds, all structures found in living organisms can be constructed. A complete explanation of how this is done is provided in Chapter 3. In that chapter, we will actually break the code—the instruction set for all life forms on earth.

The bases are attached to a sugar. The five-carbon sugar is ribose, for RNA. A similar sugar, except that there is one less oxygen, is deoxyribose, for DNA. When a base is linked with the sugar, the resulting molecule is known as a nucleoside. The sugar picks up a phosphate on the fifth carbon, which makes the whole thing acidic. At this point, the molecule is known as a nucleotide. If you can only remember one of these names, remember nucleotide—base and phosphorylated sugar. The sugars link together when an oxygen molecule from the phosphate group on one sugar bonds with a carbon molecule on another sugar. This is called a phosphodiester linkage. The second sugar is phosphorylated and links with a third sugar, and so on. The linkage of the phosphorylated sugars results in a scaffolding that support the organic bases. The sugar structure forms the railing for the familiar spiral staircase that is DNA. The bases stick to the inside, forming the steps of the staircase.

Let's build it, as shown in Figures 1-10 through 1-13. First, Figure 1-10 shows your sugar. Figure 1-11 adds a phosphate group. Figure 1-12 then links the sugars together, and finally, Figure 1-13 takes a base and attaches it to the sugar.

Figure 1-10 Ribose and deoxyribose

Figure 1-11 Phosphorylated deoxyribose

Figure 1-12 The scaffolding for DNA

Figure 1-13 The organic bases

Again, as the phosphorylated sugars form the backbone of the molecule, the bases stick to the inside like steps in a staircase, as in Figure 1-14. You need to remember that most DNA is an association of two strands, so there is scaffolding on the right and left and the bases form pair on the inside.

Like most of the phenomena we have discussed, the success of this operation depends on the operatives fitting together physically. Remember that these molecules