Biotechnology

By David P. Clark and Nanette J. Pazdernik

Biotechnology, Second Edition approaches modern biotechnology from a molecular basis, which has grown out of increasing biochemical understanding of genetics and physiology. Using straightforward, less-technical jargon, Clark and Pazdernik introduce each chapter with basic concepts that develop into more specific and detailed applications. This up-to-date text covers a wide realm of topics including forensics, bioethics, and nanobiotechnology using colorful illustrations and concise applications. In addition, the book integrates recent, relevant primary research articles for each chapter, which are presented on an accompanying website. The articles demonstrate key concepts or applications of the concepts presented in the chapter, which allows the reader to see how the foundational knowledge in this textbook bridges into primary research. This book helps readers understand what molecular biotechnology actually is as a scientific discipline, how research in this area is conducted, and how this technology may impact the future.

• Up-to-date text focuses on modern biotechnology with a molecular foundation
• Includes clear, color illustrations of key topics and concept
• Features clearly written without overly technical jargon or complicated examples
• Provides a comprehensive supplements package with an easy-to-use study guide, full primary research articles that demonstrate how research is conducted, and instructor-only resources

• Language: English
• Publisher: Elsevier Science
• Release date: May 16, 2015
• ISBN: 9780123850164

David P. Clark

David P. Clark did his graduate work on bacterial antibiotic resistance to earn his Ph.D. from Bristol University, England. He later crossed the Atlantic to work as a postdoctoral researcher at Yale University and then the University of Illinois. Dr Clark recently retired from teaching Molecular Biology and Bacterial Physiology at Southern Illinois University which he joined in 1981. His research into the Regulation of Alcohol Fermentation in E. coli was funded by the U.S. Department of Energy, from 1982 till 2007. In 1991 he received a Royal Society Guest Research Fellowship to work at Sheffield University, England while on sabbatical leave.

Book preview

Biotechnology

Second Edition

David P. Clark

Department of Microbiology, Southern Illinois University, Carbondale, Illinois, USA

Nanette J. Pazdernik

Washington University School of Medicine, St. Louis, Missouri, USA

Table of Contents

Cover image

Title page

Copyright

Online Study Guide

Dedication

Academic Cell

Preface

Acknowledgments

Introduction

Chapter 1. Basics of Biotechnology

Advent of the Biotechnology Revolution

Chemical Structure of Nucleic Acids

Packaging of Nucleic Acids

Bacteria as the Workhorses of Biotechnology

Escherichia coli Is the Model Bacterium

Many Bacteria Contain Plasmids

Other Bacteria in Biotechnology

Basic Genetics of Eukaryotic Cells

Yeast and Filamentous Fungi in Biotechnology

Yeast Mating Types and Cell Cycle

Multicellular Organisms as Research Models

Animal Cell Culture in Vitro

Arabidopsis thaliana, a Model Flowering Plant

Viruses Used in Genetics Research

Subviral Infectious Agents and Other Gene Creatures

Summary

Chapter 2. DNA, RNA, and Protein

The Central Dogma of Molecular Biology

Transcription Expresses Genes

Making RNA

Transcription Stop Signals

The Number of Genes on an mRNA Varies

Eukaryotic Transcription is More Complex

Regulation of Transcription in Prokaryotes

Regulation of Transcription in Eukaryotes

Eukaryotic mRNA is Processed Before Making Protein

Translating the Genetic Code into Proteins

Differences between Prokaryotic and Eukaryotic Translation

Mitochondria and Chloroplasts Synthesize Their Own Proteins

Summary

Chapter 3. Recombinant DNA Technology

DNA Isolation and Purification

Electrophoresis Separates DNA Fragments by Size

Restriction Enzymes Cut DNA; Ligase Joins DNA

Methods of Detection for Nucleic Acids

Complementary Strands Melt Apart and Reanneal

Hybridization of DNA or RNA in Southern and Northern Blots

Fluorescence in Situ Hybridization (FISH)

General Properties of Cloning Vectors

Specific Types of Cloning Vectors

Getting Cloned Genes into Bacteria by Transformation

Constructing a Library of Genes

Screening the Library of Genes by Hybridization

Eukaryotic Expression Libraries

Features of Expression Vectors

Recombineering Increases the Speed of Gene Cloning

Gateway® Cloning Vectors

Summary

Chapter 4. DNA Synthesis In Vivo and In Vitro

Introduction

Replication of DNA

Comparing Replication in Gene Creatures, Prokaryotes, and Eukaryotes

In Vitro DNA Synthesis

Chemical Synthesis of DNA

Chemical Synthesis of Complete Genes

Polymerase Chain Reaction Uses in Vitro Synthesis to Amplify Small Amounts of DNA

Modifications of Basic PCR

Reverse Transcriptase PCR

PCR in Genetic Engineering

PCR of DNA can Determine the Sequence of Bases

Next-Generation Sequencing Technologies

Summary

Chapter 5. RNA-Based Technologies

Noncoding RNA Plays Many Roles

RNA Coordinates Genomic Integrity in Eukaryotes

RNA Protects Genomes from Invading Viruses

RNA Modulates Transcription

Noncoding RNAs Take Part in RNA Processing

Riboswitches are Controlled by Effector Molecules

RNA Catalyzes Enzyme Reactions

Summary

Chapter 6. Immune Technology

Introduction

Antibodies, Antigens, and Epitopes

The Great Diversity of Antibodies

Structure and Function of Immunoglobulins

Monoclonal Antibodies for Clinical Use

Humanization of Monoclonal Antibodies

Humanized Antibodies in Clinical Applications

Antibody Engineering

Diabodies and Bispecific Antibody Constructs

ELISA Assay

The ELISA as a Diagnostic Tool

Visualizing Cell Components using Antibodies

Fluorescence-Activated Cell Sorting

Immune Memory and Vaccination

Creating a Vaccine

Making Vector Vaccines using Homologous Recombination

Reverse Vaccinology

Identifying New Antigens for Vaccines

DNA Vaccines Bypass the need to Purify Antigens

Edible Vaccines

Summary

Chapter 7. Nanobiotechnology

Introduction

Visualization at the Nanoscale

Scanning Tunneling Microscopy

Atomic Force Microscopy

Weighing Single Bacteria and Virus Particles

Nanoparticles and Their Uses

Nanoparticles for Labeling

Quantum Size Effect and Nanocrystal Colors

Nanoparticles for Delivery of Drugs, DNA, or RNA

Nanoparticles in Cancer Therapy

Assembly of Nanocrystals by Microorganisms

Nanotubes

Antibacterial Nanocarpets

Detection of Viruses by Nanowires

Ion Channel Nanosensors

Nanoengineering of DNA

DNA Origami

DNA Mechanical Nanodevices

Controlled Denaturation of DNA by Gold Nanoparticles

Controlled Change of Protein Shape by DNA

Biomolecular Motors

Summary

Chapter 8. Genomics and Gene Expression

Introduction

Genetic Mapping Techniques

Gaps Remain in the Human Genome

Survey of the Human Genome

Noncoding Components of the Human Genome

Bioinformatics and Computer Analysis

Medicine and Genomics

DNA Accumulates Mutations over Time

Genetic Evolution

From Pharmacology to Pharmacogenetics

Gene Expression and Microarrays

Making DNA Microarrays

Hybridization ON DNA Microarrays

Monitoring Gene Expression Using Whole-Genome Tiling Arrays

Monitoring Gene Expression by RNA-Seq

Monitoring Gene Expression of Single Genes

Epigenetics and Epigenomics

Epigenomics in Higher Organisms

Summary

Chapter 9. Proteomics

Introduction

Gel Electrophoresis of Proteins

Western Blotting of Proteins

High-Pressure Liquid Chromatography Separates Protein Mixtures

Digestion of Proteins by Proteases

Mass Spectrometry for Protein Identification

Preparing Proteins for Mass Spectroscopy

Protein Quantification Using Mass Spectrometry

Protein Tagging Systems

Phage Display Library Screening

Protein Interactions: The Yeast Two-Hybrid System

Protein Interactions by Co-immunoprecipitation

Protein Arrays

Metabolomics

Summary

Chapter 10. Recombinant Proteins

Proteins and Recombinant DNA Technology

Expression of Eukaryotic Proteins in Bacteria

Insulin and Diabetes

Cloning and Genetic Engineering of Insulin

Translation Expression Vectors

Codon Usage Effects

Avoiding Toxic Effects of Protein Overproduction

Inclusion Bodies and Protein Refolding

Increasing Protein Stability

Improving Protein Secretion

Protein Fusion Expression Vectors

Protein Glycosylation

Expression of Proteins by Eukaryotic Cells

Expression of Proteins by Yeast

Expression of Proteins by Insect Cells

Expression of Proteins by Mammalian Cells

Expression of Multiple Subunits in Mammalian Cells

Comparing Expression Systems

Summary

Chapter 11. Protein Engineering

Introduction

Engineering Disulfide Bonds

Improving Stability In Other Ways

Changing Binding Site Specificity

Structural Scaffolds

Directed Evolution

Recombining Domains

DNA Shuffling

Combinatorial Protein Libraries

Creation of De Novo Proteins

Expanding the Genetic Code

Roles of Non-Natural Amino Acids

Biomaterials Design Relies on Protein Engineering

Engineered Binding Proteins

Summary

Chapter 12. Environmental Biotechnology

Introduction

Identifying New Genes with Metagenomics

Culture Enrichment for Environmental Samples

Sequence-Dependent Techniques for Metagenomics

Function- or Activity-Based Evaluation of The Environment

Ecology and Metagenomics

Natural Attenuation of Pollutants

Biofuels and Bioenergy

Microbial Fuel Cells

Summary

Chapter 13. Synthetic Biology

Introduction

Ethanol, Elephants, and Pathway Engineering

Degradation of Starch

Degradation of Cellulose

Second-Generation Biofuels

Biodiesel

Ice-Forming Bacteria and Frost

Biorefining of Fossil Fuels

Biosynthesis of β-Lactam Antibiotics

Biosynthetic Plastics are Also Biodegradable

The Integrated Circuits Approach

Synthetic Genetic Materials: xDNA And XNA

Designer Bacteria

Summary

Chapter 14. From Cell Phones to Cyborgs

Introduction

Cell Phones

Robotics

Radio-Controlled Genes

Insect Cyborgs

Soft Robotics

Summary

Chapter 15. Transgenic Plants and Plant Biotechnology

Introduction

History of Plant Breeding

Plant Tissue Culture

Genetic Engineering of Plants

Biotechnology Improves Crops

Resistance: Nature Responds to Transgenic Plants

Functional Genomics in Plants

Summary

Chapter 16. Transgenic Animals

New and Improved Animals

Creating Transgenic Animals

Larger Mice Illustrate Transgenic Technology

Recombinant Protein Production Using Transgenic Livestock

Knockout Mice for Medical Research

Alternative Ways to Make Transgenic Animals

Location Effects on Expression of the Transgene

Deliberate Control of Transgene Expression

Gene Control by Site-Specific Recombination

Transgenic Insects

Practical Transgenic Animals

Applications of RNA Technology in Transgenics

Natural Transgenics and DNA Ingestion

Summary

Chapter 17. Inherited Defects and Gene Therapy

Introduction

Hereditary Defects in Higher Organisms

Hereditary Defects Due to Multiple Genes

Defects Due to Haploinsufficiency

Dominant Mutations may be Positive or Negative

Deleterious Tandem Repeats and Dynamic Mutations

Defects in Imprinting and Methylation

Mitochondrial Defects

Identification of Defective Genes

Genetic Screening and Counseling

General Principles of Gene Therapy

Adenovirus Vectors in Gene Therapy

Cystic Fibrosis

Cystic Fibrosis Gene Therapy

Retrovirus Gene Therapy

Retrovirus Gene Therapy for Scid

Adeno-Associated Virus

Nonviral Delivery in Gene Therapy

Liposomes and Lipofection in Gene Therapy

Aggressive Gene Therapy for Cancer

Using RNA in Therapy

Antisense RNA and Other Oligonucleotides

Aptamers—Blocking Proteins with DNA or RNA

Ribozymes in Gene Therapy

RNA Interference in Gene Therapy

Gene Editing with Nucleases

Genome Editing with Engineered Nucleases

Genome Editing with CRISPR Nucleases

Summary

Chapter 18. Cloning and Stem Cells

Introduction

What is a Stem Cell?

Identifying Adult Stem Cells

The Key Features of a Stem Cell Niche

Hematopoietic Stem Cells in the Bone Marrow

Intestinal Epithelial Stem Cells

Induced Pluripotent Stem Cells

Stem Cell Therapy

Somatic Cell Nuclear Transfer

Dolly the Cloned Sheep

Practical Reasons for Cloning Animals

Improving Livestock by Pathway Engineering

Imprinting and Developmental Problems in Cloned Animals

Summary

Chapter 19. Cancer

Cancer is Genetic in Origin

Environmental Factors and Cancer

Normal Cell Division: The Cell Cycle

Cellular Communication

Receptors and Signal Transmission

Cell Division Responds to External Signals

Genes that Affect Cancer

Oncogenes and Proto-Oncogenes

Detection of Oncogenes by Transformation

Types of Mutations that Generate Oncogenes

The RAS Oncogene—Hyperactive Protein

The MYC Oncogene—Overproduction of Protein

Tumor-Suppressor Genes or Anti-Oncogenes

The p16, p21, and p53 Anti-Oncogenes

Formation of a Tumor

Inherited Susceptibility to Cancer

Cancer-Causing Viruses

Engineered Cancer-Killing Viruses

Cancer Genomics

Cancer Epigenomics

Micro RNA Regulation and Cancer

Anticancer Agents

Summary

Chapter 20. Aging and Apoptosis

Introduction

Genetic Phenomena Associated with Aging

Cellular Dysfunction and Aging

Cellular Senescence

Programmed Cell Death

Apoptosis Involves a Proteolytic Cascade

Mammalian Apoptosis

Caspases

Execution Phase of Apoptosis

Corpse Clearance in Apoptosis

Control of Apoptotic Pathways in Development

Necroptosis

Metabolic Control of Cell Death

Cancer, Aging, and Programmed Cell Death

Programmed Cell Death in Bacteria

Summary

Chapter 21. Viral and Prion Infections

Viral Infections and Antiviral Agents

Interferons Coordinate the Antiviral Response

Antiviral Therapy using RNA Interference

Influenza is a Negative-Strand Rna Virus

The AIDS Retrovirus

Chemokine Receptors Act as Co-Receptors for Hiv

Treatment of the AIDS Retrovirus

Infectious Prion Disease

Detection of Pathogenic Prions

Approaches to Treating Prion Disease

Prions in Yeast

Using Yeast Prions as Models

Amyloid Proteins in Neurological Diseases

Summary

Chapter 22. Biological Warfare: Infectious Disease and Bioterrorism

Introduction

The Natural History of Biological Warfare

Microbes Versus Man: The Rise of Antibiotic Resistance

A Brief History of Human Biological Warfare

Identifying Suitable Biological Warfare Agents

A Closer Look at Select Biological Warfare Agents

Enhancing Biological Warfare Agents with Biotechnology

Detection of Biological Warfare Agents

Summary

Chapter 23. Forensic Molecular Biology

The Genetic Basis of Identity

Blood, Sweat, and Tears

Forensic DNA Testing

DNA Fingerprinting

Using Repeated Sequences in Fingerprinting

Probability and DNA Testing

The Use of DNA Evidence

DNA is Also Used to Identify Animals

Tracing Genealogies by Mitochondrial DNA and the Y Chromosome

Identifying the Remains of the Russian Imperial Family

Gene Doping and Athletics

Genomics Drives Advances in Forensics

Summary

Chapter 24. Bioethics in Biotechnology

Introduction

Principles of Bioethics

Use of the Precautionary Principle

The Power of Information

Possible Dangers to Health from Biotechnology

Genetically Modified Organisms

Human Enhancement, Cloning, and Engineering

Ethics Changes over Time

Summary

Glossary

Index

Copyright

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Notices

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Online Study Guide

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An Online Study Guide is now available with your textbook, containing a relevant journal article with a case study to focus understanding and discussion about each chapter.

1. To access the Online Study Guide, as well as other online resources for the book, please visit: http://booksite.elsevier.com/9780123850157

2. For instructor-only materials, please visit: http://textbooks.elsevier.com/web/Manuals.aspx?isbn=9780123850157

Dedication

This book is dedicated to Donna. —DPC

This book is dedicated to my children and husband. Their patience and understanding have given me the time and inspiration to research and write this text. —NJP

Academic Cell

How We Got Here

In speaking with professors across the biological sciences and going to conferences, we, the editors at Academic Press and Cell Press, saw how often journal content was being incorporated in the classroom. We understood the benefits students were receiving by being exposed to journal articles early: to add perspective, improve analytical skills, and bring the most current content into the classroom. We also learned how much additional preparation time was required on the part of instructors finding the articles, then obtaining the images for presentations and providing additional assessment.

So we collaborated to offer instructors and students a solution, and Academic Cell was born. We offer the benefits of a traditional textbook (to serve as a reference to students and a framework to instructors), but we also offer much more. With the purchase of every copy of an Academic Cell book, students can access an online study guide containing relevant, recent Cell Press articles and providing bridge material in the form of a case study to help ease them into the articles. In addition, the images from the articles are available as zipped .jpg files and we have optional test bank questions.

We plan to expand this initiative, as future editions will be further integrated with unique pedagogical features incorporating current research from the pages of Cell Press journals into the textbook itself.

Preface

From the simple acts of brewing beer and baking bread has emerged a field now known as biotechnology. Over the ages the meaning of the word biotechnology has evolved along with our growing technical knowledge. Biotechnology began by using cultured microorganisms to create a variety of food and drinks, despite its early practitioners not even knowing of the existence of the microbial world. Today, biotechnology is still defined as any application of living organisms or bioprocesses to create new products. Although the underlying idea is unchanged, the use of genetic engineering and other modern scientific techniques has revolutionized the area.

The fields of genetics, molecular biology, microbiology, and biochemistry are merging their respective discoveries into the expanding applied field of biotechnology, and advances are occurring at a record pace. Two or three years of research can dramatically alter the approaches that are of practical use. For example, the simple discovery that double-stranded RNA can block expression of any gene with a matching sequence has revolutionized how we study and apply genetic interactions in less than a ten-year period.

This rapid increase in knowledge is very hard to incorporate into a textbook format, and often instructors who teach advanced molecular biology classes rely on the primary research to teach students novel concepts and applications. This type of teaching is difficult and requires many hours to plan and organize.

The new partnership between Academic Press and Cell Press has adopted a solution to teaching advanced molecular biology and biotechnology courses. The partnership combines years of textbook publishing experience with the most relevant and high impact research. What has emerged is a new teaching paradigm. In Biotechnology, the basic ideas and methodologies are explained using very clear and concise language. These techniques are supplemented with a wide variety of diagrams and illustrations to simplify the complex biotechnology processes.

These basics are then supported with a Biotechnology online study guide that not only tests the student’s knowledge of the textbook chapter, but also contains primary research articles. The articles are chosen from the Cell Press family of journals, which includes such high-impact journals as Cell, Molecular Cell, and Current Biology. The articles expand upon a topic presented in each chapter or provide an exemplary research paper for that particular chapter. The entire full-color research article is included online.

In addition to the article itself, the Biotechnology study guide includes a synopsis of the research paper. The synopsis includes a thorough discussion of the relevant background information. This material is often absent from primary research articles because their authors assume that readers are also experts. Then each synopsis breaks the paper into sections, explaining each individual experiment separately. Each experiment is explained by defining the underlying hypothesis or question, the methods used to study the question, and the results. The final section of the synopsis provides the overall conclusions for the paper. This approach reinforces the basic scientific method. The instructor does not have to find an article, create a presentation on the background, and then work with the student to explain each of the methods and results. The study guide synopsis provides all of this information already.

The online format ensures that only the most recent papers are associated with the chapter. The combination of the online study guide with the newest relevant research and a solid basic textbook provides the instructor with the best of both worlds. You can teach students the basic concepts using the textbook, and then use the relevant research paper to stretch the student’s knowledge of current research in the field of biotechnology.

Acknowledgments

We would like to thank the following individuals for their help in providing information, suggestions for improvement, and encouragement: Laurie Achenbach, Rubina Ahsan, Phil Cunningham, Donna Mueller, Dan Nickrent, Holly Simmonds, and Dave Pazdernik. Special thanks go to Marshall Spector for helping us understand bioethics, to Michelle McGehee for writing the questions and online supplements and to Karen Fiorino for creating most of the original artwork for the first edition. Alex Berezow was responsible for writing a major part of the following chapters: Chapter 16, Transgenic animals, Chapter 22, Biowarfare and bioterrorism, and Chapter 24, Bioethics in biotechnology.

Introduction

Modern Biotechnology Relies on Advances in Molecular Biology and Computer Technology

Traditional biotechnology goes back thousands of years. It includes the selective breeding of livestock and crop plants as well as the invention of alcoholic beverages, dairy products, paper, silk, and other natural products. Only in the past couple of centuries has genetics emerged as a field of scientific study. Recent rapid advances in this area have in turn allowed the breeding of crops and livestock by deliberate genetic manipulation rather than trial and error. The so-called green revolution of the period from 1960 to 1980 applied genetic knowledge to natural breeding and had a massive impact on crop productivity in particular. Today, plants and animals are being directly altered by genetic engineering.

New varieties of several plants and animals have already been made, and some are in agricultural use. Animals and plants used as human food sources are being engineered to adapt them to conditions that were previously unfavorable. Farm animals that are resistant to disease and crop plants that are resistant to pests are being developed in order to increase yields and reduce costs. The impact of these genetically modified organisms on other species and on the environment is presently a controversial issue.

Modern biotechnology applies not only modern genetics but also advances in other sciences. For example, dealing with vast amounts of genetic information depends on advances in computing power. Indeed, the sequencing of the human genome would have been impossible without the development of ever more sophisticated computers and software. It is sometimes claimed that we are in the middle of two scientific revolutions, one in information technology and the other in molecular biology. Both involve handling large amounts of encoded information. In one case the information is human made, or at any rate man-encoded, and the mechanisms are artificial; the other case deals with the genetic information that underlies life.

However, there is a third revolution that is just emerging—nanotechnology. The development of techniques to visualize and manipulate atoms individually or in small clusters is opening the way to an ever-finer analysis of living systems. Nanoscale techniques are now beginning to play significant roles in many areas of biotechnology.

This raises the question of what exactly defines biotechnology. To this there is no real answer. A generation ago, brewing and baking would have been viewed as biotechnology. Today, the application of modern genetics or other equivalent modern technology is usually seen as necessary for a process to count as biotechnology. Thus, the definition of biotechnology has become partly a matter of fashion. In this book, we regard (modern) biotechnology as resulting in a broad manner from the merger of classical biotechnology with modern genetics, molecular biology, computer technology, and nanotechnology.

The resulting field is of necessity large and poorly defined. It includes more than just agriculture: it also affects many aspects of human health and medicine, such as vaccine development and gene therapy. We have attempted to provide a unified approach that is based on genetic information, while at the same time indicate how biotechnology has begun to sprawl, often rather erratically, into many related fields of human endeavor.

Chapter 1

Basics of Biotechnology

Abstract

Biotechnology involves the use of living organisms in industrial processes—particularly in agriculture, food processing, and medicine. Biotechnology has been around ever since humans began manipulating the natural environment to improve their food supply, housing, and health. Biotechnology is not limited to humankind. Beavers cut up trees to build homes. Elephants deliberately drink fermented fruit to get an alcohol buzz. People have been making wine, beer, cheese, and bread for centuries. For wine, the earliest evidence of wine production has been dated to c. 6000 BC. All these processes rely on microorganisms to modify the original ingredients. Ever since the beginning of human civilization, farmers have chosen higher yielding crops by trial and error, so that many modern crop plants have much larger fruit or seeds than their ancestors.

Keywords

bacteriocins; bacteriophage; deoxyribonucleic acid; DNA polymerase; double helix; early genes; gene creatures; germline; human immunodeficiency virus; immunity protein; integrase; long terminal repeats; matrix attachment regions; mobile DNA; nucleosome; nucleotides; phosphate group; polymerase chain reaction; principle of independent assortment; principle of segregation; provirus; retroviruses; ribonucleic acid; stem cell; target sequence; virion

Advent of the Biotechnology Revolution

Chemical Structure of Nucleic Acids

Caenorhabditis elegans, a Small Roundworm

Bacteria as the Workhorses of Biotechnology

Escherichia coli Is the Model Bacterium

Many Bacteria Contain Plasmids

Other Bacteria in Biotechnology

Basic Genetics of Eukaryotic Cells

Yeast and Filamentous Fungi in Biotechnology

Yeast Mating Types and Cell Cycle

Multicellular Organisms as Research Models

Caenorhabditis elegans, a Small Roundworm

Drosophila melanogaster, the Common Fruit Fly

Zebrafish Are Models for Developmental Genetics

Mus musculus, the Mouse, Is Genetically Similar to Humans

Animal Cell Culture in Vitro

Arabidopsis thaliana, a Model Flowering Plant

Viruses Used in Genetics Research

Subviral Infectious Agents and Other Gene Creatures

Advent of the Biotechnology Revolution

Biotechnology involves the use of living organisms in industrial processes—particularly in agriculture, food processing, and medicine. Biotechnology has been around ever since humans began manipulating the natural environment to improve their food supply, housing, and health. Biotechnology is not limited to humankind. Beavers cut up trees to build homes. Elephants deliberately drink fermented fruit to get an alcohol buzz. People have been making wine, beer, cheese, and bread for centuries (Fig. 1.1). For wine, the earliest evidence of wine production has been dated to c. 6000 BC. All these processes rely on microorganisms to modify the original ingredients. Ever since the beginning of human civilization, farmers have chosen higher yielding crops by trial and error, so that many modern crop plants have much larger fruit or seeds than their ancestors (Fig. 1.2).

FIGURE 1.1  Traditional Biotechnology Products

Bread, cheese, wine, and beer have been made worldwide using microorganisms such as yeast. Photo taken by Karen Fiorino, Clay Lick Creek Pottery, IL, USA.

FIGURE 1.2  Teosinte versus Modern Corn

Since early civilization, people have improved many plants for higher yields. Teosinte (smaller cob and green seeds) is considered the ancestor of commercial corn (larger cob; a blue-seeded variety is shown). Courtesy of Wayne Campbell, Hila Science Camp.

We think of biotechnology as modern because of recent advances in molecular biology and genetic engineering. Huge strides have been made in our understanding of microorganisms, plants, livestock, as well as the human body and the natural environment. This has caused an explosion in the number and variety of biotechnology products. Face creams contain antioxidants—supposedly to fight the aging process. Genetically modified plants have genes inserted to protect them from insects, thus increasing the crop yield while decreasing the amount of insecticides used. Medicines are becoming more specific and compatible with our physiology. For example, insulin for diabetics is now genuine human insulin, although produced by genetically modified bacteria. Almost everyone has been affected by the recent advances in genetics and biochemistry.

Mendel’s early work that described how genetic characteristics are inherited from one generation to the next was the beginning of modern genetics (see Box 1.1). Next came the discovery of the chemical material of which genes are made—DNA (deoxyribonucleic acid). This in turn led to the central dogma of genetics: the concept that genes made of DNA are expressed as an RNA (ribonucleic acid) intermediary that is then decoded to make proteins. These three steps are universal, applying to every type of living organism on earth. Yet these three steps are so malleable that life is found in almost every available niche on our planet.

Biotechnology affects all of our lives and has altered everything we encounter in life.

FIGURE A  Relationship of Genotype and Phenotype

(A) Each parent has two alleles, either two yellow or two green. Any offspring will be heterozygous, each having a yellow and a green allele. Since the yellow allele is dominant, the peas look yellow. (B) When the heterozygous F1 offspring self-fertilize, the green phenotype re-emerges in one-fourth of the F2 generation.(B) When the heterozygous F1 offspring self-fertilize, the green phenotype re-emerges in one-fourth of the F2 generation.

Box 1.1

Gregor Johann Mendel (1822–1884): Founder of Modern Genetics

As a young man, Mendel spent his time doing genetics research and teaching math, physics, and Greek to high school children in Brno (now in the Czech Republic). Mendel studied the inheritance of various traits of the common garden pea, Pisum sativum, because he was able to raise two generations a year. He studied many different physical traits of the pea, such as flower color, flower position, seed color and shape, and pod color and shape. Mendel grew different plants next to each other, looking for traits that mixed together. Luckily, the traits he studied were each due to a single gene that was either dominant or recessive, although he did not know this at the time. Consequently, he never saw them mix. For example, when he grew yellow peas next to green peas, the offspring looked exactly like their parents. This showed that traits do not blend in the offspring, which was a common theory at the time.

Next Mendel moved pollen from one plant to another with different traits. He counted the number of offspring that inherited each trait and found that they were inherited in specific ratios. For example, when he cross-pollinated the yellow and green pea plants, their offspring, the F1 generation, was all yellow. Thus, the yellow trait must dominate or mask the green trait. He then let the F1 plants produce offspring, and grew all of the seeds. These, the F2 generation, segregated into 3/4 yellow and 1/4 green. When green seeds reappeared after skipping a generation, Mendel concluded that a factor for the trait—what we call a gene today—must have been present in the parent, even though the trait was not actually displayed.

Mendel demonstrated many principles that form the basis of modern genetics. First, units or factors (now called genes) for each trait are passed on to successive generations. Each parent has two copies of each gene but contributes only one copy of the gene to each offspring. This is called the principle of segregation. Second, the principle of independent assortment states that different offspring from the same parents can get separate sets of genes. The same phenotype (the observable physical traits) can be represented by different genotypes (combinations of genes). In other words, although a gene is present, the corresponding trait may not be seen in each generation. When Mendel began these experiments, he used purebred pea plants; that is, each trait always appeared the same in each generation. So when he first crossed a yellow pea with a green pea, each parent had two identical copies or alleles of each gene. The green pea had two green alleles, and the yellow pea had two yellow alleles. Consequently, each F1 offspring received one yellow allele and one green allele. Despite this, the F1 plants all had yellow peas. Thus, yellow is dominant to green. Finally, when the F1 generation was self-pollinated, the F2 plants included some that inherited two recessive green alleles and had a green phenotype (Fig. A).

Mendel published these results, but no one recognized the significance of his research until after his death. Later in life he became the abbot of a monastery and did not pursue his genetics research.

Chemical Structure of Nucleic Acids

The upcoming discussions introduce the organisms used extensively in molecular biology and genetics research. Each of these has genes made of DNA that can be manipulated and studied. Thus, a discussion of the basic structure of DNA is essential. The genetic information carried by DNA, together with the mechanisms by which it is expressed, unifies every creature on earth and is what determines our identity.

Nucleic acids include two related molecules: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA and RNA are polymers of subunits called nucleotides, and the order of these nucleotides determines the information content. Nucleotides have three components: a phosphate group, a five-carbon sugar, and a nitrogen-containing base (Fig. 1.3). The five-carbon sugar, or pentose, is different for DNA and RNA. DNA has deoxyribose, whereas RNA uses ribose. These two sugars differ by one hydroxyl group. Ribose has a hydroxyl at the 2′ position that is missing in deoxyribose. There are five potential bases that can be attached to the sugar. In DNA, guanine, cytosine, adenine, or thymine is attached to the sugar. In RNA, thymine is replaced with uracil (see Fig. 1.3).

Each phosphate connects two sugars via a phosphodiester bond. This connects the nucleotides into a chain that runs in a 5′ to 3′ direction. The 5′-OH of the sugar of one nucleotide is linked via oxygen to the phosphate group. The 3′-OH of the sugar of the following nucleotide is linked to the other side of the phosphate.

The nucleic acid bases jut out from the sugar phosphate backbone and are free to form connections with other molecules. The most stable structure occurs when another single strand of nucleotides aligns with the first to form a double-stranded molecule, as seen in the DNA double helix. Each base forms hydrogen bonds to a base in the other strand. The two strands are antiparallel; that is, they run in opposite directions with the 5′ end of the first strand opposite the 3′ end of its partner and vice versa.

FIGURE 1.3  Nucleic Acid Structure

(A) DNA has two strands antiparallel to each other. The structure of the subcomponents is shown to the sides. (B) RNA is usually single-stranded and has two chemical differences from DNA. First, an extra hydroxyl group (-OH) is found at the 2′ position of ribose, and second, thymine is replaced by uracil.

The bases are of two types: purines (guanine and adenine) and pyrimidines (cytosine and thymine). Each base pair consists of one purine connected to a pyrimidine via hydrogen bonds. Guanine pairs only with cytosine (G-C) via three hydrogen bonds. Adenine pairs only with thymine (A-T) in DNA or uracil (A-U) in RNA. Because an adenine–thymine (A-T) or adenine–uracil (A-U) base pair is held together with only two hydrogen bonds, it requires less energy to break the connection between the bases than in a G-C pair.

Double-stranded DNA takes the three-dimensional shape that has the lowest energy constraints. The most stable shape is a double-stranded helix. The helix turns around a central axis in a clockwise manner and is considered a right-handed helix. One complete turn is 34 Å in length and has about 10 base pairs. DNA is not static but can alter its conformation in response to various environmental changes. The typical conformation just described is the B-form of DNA and is most prevalent in aqueous environments with low salt concentrations. When DNA is in a high-salt environment, the helix alters, making an A-form that has closer to 11 base pairs per turn. Another conformation of DNA is the Z-form, which has a left-handed helix with 12 base pairs per turn. This form occurs when certain proteins bind to the DNA in regions around genes and induce the change in shape. In this form, the phosphate backbone has a zigzag conformation. These forms are biologically relevant under certain conditions, but the exact role the shape of DNA plays in cellular function is still under investigation.

DNA and RNA are both structures with alternating phosphate and sugar residues linked to form a backbone. Base residues attach to the sugar and stick out from the backbone. These bases can base-pair with another strand to form double-stranded helices.

Packaging of Nucleic Acids

Most bacteria have just a few thousand genes, each approximately 1000 nucleotides long. These are carried on a chromosome that is a single giant circular molecule of DNA, although there are exceptions. A single DNA double helix with this many genes is about 1000 times too long to fit inside a bacterial cell without being condensed somehow in order to take up less space.

In bacteria, the double helix undergoes supercoiling to condense it. Supercoiling is induced by the enzyme DNA gyrase, which twists the DNA in a left-handed direction so that about 200 nucleotides are found in one supercoil. The twisting causes the DNA to condense. Extra supercoils are removed by topoisomerase I. The supercoiled DNA forms loops that connect to a protein scaffold (see Fig. 1.4).

In humans and plants, much more DNA must be packaged, so just adding supercoils is not sufficient. Eukaryotic DNA is wound around proteins called histones first. Histones have a positive charge to them, and this neutralizes the negatively charged phosphate backbone. DNA plus histones looks like beads on a string and is called chromatin. Each bead, or nucleosome, has about 200 base pairs of DNA and nine histones—two H2A, two H2B, two H3, two H4, and one H1. All the histones form the bead except for H1, which connects the beads by holding the DNA in the linker region. The histones are highly conserved proteins that are found in all eukaryotes and, in simplified form, in archaebacteria. Histone tails stick out from the nucleosome and are important in regulation. In regions of DNA that are expressed, the histones are loose, allowing regulatory proteins and enzymes access to the DNA. In regions that are not expressed, the histones are condensed, preventing other proteins from accessing the DNA (this structure is called heterochromatin).

FIGURE 1.4  Packaging of DNA in Bacteria and Eukaryotes

(A) Bacterial DNA is supercoiled and attached to a scaffold to condense its size to fit inside the cell. (B) Eukaryotic DNA is wrapped around histones to form a nucleosome. Nucleosomes are further condensed into a 30-nm fiber attached to proteins at MAR sites.

Chromatin is not condensed enough to fit the entire eukaryotic DNA genome into the nucleus. It is coiled into a helical structure, the 30-nanometer fiber, which has about six nucleosomes per turn. These fibers loop back and forth, and the ends of the loops are attached to a protein scaffold or chromosome axis. These attachments occur at matrix attachment regions (MAR) and are mediated by MAR proteins. These sites are 200–1000 base pairs in length and have 70% A/T. The structure of A/T-rich DNA is slightly bent, and these bends promote the connection between proteins in the matrix and the DNA. Often, enhancer and regulatory elements are also found at these regions, suggesting that the structure here may favor the binding of protein activators or repressors. This structure refers to chromosomes during normal cellular growth. When a eukaryotic chromosome readies for mitosis and cell division, it condenses even more. The nature of this condensation is still uncertain.

DNA must be condensed by supercoiling and wrapping around nucleosomes to form chromatin, and finally attached to protein scaffolds in order to fit into the nucleus.

Bacteria as the Workhorses of Biotechnology

DNA is the common thread of life. DNA is found in every living organism on Earth (and even in some entities that are not considered living such as viruses—see later discussion). Only a tiny selection of living organisms has been studied in the molecular biology laboratory. These few chosen species have special traits or features that make them easy to grow, study, and manipulate genetically. Each of the model organisms has had its entire genome sequenced. The model organisms are used both as a guide to understand other related organisms not investigated in detail and for various more practical biotechnological purposes.

Bacteria are the workhorse of model organisms. Bacteria live everywhere on the planet and are an amazing part of the ecosystem. There are an estimated 5 × 10³⁰ bacteria on the Earth, with about 90% of these living in the soil and the ocean subsurface. If this estimate is accurate, then about 50% of all living matter is microbial. Bacteria have been found in every environmental niche. Some bacteria live in icy lakes of Antarctica that only thaw a few months each year. Others live in extremely hot environments such as hot sulfur springs or the thermal vents at the bottom of the ocean (Fig. 1.5).

FIGURE 1.5  Hydrothermal Vent

Mineral-rich fluid is escaping from an opening in the bottom of the ocean along the East Pacific Rise, which has temperatures as high as 403°C. Surprisingly, bacteria are able to survive in this high-heat environment. The vent base is covered with a bed of tube worms, and a probe surrounds the vent. Photo courtesy of NOAA PMEL EOI program and obtained from http://www.pmel.noaa.gov/eoi/gallery/.

There has been great interest in these extreme microbes because of their physiological differences. For example, Thermus aquaticus, a bacterium from hot springs, can survive at temperatures near boiling point and at a pH near 1. Like others, this bacterium replicates its DNA using the enzyme DNA polymerase. The difference is that T. aquaticus DNA polymerase has to function at high temperatures and is therefore considered thermostable. Molecular biologists have exploited this enzyme for procedures like polymerase chain reaction or PCR (see Chapter 4), which is carried out at high temperatures. Other bacteria from extreme environments provide interesting proteins and enzymes that may be used for new biotechnological applications. Hydrothermal vents found on the ocean floor have revealed a fascinating array of novel organisms (see Fig. 1.5). Water temperatures in different vents range from 25°C to 450°C.

Bacteria are highly evolved into every niche of the planet and provide researchers with many unique properties.

Escherichia coli Is the Model Bacterium

Although extreme bacteria are interesting and useful, more typical bacteria are the routine workhorses for research in molecular biology and biotechnology. The most widely used is Escherichia coli, a rod-shaped bacterium about 1 by 2.5 microns in size. E. coli normally inhabits the colon of mammals including humans (Fig. 1.6). E. coli is a Gram-negative bacterium that has an outer membrane, a thin cell wall, and a cytoplasmic membrane surrounding the cellular components. Like all prokaryotes, E. coli does not have a nucleus or nuclear membrane, and its chromosome is free in the cytoplasm. The outer surface of E. coli carries about 10 flagella that propel the bacteria to different locations, and thousands of pili that allow the cells to attach to surfaces.

FIGURE 1.6  Subcellular Structure of Escherichia coli

(A) Scanning electron micrograph of E. coli. The rod-shaped bacteria are approximately 0.6 microns by 1–2 microns. Courtesy of Rocky Mountain Laboratories, NIAID, NIH.

(B) Gram-negative bacteria have three structural layers surrounding the cytoplasm. The outer membrane and cytoplasmic membrane are lipid bilayers, and the cell wall is made of peptidoglycan. Unlike eukaryotes, no membrane surrounds the chromosome, leaving the DNA readily accessible to the cytoplasm.

Although the media often report about E. coli-contaminated food, E. coli is usually harmless. However, occasional strains of E. coli are pathogenic and secrete toxins that cause diarrhea by damaging the intestinal wall. This results in fluid being released into the colon rather than being extracted. E. coli O157:H7 is a particularly potent pathogenic strain of E. coli with two toxin genes that can cause bloody diarrhea. It is especially dangerous to young children, the elderly, and those with compromised immune systems.

Bacteria provide many advantages for research. Bacteria have growth characteristics that are very useful when large numbers of identical cells are needed. A culture of bacteria can be grown in a few hours and can contain up to 109 bacterial cells per milliliter. Growth can be strictly controlled; that is, the amount and types of nutrients, temperature, and time may all be adjusted based on the desired result. E. coli are so easy to grow that they can grow in mineral salts, water, and a sugar source. The cells can be grown in liquid cultures or as solid cultures on agar plates (Fig. 1.7). Liquid cultures can be stored in a refrigerator for weeks, and the bacteria will not be harmed. Additionally, bacteria can be frozen at −70°C for 20 years or more, so different strains can be maintained without having to constantly culture them. E. coli are normally grown in air but can grow anaerobically if an experiment requires that oxygen be eliminated.

FIGURE 1.7  Bacteria Are Easy to Grow

(A) Bacteria growing in liquid culture. (B) Bacteria growing on agar. This photo shows a mixture of bacterial colonies from the blue/white method for screening plasmid insertions. (C) Fast-growing bacteria can double in numbers in short periods. Here, the number of bacteria double after approximately 45 minutes and reach a density of 5 × 10⁹ cells/mL in about 5 hours.

Bacteria are single-celled organisms. The cells in a bacterial culture are identical in contrast to mammalian cells where even a single tissue contains many different types of cells. Each E. coli has one circular chromosome with one copy each of about 4000 genes. This is significantly fewer than in humans, who have two copies each of about 25,000 genes on 46 chromosomes. This makes genetic analysis much easier in bacteria (Fig. 1.8).

Escherichia coli is the model bacterial organism used in basic molecular biology and biotechnology research. The organism is simple in structure, grows easily in the laboratory, and contains very few genes.

Many Bacteria Contain Plasmids

Because many different types of bacteria are found in every environment, competition for nutrients and habitat occurs regularly. Many bacteria compete using a form of biological warfare and secrete toxins, called bacteriocins, which kill neighboring bacteria. For example, nisin, a bacteriocin from Lactococcus lactis, kills other food-borne pathogens such as Listeria monocytogenes and Staphylococcus aureus. E. coli also produce bacteriocins, called colicins. Bacteriocin is a general term, whereas colicin specifically refers to toxins produced by E. coli. (Sometimes colicin is used as a general term, but this is not strictly correct.) E. coli makes different types of colicins, such as colicin E1 or colicin M, to kill neighboring cells. Colicins act by two main mechanisms. Some puncture the cell membrane, allowing vital cellular ions to leak out, and destroying the proton motive force that drives ATP production. Others encode nucleases that degrade DNA and RNA. These toxins do not affect their producer cells because the cell that makes the toxin also makes an immunity protein that recognizes the toxin and neutralizes it.

FIGURE 1.8  The E. coli Chromosome

The E. coli chromosome is divided into 100 map units, arbitrarily starting at the thrABC operon. Various genes and their locations are shown. The replication origin (oriC  ) and termination zone (terB and terC  ) are indicated.

The ability to make colicin is due to the presence of an extrachromosomal genetic element called a plasmid. These are small rings of DNA that exist within the cytoplasm of bacteria and some eukaryotes such as yeast. A colicin-producing plasmid has several genes: the gene for the colicin, the gene for the immunity protein, and genes that control plasmid replication and copy number. In addition, all plasmids contain an origin for DNA replication. When the host cell divides, the plasmid divides in step (Fig. 1.9). These colicin plasmids are used extensively for molecular biology. The colicin genes have been removed, and the remaining segments have been greatly modified so that other genes can be expressed efficiently in bacteria. The resulting recombinant plasmids are the crux of all molecular biology. All the modern advances in biotechnology started with the ability to express heterologous proteins in bacteria (see Chapter 3 for cloning vectors).

Another useful trait of E. coli is the presence of extrachromosomal elements called plasmids. These small rings of DNA are easily removed from the bacteria, modified by adding or modifying genes, and reinserted into a new bacterial cell where new genes can be evaluated.

Other Bacteria in Biotechnology

Other bacteria besides E. coli are used to produce biotechnology products. Bacillus subtilis is a Gram-positive bacterium that is used as a research organism to study the biology and genetics of Gram-positive organisms. Bacillus can form hard spores that can survive almost indefinitely. It is also used in biotechnology. For industrial production, secreting proteins through the single membrane of Gram-positive bacteria is much easier than secreting them through the double membrane of Gram-negative bacteria; therefore, Bacillus strains are used to make extracellular enzymes such as proteases and amylases on a large scale.

Pseudomonas putida is a bacterium that normally lives in water. It is a Gram-negative bacterium like E. coli but is commonly used in environmental studies because it is able to degrade many aromatic compounds. Streptomyces coelicolor is a soil bacterium that is Gram positive. This organism degrades cellulose and chitin, and also produces a large number of different antibiotics. Another example of a common industrial microorganism is Corynebacterium glutamicum, which is used to produce L-glutamic acid and L-lysine for the biotechnology industry.

Many different bacteria are used for biotechnology research because of their unique qualities.

Basic Genetics of Eukaryotic Cells

Most eukaryotes are diploid; that is, they have two homologous copies of each chromosome. This is the case for humans, mice, zebrafish, Drosophila, Arabidopsis, Caenorhabditis elegans, and most other eukaryotes. Having more than two copies of the genome is extremely rare in animals, and only one rat from Argentina has been discovered with four copies of its genome. On the other hand, many plants, especially crop plants, are polyploid and contain multiple copies of their genomes. For example, ancestral wheat has seven pairs of chromosomes (i.e., its diploid state = 2n = 14), whereas the wheat grown for food today has 42 chromosomes. Thus, modern wheat is hexaploid. Domestic oats, peanuts, sugar cane, white potato, tobacco, and cotton also have four to six copies of their genome. This makes genetic analysis very difficult!

FIGURE 1.9  Plasmids Encode the Genes for Colicin

ColE1 plasmids are extrachromosomal DNA elements that are maintained by bacteria for producing a toxin (cea gene). They also carry genes for toxin release and immunity. These plasmids have been modified to carry genes useful in genetic engineering.

In animals, there is a division between germline and somatic cells. Germline cells are the only ones that divide to give haploid descendents. Diploid germline cells give rise to haploid gametes—the eggs and sperm that propagate the species—by undergoing meiosis. After mating, the two haploid cells fuse to become diploid (forming the zygote). Somatic cells, on the other hand, are normally diploid and make up the individual. Any mutations in a somatic cell disappear when the organism dies, whereas a mutation in a germline cell is passed on to the next generation (Fig. 1.10).

If a somatic cell is mutated early in development, all the somatic cells derived from this ancestral cell will receive the defect. Suppose this ancestral cell is the precursor of the left eye and that this defect prevents the manufacture of the brown pigment responsible for brown eyes. The right eye will be brown, but the mutant left eye will be blue (Fig. 1.11). Blue eyes are not due to blue pigment; they simply lack the brown pigment. People or animals with eyes that don’t match are unusual but not incredibly rare. Such events are known as somatic mutations. They are not passed on to the offspring. Nonetheless, mutations in somatic cells can cause severe problems, as they are the cause of most cancers (see Chapter 19).

In plants, the division between germline and somatic cells is less distinct because many plant cells are totipotent. A single plant cell has the ability to form any part of the plant, reproductive or not. This is not true for the majority of animal cells. Nevertheless, many animal cells do have the potential to form several different types of cells. A cell able to differentiate into multiple cell types is called a stem cell. Research on embryonic stem cells has become a hot political topic because of the potential ability to form an embryo. However, researching adult stem cells holds much promise (see Chapter 18). For example, researchers are hoping to identify stem cells that can form new neurons so that patients with spinal cord injuries can be cured.

Eukaryotic cells are more complex than bacteria. Eukaryotic cells are also specialized; that is, some cells are for reproduction, some cells are stem cells that can differentiate into somatic cells, and some cells are specialized in function and shape.

Yeast and Filamentous Fungi in Biotechnology

Fungi are incredibly useful microorganisms in the world of biotechnology. Anyone who has grown mold on a loaf of bread understands the ease with which these are cultured. Fungi are traditionally used in food applications. Yeasts are used in baking and brewing and other fungi in cheese making, mushroom cultivation, and making foods such as soy sauce. Cheese production uses a variety of fungi. For example, a mold called Penicillium roqueforti makes the blue veins in cheeses such as Roquefort, and Penicillium candidum, Penicillium caseicolum, and Penicillium camemberti make the hard surfaces of Camembert and Brie cheeses. Soy sauce is made from soybeans that are fermented with Aspergillus oryzae.

Fungi are responsible for the production of many industrial chemicals and pharmaceuticals. The most famous is penicillin, which is manufactured by Penicillium notatum, in large tanks called bioreactors. Citric acid is a chemical additive to food that occurs naturally in lemons. It gives the fruit their sour taste. Rather than extracting citric acid from lemons, it has been manufactured since about 1923 by culturing Aspergillus niger.

Much like bacteria, yeast has a two-fold purpose in biotechnology. It offers many of the same advantages as bacteria with the additional advantage of being a eukaryote. Yeasts are also important for production of biotechnological products. The most common research strain of yeast is brewer’s or baker’s yeast, Saccharomyces cerevisiae. This is the same little creature that makes the alcohol in beer and makes bread soft and fluffy by releasing carbon dioxide bubbles that get trapped in the dough.

FIGURE 1.10  Somatic versus Germline Cells

During development, cells either become somatic cells, which form the body, or germline cells, which form either eggs or sperm. The germline cells are the only cells whose genes are passed on to future generations.

Yeast is a single-celled eukaryote that has its cellular components compartmentalized (Fig. 1.12). Like all eukaryotes, yeasts have their genomes encased in a nuclear envelope. The nucleus and cytoplasm are separated, but they communicate with each other through gated channels called nuclear pores. Saccharomyces cerevisiae has 16 linear chromosomes that have telomeres and centromeres, two features not found in bacteria. The yeast genome was the first eukaryotic genome sequenced in its entirety. It has 12 Mb of DNA with about 6000 different genes. Unlike higher eukaryotes, yeast genes have very few intervening sequences or introns (see Chapter 2). Outside the nucleus, yeast has organelles including the endoplasmic reticulum, Golgi apparatus, and mitochondria to carry out vital cellular functions.

Like bacteria, yeast grow as single cells. A culture of yeast has identical cells, making genetic and biochemical analysis easier. The culture medium can either be liquid or solid, and the amount and composition of nutrients can be controlled. The temperature and time of growth may also be controlled. Under ideal circumstances, yeast doubles in number in about 90 minutes, as opposed to E. coli, which doubles in 20 minutes. Although slower than bacteria, the growth of yeast is fast in comparison to other eukaryotes. Like bacteria, yeast cells can be stored for weeks in the refrigerator and may be frozen for years at −70°C.

Much like bacteria, some yeast cells also have extrachromosomal elements within their nuclei. The most common element is a plasmid called the 2-micron circle. Like the chromosomes of all eukaryotes, the DNA of this plasmid is also wound around histones. This element has been exploited as a cloning vector (see Chapter 3) to express heterologous genes in yeast. The plasmid has two perfect DNA repeats (FRT sites) on opposite sides of the circle. The plasmid also has a gene for Flp protein, also called Flp recombinase or flippase. This enzyme recognizes the FRT sites and flips one half of the plasmid relative to the other via DNA recombination (Fig. 1.13). Flippase recombines any DNA segments carrying FRT sites, no matter what organism they are in. Consequently, flippase is used in transgenic engineering in higher organisms (see Chapter 16). In plants, a related system, Cre (recombinase) plus LoxP sites, is used in a similar way (see Chapter 15).

FIGURE 1.11  Somatic Mutations

The early embryo has the same genetic information in every cell. During division of a somatic cell, a mutation may occur that affects the organ or tissue it gives rise to. Because the mutation was isolated in a single precursor cell, other parts of the body and the germline cells will not contain the mutation. Consequently, the mutation will not be passed on to any offspring.

FIGURE 1.12  Structure of Yeast Cell

This yeast cell, undergoing division, is starting to partition components into the bud. Eventually, the bud will grow in size and be released from the mother (lower oval), leaving a scar on the surface of the cell wall.

Yeast offer a variety of advantages to biotechnology. They are single-celled organisms that grow fast. Yeast are eukaryotes with chromosomes that have telomeres and centromeres, like the human genome.

Yeast cells have extrachromosomal elements similar to plasmids that allow researchers to study new genes.

Yeast Mating Types and Cell Cycle

Yeast cells grow and divide by budding. Cellular organelles such as mitochondria and some cellular proteins are partitioned into the growing bud. Finally, mitosis creates another nucleus, and when the bud has reached a sufficient size, the new daughter cell is released, leaving a scar on the surface of the mother cell. Budding creates genetically identical cells because the genome divides by mitosis.

FIGURE 1.13  The 2-Micron Plasmid of Yeast

Two different forms of the 2-micron plasmid are shown. The enzyme Flp recombinase recognizes the FRT sites and recombines them, thus flipping one half of the plasmid relative to the other half.

Yeast has diploid and haploid phases in its life cycle, greatly simplifying genetic analysis. Most yeast found in the environment is diploid, having two copies of its genome. Under poor environmental conditions, yeast can undergo meiosis, creating four haploid spores, called ascospores, contained within an ascus. These are released to find a new environment with more nutrients. If the spores find a better environment, they germinate. In the laboratory, the haploid cells can be isolated and grown separately, but in the wild, haploid cells quickly fuse with another, forming diploid cells again (Fig. 1.14). This life cycle allows individual genes to be followed during segregation and inheritance patterns to be analyzed much as with Mendel’s peas. However, the shorter life cycle of yeast allows greater numbers to be analyzed.

Just as meiosis creates haploid male and female gametes in humans, meiosis in yeast creates haploid cells of two different mating types. Because they are structurally the same, rather than male and female, the yeast mating types are called a and α. Fusion may occur only between different mating types; that is, only an a plus an α cell can merge forming a diploid. Each mating type expresses a distinct mating pheromone that binds to receptors on the opposite mating type. The pheromones are secreted into the environment. For example, when an a cell encounters the α pheromone, a cell surface receptor, the α receptor, binds the α pheromone, readying the yeast for fusion. Conversely, when α cells encounter an a pheromone, the cell surface a receptor binds the a pheromone and readies the cell for mating. The two cells then fuse, combining two different genomes into one. The exchange of genes during sex is important for evolution, as it forms new genetic combinations that may have an advantage in different environments.

Diploid yeast will also form genetic clones by budding when plenty of nutrients are available for growth.

Yeast, like other eukaryotic organisms, can create new genetic combinations with sexual reproduction. The two forms of haploid yeast are a and α, which mate to form a new genetically unique diploid cell.

Multicellular Organisms as Research Models

Single-celled creatures offer many advantages, but understanding human physiology requires information about cellular interactions. Although single-celled organisms interact with each other, this is not the same as multicellular organisms where one cell is surrounded by other cells on all sides. The location of cells affects both their role and development.