The Crucial Importance of Model Systems in the Biological Sciences
Scientists who make significant contributions to understanding the nature of the biological universe consciously or sometimes by chance make three very important decisions about how they conduct their investigations. First, they focus on understanding some fundamental aspect of how a living system functions or develops. Second, they formulate questions, well defined and limited in scope, that have a high probability of being answered. This is an important point, because it is generally easy to pose profound questions about the unknown, but it is very difficult to plan and execute a realistic approach to answers for those questions. The third, and equally important decision, is to choose an experimental system that makes answering the questions feasible.
Consider, for example, the work of Gregor Mendel, who, seeking to learn about the inheritance of specific plant characteristics, chose to hybridize different strains of the garden pea. In Mendel's own words, Experiments of artificial fertilization, such as is effected with ornamental plants in order to obtain new variations in colour, has led to the experiments which will here be discussed. He chose his questions and experimental material well, for his results from those experiments form the foundation of modern genetic principles. See also Mendel, Gregor Johann
Mendel corresponded with Nägeli, Professor of Botany at Munich, who failed to grasp the significance of Mendel's work. It is evident from the letters to Nägeli that Mendel was continuing his experiments using other plants, including Hieracium. Though Mendel never published the results of the Hieracium experiments, it is evident that he was able with great difficulty to obtain several hybrids. He was probably quite disappointed when all of those hybrids bred true, unlike the results from the pea plants. Later, it became known that Hieracium seeds are usually produced by apomixis, in which sexual reproduction is replaced by asexual reproduction. In Hieracium, meiosis and the formation and fusion of gametes are usually skipped. The offspring are completely maternal in origin. Heterozygous genotypes, therefore, are preserved and hybrid plants breed true. Clearly, Hieracium was not a model system in which to study gene segregation and independent assortment. Despite the failure with Hieracium, Mendel did reproduce the results from the pea experiments using other plant species such as Mirabilis and maize. See also Apomixis, and History of Classical Genetics
Overview of Experimental Organisms
Most of the organisms that have risen to the rank of model experimental systems have done so primarily because they are open to genetic experimentation. Why are genetic studies so important for understanding fundamental processes in biology? It is because genes encode proteins and proteins are the primary structural components and regulators of biological systems. With modern experimental techniques, the most direct way of linking a protein with its function is through the analysis of the gene that encodes it. As we focus on some important experimental models, it will become evident that experiments using these organisms contribute significantly to the advancement of almost all fields of biology.
This virus, which infects Escherichia coli cells, has two options in its infectious cycle (Figure 1). It often chooses a lytic pathway in which many copies of the viral DNA are synthesized and packaged in protein coats. The host cell bursts, releasing mature viral particles, which can then infect new host cells. Alternatively, the virus may choose a lysogenic cycle, where the lambda chromosome becomes inserted into a special region of the bacterial chromosome. The host cell then does not lyse; rather it becomes immune to further viral infection and grows normally, producing many cells, each of which carries an integrated copy of the lambda chromosome. Genetic screens have been used to identify the genes that control entrance of the virus into one or the other of these two pathways. The proteins that those genes encode have been identified and the analysis of the function of the normal versus mutant molecules has led to understanding how the switch for entry to one or the other pathway is regulated. See also Bacteriophage Lambda and its Relatives
|Figure 1. Diagram depicting alternative lytic and lysogenic paths for lambda virus following its infecting an E. coli host cell.|
Lysis of a lysogenized culture can be induced by exposing it to ultraviolet light. Lambda DNA then excises from the host chromosome, replicates many copies, each of which is packaged in a protein coat. The release of very large numbers of viral particles from a lysed bacterial culture makes feasible the recognition and recovery of rare mutations or recombinants.
A variety of mutants of lambda affecting its structure and life cycle are known. They have been grouped as to function by complementation tests and mapped in the phage chromosome by recombination between different phage types. These tests are done by infecting host cells with two genetically different particles by manipulating multiplicity of infection and various aspects of the host cell types and growth conditions.
Lambda is also very useful for the study of its host genetics and functions. When the viral DNA exits the host chromosome it occasionally will carry with it some E. coli chromosome material on either side of its insertion site, leaving behind some of its own DNA. Such a defective particle can infect and carry over the bacterial gene to a new host cell. This is called transduction, a process that has proved to be invaluable for analysing bacterial genes.
Lambda also plays a very important role in the molecular analysis of eukaryotic organisms. It is one of the vectors used to create molecular libraries of eukaryotic chromosomes. Eukaryotic DNA fragments can be cloned into modified lambda chromosomes, replacing some lambda genes not essential for infection and growth. Such libraries can be screened by molecular techniques to identify viruses containing a particular piece of the eukaryote's genome. Once having identified and isolated a desired virus, that virus can be grown to billions of copies to produce a large quantity of the eukaryotic DNA for analysis. This is a very useful way of molecularly purifying eukaryotic genes for mapping and sequencing. See also Bacteriophage Lambda Gene Regulation
Bacterial model systems
Two species of bacteria, the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis, are model organisms for studying the genetics and metabolism of prokaryotes. Both normally lack pathogenicity and are easily cultured on defined media. The cell cycle is very short and growth can be synchronized to obtain cells all in the same stage. Small size makes possible the analysis of very large numbers of cells, which aids in detecting specific types of mutations and other rare genetic events. The discovery of sexual recombination in E. coli in 1947 vaulted this bacterium into the premier position of genetic analysis of prokaryotes. The single circular DNA chromosome of E. coli, only 4.6 × 10
Examples of major advances in understanding the function and evolution of prokaryotes come from the study of mutagenesis and DNA repair mechanisms in E. coli. In response to the threat of DNA damage caused by a wide variety of environmental agents, organisms have evolved important response mechanisms. One is an error-free process of nucleotide excision repair, by which a damaged nucleotide is detected, excised and replaced using the complementary DNA strand as a template. When error-free repair cannot be accomplished, however, E. coli and other bacteria utilize an inducible system termed the SOS response. The mechanistic basis of SOS mutagenesis is the alteration of DNA polymerase III to allow it to replicate DNA that contains miscoding and noncoding lesions. This is an error-prone process but it circumvents a lethal interruption of replication by allowing synthesis to continue through such lesions. The study of this system has produced insights into the regulation of gene expression in response to DNA damage and has contributed to the understanding of the mutation process itself. Recently, there is evidence that one of the operons in the SOS system may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred. See also DNA Damage
Fungal model systems
The fungi bring some powerful tools to eukaryotic genetic analysis. Saccharomyces cerevisiae is the best studied of the group. Its life cycle (Figure 2) has both haploid and diploid phases, both of which can reproduce vegetatively. In the wild, yeasts are diploid, but in the laboratory haploid strains are easily cultured. Sexual reproduction is accomplished by fusion of two haploid cells of different mating types (a and ) to create the diploid form. Meiosis results in four ascospores (a tetrad) packaged in an ascus sac. Tetrad analysis, the isolation and genetic analysis of the four meiotic products, is a very powerful method for studying the mechanics and molecular bases of meiotic recombination. See also Saccharomyces Cerevisiae as an Experimental Organism
|Figure 2. Life cycle of Saccharomyces cerevisiae depicting diploid and haploid vegetative cycles. Meiosis results in a tetrad of haploid ascospores of two mating types, a and alpha.|
Another analytical tool is the use of retrotransposons, particularly Ty1, that can infect yeast cells. These are mobile elements capable of moving in and out of yeast chromosomes. They can create mutations by inserting into a gene, or by creating a deficiency when excising. Ty1 elements can also be engineered molecularly to contain selected yeast genes, which can then be transported into a host cell and integrated into one of its chromosomes. In this fashion the yeast genome can be manipulated for precise molecular analysis. See also Transposons as Natural and Experimental Mutagens
Of primary importance is that the entire yeast genome has now been cloned and sequenced. Data banks relating to genes, their products and their functions are extensive, making the answers to complex questions such as signal transduction, the mechanisms of recombination or the process of DNA repair much more accessible than if starting afresh with a new system.
Yeast artificial chromosomes (YACs) are now being employed as vectors for cloning other eukaryotic DNAs. The YAC consists of a yeast chromosome centromere, telomeres and an origin of replication. Into this construct fragments of foreign DNAs of the order of 2001000 kb can be inserted. These artificial chromosomes can be grown in yeast cells to produce large quantities of purified DNA. Such large inserts of DNA often undergo rearrangements or excisions, so special precautions with regard to host cell types and growth conditions need to be taken to reduce recombination and excision. See also Yeast Artificial Chromosomes, and Artificial Chromosomes
Plant model systems
Three species are extensively used to study the structure and function of members of the plant kingdom: a unicellular alga, Chlamydomonas reinhardtii, and two flowering plants, Arabidopsis thaliana (a dicot) and Zea mays (a monocot). A dicot is a plant that produces seeds having two primary seed leafs (cotyledons) in the embryo. A monocot is a plant that produces one cotyledon.
This haploid single-celled green alga is a flagellated free-swimming eukaryote. There are two mating types that under certain growth conditions will pair and fuse. A diploid zygote then forms and undergoes meiosis upon maturation. The four products of meiosis can be manipulated and grown on defined culture media on a Petri dish. This allows tetrad analysis much like that done with yeast. Its single-celled nature makes it easy to deal with large numbers of individuals. Because Chlamydomonas can be cultured on defined growth media, mutations in metabolic pathways are easily obtained and open to analysis. Also very important are the studies on mutations that block steps in photosynthesis. Mutations that affect cellular structures such as the flagella are also useful for understanding the formation and function of such organelles.
This flowering plant is a recent entry into the model organism class. It is a very small plant, generally considered a weed, in the mustard family. It is easily cultured in the laboratory on artificial defined medium, and plants can be regenerated vegetatively from protoplasts. Its genome of five chromosomes consists of only 7 × 10
Many mutations are known that affect plant morphology, including the control of flower development. Mutations affecting photorespiratory functions, chlorophyll binding, gibberellin metabolism, auxin resistance, nitrate reduction are just some of the other important classes of genetic change that are being used to discover how plant cells develop and function. See also Arabidopsis: Flower Development and Patterning
The corn plant has been the object of genetic engineering for centuries. Its progenitor is thought to be the wild grass, teosinte. Its domestication and selection by natives of the Western world resulted in forms used as a staple food crop. The application of Mendelian genetic principles for the improvement of the grain began early in the 1900s. One result of that breeding programme is the hybrid corn varieties grown today. See also Plant Breeding and Crop Improvement
Corn is not an ideal genetic model organism because of its large size, long life cycle and considerable space for cultivation. Despite these drawbacks, experiments with corn have produced important discoveries about the structure of chromosomes and the mechanisms of recombination. Possibly the most notable research using the corn plant was done by Barbara McClintock, who discovered a variety of active mobile genetic elements in corn. Their movements into and out of chromosomes cause mutations that are often unstable, i.e. the elements responsible for mutations continue to move to new locations creating still other mutations. McClintock's work marked the beginnings of studies characterizing the genetic behaviour and the molecular structures of transposons. These elements are widespread throughout both prokaryotes and eukaryotes, and have proven to be very effective tools for genetic analysis of gene structure and expression. See also Transposons: Eukaryotic, and Transposons: Prokaryotic
Animal model systems
Useful animal models for genetic and developmental studies include the round worm (Caenorhabditis elegans), an insect (Drosophila melanogaster) and a vertebrate (Mus musculus). Humans also must be included despite the considerable difficulty of applying many of the modern techniques of genetic analysis.
Other animal models that have contributed significantly to understanding vertebrate development are the chicken, Zebra fish and amphibians such as Xenopus laevis. For the descriptive embryology tracing cell migrations and tissue interactions, the developing embryos of these animals are unsurpassed. Also, surgical manipulation transplanting tissues into new positions at different developmental stages provided evidence for the transmission of developmental signals between cells and tissue layers. Transplantation experiments using Xenopus cell nuclei injected into enucleated eggs showed that though most such hybrids failed to develop, a few did indeed complete development to normal larval stages. This is a forerunner of recent cloning of animals such as sheep by nuclear transplant from reportedly differentiated donor cells.
This very small free-living nematode can be grown in large numbers on Petri dishes. The genome size is approximately 8 × 10
A great advantage for studying the worm's developmental programmes is that all adult structures develop from founder cells that have rigid developmental fates. The entire cell lineage has been worked out for the 671 cells generated during embryogenesis. Some (113 in hermaphrodites, 111 in males) undergo programmed cell death; the remainder differentiate terminally in a very invariant pattern, or become blast cells.
A useful tool for obtaining mutations and manipulating genes in the chromosomes of Caenorhabditis is the existence of the mobile element Tc1. However, some of the first mutations found were identified simply by observing and selecting animals exhibiting uncoordinated movement. Many of those mutations were shown to affect muscle or nerve system development. Mutations in such genes and others that regulate developmental steps, such as programmed cell death, make it possible to discover what proteins those genes encode, what the functions of those proteins are, and equally important, how such genes receive and respond to developmental signals. See also Genome Mapping, and Apoptosis: Molecular Mechanisms
The contributions advancing the genetics and development of multicellular animals made through studies of Drosophila are unsurpassed by those using any other organism. Beginning in 1910 with the discovery of the first white eye mutant by T. H. Morgan and his students, this small fly has been used to establish many of the fundamental principles of inheritance. The Morgan group demonstrated sex linkage, established that genes exist on chromosomes, showed that homologous chromosomes undergo exchange during meiosis and worked out linkage maps. In 1927, H. J. Muller, using Drosophila, showed that X-rays cause mutations in genes and break chromosomes. Over the years, the characterization of a large number of gene mutations in Drosophila, beyond extending the knowledge of genetic mechanisms, has resulted in remarkable advances in understanding how genes regulate developmental processes in multicellular animals. See also Morgan, Thomas Hunt, Eukaryotic Chromosomes, and Linkage and Crossing over
The characteristics of this fly that make it so useful as an experimental system are its small size, ease of culturing, a short life cycle of about 2 weeks, a small genome size (four pairs of chromosomes, composed of 1.4 × 10
Mobile genetic elements have been exploited to great advantage for genetic analysis in Drosophila. New mutants created by the insertion of a transposon are genetically and molecularly tagged by virtue of the presence of the transposon inside the gene. Such a tag makes locating the gene in the polytene chromosomes very easy by in situ hybridization with radioactive transposon DNA. Also important, cloning the mutated gene is straightforward because any DNA fragment containing the transposon will probably be flanked by parts of the mutated gene.
By far the most important use of a transposon in Drosophila is the development of a transformation system using the mobile genetic element, P. Modified P elements carrying selected sequences of Drosophila DNA, when injected into embryos, will in a small proportion be taken up by the embryonic cells and incorporated into a chromosome. This creates a transformant that is mosaic for the transgene. Should the mosaicism include cells in the germline, a transformed line can be established. This system has been invaluable for identifying and molecularly cloning genes that are strongly expressed in particular tissues and at specific stages of development. For example, in a genetic screen known as an enhancer trap, a reporter gene encoding beta-galactosidase is placed under the control of a weak promoter element. This construct when placed into a transposable element can be hopped about in the genome. When the construct lands near a strong enhancer element, the reporter gene will be strongly expressed. That expression can be detected by staining with X-Gal, a substrate that produces a colour reaction when acted upon by beta-galactosidase. See also Transgenic Animals
The mouse is an excellent model organism for the study of genetics and development in vertebrates. Though it is relatively expensive to grow, it adapts readily to laboratory conditions. Inbred strains have been developed so that genetic experiments can be conducted in geneotypically uniform animals. More than 700 mutant strains are available for study. Mutant traits involve almost every aspect of development and metabolism, such as coat colour, skeletal structure, haematology, endocrine and immune systems, neurological and behavioural characteristics, and viral, disease and tumour susceptibility or resistance. See also Mice as Experimental Organisms
Possibly the most important aspect of the mouse model system is the ability to effect gene transfers into host animals. There are at present four different methods for placing engineered DNA into a mouse.
- Cloned DNA can be microinjected into a fertilized egg and the egg then implanted in a surrogate mother for development.
- A recombinant retrovirus vector carrying mouse DNA can be used to infect a pre- or post-implantation embryo.
- Embryonic stem cells can be transformed with cloned DNA. Those cells are then injected into a blastocyst, resulting in a mosaic animal at birth (Figure 3). Should the transformed cells happen to be included among the germline stem cells, the transformed genotype can be transmitted to offspring to establish a stable strain.
Figure 3. Diagram depicting one method for creating mosaic transgenic mice using transformed embryonic stem cells (ES cells) injected into a host blastocyst, which is placed in a surrogate mother for development.
- Bone marrow cells can be infected with a recombinant retrovirus to allow expression of the transferred gene in the infected animal.
Often the object of gene transfer experiments is to accomplish what is known as a gene knockout. By replacing a normal gene with a defective or slightly modified copy, the researcher can create a model for studying a mutant condition such as a developmental abnormality or a cancer-causing genetic change. See also Targeted Mutagenesis in the Immune System
Humans are not good model experimental organisms. However, medical practices demand investigations into human health and the basis of human diseases. Genetic characteristics of humans are best worked out through family pedigrees because there are very few direct experimental manipulations that can be applied. A majority of human diseases have, to a considerable degree, a genetic basis. Therefore, the discovery of the genes involved often come from studies of other animal models, such as the mouse. This is because many of the genes that regulate basic developmental and metabolic pathways are conserved to a considerable degree. In fact, some major developmental steps such as the establishment of segmental pattern are conserved across several phyla of metameric animals. This makes it possible to recognize and clone human homologues of genes controlling similar functions in the mouse. A major effort is currently underway to sequence the entire human genome, which could in theory make it possible to determine the function of every human gene and how its interactions with other genes are regulated. See also Human Disease: Mouse Models, Pedigree Analysis, Human Genome Project, Medical Relevance of, and Bioethics: ELSI
This survey of model systems, ranging from lambda to the mouse, with humans as an honorary member, illustrates that they have several characteristics in common. Most are small sized and easily grown in the laboratory, and genetic manipulation is almost essential, except for strictly descriptive or taxonomic studies. To be genetically useful, an organism must be amenable to tests for dominance, complementation and mapping by recombination. It is helpful if its genome is small and has been or is in the process of being sequenced. Furthermore, some system for transforming it with DNA is almost essential. With these tools the experimenter can discover genes, connect them to their protein (or in some cases RNA) products and determine their function and regulation. Most fundamental questions in biology can be investigated effectively with this approach. An important point is that the more an organism is used as an experimental model the more valuable it is likely to become.
- In prokaryotes, a group of contiguous structural genes under coordinate expression.
- Any of the enzymes that catalyse the assembly of nucleotides or deoxyribonucleotides into RNA or DNA on a DNA or RNA template.
- Any process that gives rise to cells or individuals associating in new combinations two or more genes by which their parents differed.
- The sets of repeated DNA sequences found at the ends of eukaryotic chromosomes.
- The transfer of genetic information, either intra- or interspecific by means of naked extracellular DNA.
- Any of several families of mobile genetic elements usually transmitted vertically from a cell to daughter cells, that are capable of causing mutations, including chromosome breaks by integrating into or excising from a host chromosome.