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Fly Life: Why fruit flies are a good model organism for research
Since this is my first post, I’d like to start by talking about the basics: Why are we doing research with fruit flies, or even animal models in general? How can these simple organisms possibly provide us with relevant data for future human research? Generally speaking, animal models are important for biological research because they allow scientists to reproduce human diseases or abnormal behavior without the ethical concerns inherent in human studies. Although there are obvious differences between humans and other animals, there are many molecular and cellular processes that are shared among all species through evolution. Many complex human behaviors are also seen in animals, such as aggression, circadian rhythms, sleep, learning and memory, and mating. By studying these processes and behaviors in animal models, researchers gain an understanding of the basic biology underlying them and can use this knowledge to figure out how diseases occur when things go awry (and then, how to fix it).
One important feature of an animal model is the ability to manipulate its genome and investigate the function of specific genes. For example, scientists know that a mutation in the Pink1 gene is responsible for early-onset Parkinson’s disease in humans 1 , but they don’t know what the gene does. Researchers need to know the gene’s function before they can investigate how the mutation leads to Parkinson’s disease and how to treat it. In animal models, researchers can study the gene’s function by manipulating the relevant gene in the animal’s genome. One important type of genetic modification is a gene “knock-out”, which means they induce mutations in a specific gene so that it becomes inactive or non-functional. By observing the resulting change in physiological processes or behavior, researchers can determine what role the gene played in normal functioning. In our example, scientists can knock-out the Pink1 gene in fruit flies or mice and observe on a cellular level which processes fail. As an added benefit, researchers can also observe the animals themselves to see if they display the same behavioral phenotypes as patients with Parkinson’s disease, such as tremors and slow movement.
Another type of genetic manipulation is called a “knock-in”, where researchers instead insert a gene from another animal into the genome (or, in many cases, replace the endogenous gene with another version). For example, researchers often insert a mutated version of a human gene implicated in a disease, which allows them to determine what effect that particular mutation has on the gene’s function. To return to our previous example, scientists can insert the specific human Pink1 mutation that leads to early-onset Parkinson’s disease in an animal’s genome instead of knocking it out completely. It also allows them to test the effectiveness (and side effects) of various promising therapeutic drugs before going to human trials.
So what’s this got to do with fruit flies? On the About page on this site, I gave a few of the basic reasons why Drosophila melanogaster are a good model organism for research. They have been used for research at all levels of biology, but genetic research is where these organisms really shine. Genetic manipulations are so much easier in fruit flies because they have a smaller genome which was fully sequenced in March 2000 2 . Their short life cycle and large number of offspring are also advantageous for genetic research because new fly lines are quick and easy to make. As a result, although the manipulations I mentioned above can be performed in other animal models such as mice, mutations in flies can be generated much more easily. Making a new line of flies usually only takes about six weeks and costs less than $300, whereas a new mouse line takes months and can costs thousands of dollars.
But how do researchers initially identify the genes they’re interested in studying? In order to study a process or behavior using specific gene mutations such as those described above, the gene of interest must already be identified. Traditionally, researchers perform genetic “screens”, in which they use mutagenic chemicals or radiation to cause random mutations in animal models, and then screen offspring for abnormalities of interest and identify the mutated gene. But this process is difficult and time-consuming, and often based on luck. Enter Drosophila melanogaster! Using this animal model, researchers can conduct large-scale screens relatively quickly. For example, to find genes that contribute to sensing heat, fly researchers can test hundreds of mutant flies for impaired heat avoidance within a few months. Identifying those relevant gene(s) will then provide a starting point for studying sensory abnormalities in mammals.
Figure 1. Example of GAL4/UAS system being used to express a green fluorescent marker in eye cells. Photo by Wellcome Images / CC by-nc-nd 2.0
Over the years, fly researchers have also developed an impressive array of genetic tools that make Drosophila melanogaster an even better animal model for research. The list is too long to cover in this post, but there is one type of tool I want to introduce called binary expression systems. These systems allow fly researchers to insert a specific gene into a specific set of cells, and even activate or deactivate the gene at specific times. One example binary expression system is called the GAL4/UAS system. In this system, a fly line with genetic instructions for where something should be inserted (GAL4) is mated with a fly line with genetic instructions for what should be inserted (UAS). For example, the GAL4 instructions might define the cells of the eyes while the UAS might be a green fluorescent marker. The individual lines have no abnormal phenotypes, but the offspring will have green glowing eyes! (Figure 1) In practice, if a researcher wants to know in which brain structures a particular gene is expressed, such as Pink1, they can combine a GAL4 that targets “cells with an active Pink1 gene” with the green UAS. They can then view a dissected fly brain under a microscope and see where cells expressing the Pink1 gene are located in the brain. Alternatively, the UAS instructions might describe a gene to be deactivated instead of inserted, so that it is “knocked out” in a very specific set of cells. In this way, fly researchers can investigate the gene’s function in a relevant region without affecting the overall health of the fly, which is important for reproducing human diseases that target specific cell-types. And because genetic modification is so comparatively easy in the fruit fly, the fly community has created a collection of thousands of fly lines for these systems, which researchers are more than willing to share with each other. As a result, it is often very likely that a specific combination is already available for use.
Ultimately, every animal model has its advantages and disadvantages. Researchers wouldn’t want to use monkeys for a genetic screen, and they wouldn’t use fruit flies to study complex emotions. But although fruit flies may seem very different from us, an estimated 75% of known human disease genes have a match in the fruit fly genome 3-4 . They are already being used as a genetic model for several human diseases, including Parkinson’s disease, Alzheimer’s disease, Fragile X syndrome, and Rett’s syndrome, in addition to the basic research needed to advance our general understanding of biology and how we work. So the next time you see a fruit fly in your kitchen, make sure to say “thank you” before you swat it away.
Introduction and Goals
This tutorial emphasizes the work of Gregor Mendel, the father of modern genetics. Mendel was the first scientist to examine, in a quantitative manner, the behavior of traits between generations. By looking at the proportions of progeny, he was able to infer the basic tenets of modern genetics. By the conclusion of this tutorial you should have a basic understanding of:
- Mendel and modern genetics
- The distinction between characters and traits
- Meiosis and the segregation of alleles
- The molecular relationship between genotype and phenotype
- The laws of segregation and independent assortment
Characteristics and Traits
The seven characteristics that Mendel evaluated in his pea plants were each expressed as one of two versions, or traits. The physical expression of characteristics is accomplished through the expression of genes carried on chromosomes. The genetic makeup of peas consists of two similar or homologous copies of each chromosome, one from each parent. Each pair of homologous chromosomes has the same linear order of genes. In other words, peas are diploid organisms in that they have two copies of each chromosome. The same is true for many other plants and for virtually all animals. Diploid organisms utilize meiosis to produce haploid gametes, which contain one copy of each homologous chromosome that unite at fertilization to create a diploid zygote.
For cases in which a single gene controls a single characteristic, a diploid organism has two genetic copies that may or may not encode the same version of that characteristic. Gene variants that arise by mutation and exist at the same relative locations on homologous chromosomes are called alleles. Mendel examined the inheritance of genes with just two allele forms, but it is common to encounter more than two alleles for any given gene in a natural population.
Phenotypes and Genotypes
Two alleles for a given gene in a diploid organism are expressed and interact to produce physical characteristics. The observable traits expressed by an organism are referred to as its phenotype. An organism’s underlying genetic makeup, consisting of both physically visible and non-expressed alleles, is called its genotype. Mendel’s hybridization experiments demonstrate the difference between phenotype and genotype. When true-breeding plants in which one parent had yellow pods and one had green pods were cross-fertilized, all of the F1 hybrid offspring had yellow pods. That is, the hybrid offspring were phenotypically identical to the true-breeding parent with yellow pods. However, we know that the allele donated by the parent with green pods was not simply lost because it reappeared in some of the F2 offspring. Therefore, the F1 plants must have been genotypically different from the parent with yellow pods.
The P0 plants that Mendel used in his experiments were each homozygous for the trait he was studying. Diploid organisms that are homozygous at a given gene, or locus, have two identical alleles for that gene on their homologous chromosomes. Mendel’s parental pea plants always bred true because both of the gametes produced carried the same trait. When P0 plants with contrasting traits were cross-fertilized, all of the offspring were heterozygous for the contrasting trait, meaning that their genotype reflected that they had different alleles for the gene being examined.
Dominant and Recessive Alleles
Our discussion of homozygous and heterozygous organisms brings us to why the F1 heterozygous offspring were identical to one of the parents, rather than expressing both alleles. In all seven pea-plant characteristics, one of the two contrasting alleles was dominant, and the other was recessive. Mendel called the dominant allele the expressed unit factor the recessive allele was referred to as the latent unit factor. We now know that these so-called unit factors are actually genes on homologous chromosome pairs. For a gene that is expressed in a dominant and recessive pattern, homozygous dominant and heterozygous organisms will look identical (that is, they will have different genotypes but the same phenotype). The recessive allele will only be observed in homozygous recessive individuals (Table 4).
|Table 4. Human Inheritance in Dominant and Recessive Patterns|
|Dominant Traits||Recessive Traits|
|Huntington’s disease||Duchenne muscular dystrophy|
|Widow’s peak||Sickle-cell anemia|
|Wooly hair||Tay-Sachs disease|
Several conventions exist for referring to genes and alleles. For the purposes of this chapter, we will abbreviate genes using the first letter of the gene’s corresponding dominant trait. For example, violet is the dominant trait for a pea plant’s flower color, so the flower-color gene would be abbreviated as V (note that it is customary to italicize gene designations). Furthermore, we will use uppercase and lowercase letters to represent dominant and recessive alleles, respectively. Therefore, we would refer to the genotype of a homozygous dominant pea plant with violet flowers as VV, a homozygous recessive pea plant with white flowers as vv, and a heterozygous pea plant with violet flowers as Vv.
The Punnett Square Approach for a Monohybrid Cross
When fertilization occurs between two true-breeding parents that differ in only one characteristic, the process is called a monohybrid cross, and the resulting offspring are monohybrids. Mendel performed seven monohybrid crosses involving contrasting traits for each characteristic. On the basis of his results in F1 and F2 generations, Mendel postulated that each parent in the monohybrid cross contributed one of two paired unit factors to each offspring, and every possible combination of unit factors was equally likely.
To demonstrate a monohybrid cross, consider the case of true-breeding pea plants with yellow versus green pea seeds. The dominant seed color is yellow therefore, the parental genotypes were YY for the plants with yellow seeds and yy for the plants with green seeds, respectively. A Punnett square, devised by the British geneticist Reginald Punnett, can be drawn that applies the rules of probability to predict the possible outcomes of a genetic cross or mating and their expected frequencies. To prepare a Punnett square, all possible combinations of the parental alleles are listed along the top (for one parent) and side (for the other parent) of a grid, representing their meiotic segregation into haploid gametes. Then the combinations of egg and sperm are made in the boxes in the table to show which alleles are combining. Each box then represents the diploid genotype of a zygote, or fertilized egg, that could result from this mating. Because each possibility is equally likely, genotypic ratios can be determined from a Punnett square. If the pattern of inheritance (dominant or recessive) is known, the phenotypic ratios can be inferred as well. For a monohybrid cross of two true-breeding parents, each parent contributes one type of allele. In this case, only one genotype is possible. All offspring are Yy and have yellow seeds (Figure 4).
Figure 4. In the P0 generation, pea plants that are true-breeding for the dominant yellow phenotype are crossed with plants with the recessive green phenotype. This cross produces F1 heterozygotes with a yellow phenotype. Punnett square analysis can be used to predict the genotypes of the F2 generation.
A self-cross of one of the Yy heterozygous offspring can be represented in a 2 × 2 Punnett square because each parent can donate one of two different alleles. Therefore, the offspring can potentially have one of four allele combinations: YY, Yy, yY, or yy (Figure 4). Notice that there are two ways to obtain the Yy genotype: a Y from the egg and a y from the sperm, or a y from the egg and a Y from the sperm. Both of these possibilities must be counted. Recall that Mendel’s pea-plant characteristics behaved in the same way in reciprocal crosses. Therefore, the two possible heterozygous combinations produce offspring that are genotypically and phenotypically identical despite their dominant and recessive alleles deriving from different parents. They are grouped together. Because fertilization is a random event, we expect each combination to be equally likely and for the offspring to exhibit a ratio of YY:Yy:yy genotypes of 1:2:1 (Figure 4). Furthermore, because the YY and Yy offspring have yellow seeds and are phenotypically identical, applying the sum rule of probability, we expect the offspring to exhibit a phenotypic ratio of 3 yellow:1 green. Indeed, working with large sample sizes, Mendel observed approximately this ratio in every F2 generation resulting from crosses for individual traits.
Mendel validated these results by performing an F3 cross in which he self-crossed the dominant- and recessive-expressing F2 plants. When he self-crossed the plants expressing green seeds, all of the offspring had green seeds, confirming that all green seeds had homozygous genotypes of yy. When he self-crossed the F2 plants expressing yellow seeds, he found that one-third of the plants bred true, and two-thirds of the plants segregated at a 3:1 ratio of yellow:green seeds. In this case, the true-breeding plants had homozygous (YY) genotypes, whereas the segregating plants corresponded to the heterozygous (Yy) genotype. When these plants self-fertilized, the outcome was just like the F1 self-fertilizing cross.
The Test Cross Distinguishes the Dominant Phenotype
Beyond predicting the offspring of a cross between known homozygous or heterozygous parents, Mendel also developed a way to determine whether an organism that expressed a dominant trait was a heterozygote or a homozygote. Called the test cross, this technique is still used by plant and animal breeders. In a test cross, the dominant-expressing organism is crossed with an organism that is homozygous recessive for the same characteristic. If the dominant-expressing organism is a homozygote, then all F1 offspring will be heterozygotes expressing the dominant trait (Figure 5). Alternatively, if the dominant expressing organism is a heterozygote, the F1 offspring will exhibit a 1:1 ratio of heterozygotes and recessive homozygotes (Figure 5). The test cross further validates Mendel’s postulate that pairs of unit factors segregate equally.
Figure 5. A test cross can be performed to determine whether an organism expressing a dominant trait is a homozygote or a heterozygote.
In pea plants, round peas (R) are dominant to wrinkled peas (r). You do a test cross between a pea plant with wrinkled peas (genotype rr) and a plant of unknown genotype that has round peas. You end up with three plants, all which have round peas. From this data, can you tell if the round pea parent plant is homozygous dominant or heterozygous? If the round pea parent plant is heterozygous, what is the probability that a random sample of 3 progeny peas will all be round?
Many human diseases are genetically inherited. A healthy person in a family in which some members suffer from a recessive genetic disorder may want to know if he or she has the disease-causing gene and what risk exists of passing the disorder on to his or her offspring. Of course, doing a test cross in humans is unethical and impractical. Instead, geneticists use pedigree analysis to study the inheritance pattern of human genetic diseases (Figure 6).
Figure 6. Pedigree Analysis for Alkaptonuria
Alkaptonuria is a recessive genetic disorder in which two amino acids, phenylalanine and tyrosine, are not properly metabolized. Affected individuals may have darkened skin and brown urine, and may suffer joint damage and other complications. In this pedigree, individuals with the disorder are indicated in blue and have the genotype aa. Unaffected individuals are indicated in yellow and have the genotype AA or Aa. Note that it is often possible to determine a person’s genotype from the genotype of their offspring. For example, if neither parent has the disorder but their child does, they must be heterozygous. Two individuals on the pedigree have an unaffected phenotype but unknown genotype. Because they do not have the disorder, they must have at least one normal allele, so their genotype gets the “A?” designation.
What are the genotypes of the individuals labeled 1, 2 and 3?
Laws of Inheritance
The seven characteristics that Mendel evaluated in his pea plants were each expressed as one of two versions, or traits. Mendel deduced from his results that each individual had two discrete copies of the characteristic that are passed individually to offspring. We now call those two copies genes, which are carried on chromosomes. The reason we have two copies of each gene is that we inherit one from each parent. In fact, it is the chromosomes we inherit and the two copies of each gene are located on paired chromosomes. Recall that in meiosis these chromosomes are separated out into haploid gametes. This separation, or segregation, of the homologous chromosomes means also that only one of the copies of the gene gets moved into a gamete. The offspring are formed when that gamete unites with one from another parent and the two copies of each gene (and chromosome) are restored.
For cases in which a single gene controls a single characteristic, a diploid organism has two genetic copies that may or may not encode the same version of that characteristic. For example, one individual may carry a gene that determines white flower color and a gene that determines violet flower color. Gene variants that arise by mutation and exist at the same relative locations on homologous chromosomes are called alleles. Mendel examined the inheritance of genes with just two allele forms, but it is common to encounter more than two alleles for any given gene in a natural population.
3.11: Peas and flies- basics of inheritance - Biology
In this section, you will explore the following questions:
- How does the structure of DNA provide for the process of replication?
- How did the Meselson and Stahl experiments support the semi-conservative nature of replication?
Connection for AP ® Courses
The Watson and Crick model suggested a way in which DNA could be replicated during cell division. Basically, the two strands unwind and separate where the hydrogen bonds connect the nucleotides. Each parental strand then serves as a template for a new, complementary daughter strand. Replication is said to be semi-conservative because the original information encoded in each parental strand is conserved (kept) in the daughter molecules. Thus, a newly replicated molecule of DNA consists of one “old” strand and one “new” strand. Meselson and Stahl used density differences in nitrogen isotopes to investigate replication, and their experiments supported the semi-conservative model. However, the process of replication is more complex than their model’s simple description.
Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 and Big Idea 4 of the AP ® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® exam questions. A Learning Objective merges required content with one or more of the seven Science Practices.
|Big Idea 3||Living systems store, retrieve, transmit and respond to information essential to life processes.|
|Enduring Understanding 3.A||Heritable information provides for continuity of life.|
|Essential Knowledge||3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.|
|Science Practice||1.2 The student can describe representations and models of natural or man-made phenomena and systems in the domain.|
|Learning Objective||3.3 The student is able to describe representations and models that illustrate how genetic information is copied for transmission between generations.|
The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 2.34][APLO 3.3][APLO 4.1]
The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested (Figure 14.12): conservative, semi-conservative, and dispersive.
In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands act as a template for new DNA to be synthesized after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.
Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15 N) that gets incorporated into nitrogenous bases, and eventually into the DNA (Figure 14.13).
The E. coli culture was then shifted into medium containing 14 N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in 14 N and spun again. During the density gradient centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in 15 N will band at a higher density position than that grown in 14 N. Meselson and Stahl noted that after one generation of growth in 14 N after they had been shifted from 15 N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15 N and 14 N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14 N formed two bands: one DNA band was at the intermediate position between 15 N and 14 N, and the other corresponded to the band of 14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.
During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.
LINK TO LEARNING
Click through this tutorial on DNA replication.
- Aging causes accumulation of DNA mutations and DNA damage of only the nuclear DNA and the mistakes will be passed down to new cells causing age related diseases.
- Aging results in ineffective DNA repair mechanism so that the mistakes in the DNA will be passed down to new cells. This could lead to the development of age-related diseases.
- Aging causes DNA polymerase to function abnormally. This is the sole reason which causes defects in DNA replication.
- DNA replication of only fast growing cells is affected by aging.
SCIENCE PRACTICE CONNECTION FOR AP® COURSES
Design (but do not implement) an experiment to test the three models of DNA replication. Summarize the results you would expect if each of the three models of DNA replication were correct. Assume you have access in a laboratory to the following: an experimental organism such as E. coli, an unlimited variety of isotopes, test tube and centrifuge, and organic growth media.