Assignments for majors
Twenty-two different mutations of human genes that produce actual genetic diseases are included in PedigreeLab. Some of these mutations produce diseases that you have probably heard about and already studied; in other cases, rarer genetic mutations and diseases are presented. You will use PedigreeLab to study a number of different pedigrees to develop a hypothesis to explain the mode of inheritance for the trait that you selected. Once you have done this, your goal is to study recombination between the mutant gene and markers that you will select, using RFLP analysis to help you determine the chromosome containing the mutant gene, and the location of the mutated gene relative to genetic markers on that chromosome.
For your ease in completing each assignment, the background text relevant to the experiment that you will perform is blue, instructions for each assignment are indicated by plain text, and questions or activities that you will be asked to provide answers for are indicated by bold text.
The following assignment is designed to help you become familiar with the operation of PedigreeLab.
Getting to Know PedigreeLab: Inheritance of Amyotrophic Lateral Sclerosis (ALS)
first screen that appears in PedigreeLab is the Information view, beginning with
The first screen that appears in PedigreeLab is the Information view, beginning with Alzheimers disease.
Click on the popup menu for the mutation list. A list of abbreviations for gene mutations of various human genetic disorders appears. Scroll down this list and view the different mutations and diseases presented. Note that for each mutation you are provided with a short narrative of background information describing the history, phenotypes, and other symptoms created by the mutation.
You are not provided with any clues, however, about the location of the gene for this condition. It is your job to hunt for the chromosomal location of each mutation that you choose!
Choose the ALS abbreviation for the mutated gene that results in amyotrophic lateral sclerosis, also commonly known as Lou Gehrigs disease, named after the famous New York Yankees baseball player who died from the condition. Read the information panel to learn more about ALS.
You may have studied ALS before, and you may also know that another former Yankee, Jim "Catfish" Hunter, was recently diagnosed with ALS. This first assignment is designed to introduce you to the process and intricacies of using PedigreeLab by studying the inheritance of ALS and mapping the locus of the ALS gene to its correct chromosome. The other assignments in this laboratory are based on your ability to use your understanding of genetics, and the basic functions of PedigreeLab that you will learn in this first assignment, to map the mutations in PedigreeLab.
To determine the pattern of inheritance for a particular mutation, click on the Full Pedigree tab at the top of the screen. The pedigree that is displayed on this page if one of 100 different sample pedigrees for ALS available in PedigreeLab. To choose a different pedigree, click on one of up or down arrows in the upper right corner of the screen. The purpose of this Full Pedigree function is to provide you will a large number of different pedigrees that you can evaluate to develop a hypothesis for whether a particular mutation is inherited by autosomal recessive, autosomal dominant, or sex-linked inheritance.
Identifying the chromosome that contains the mutation that you are studying and mapping the locus for this mutation using PedigreeLab involves your ability to apply what you learned about the mode of inheritance for the mutation, and your ability interpret the results of RFLP and recombination data. This process is part of the challenge and fun of PedigreeLab.
Using the Large Family function is the key to the gene mapping functions of PedigreeLab. This function simulates databases that present family pedigrees for individuals with different genetic disorders that have been subjected to RFLP analysis using one of 17 different probes for markers on human chromosomes. The pedigrees are arranged according to genotypes for the RFLPs; phenotypes are also shown with these pedigrees. The probes in PedigreeLab do not correspond to actual marker loci in human chromosomes.
You will then pick an appropriate pedigree that will allow you to look for recombination between the marker sequence of DNA recognized by the probe and the mutation. Based on recombination frequencies that you generate, and LOD scores, you will then decide whether the probe and mutation are linked to the same chromosome. If they are linked, you will then use the Chromosome View function to pinpoint the locus for your trait based on recombination frequencies. All of the traits presented in PedigreeLab will map to their actual chromosomes as found in real life. No shortcuts or hints are given to determine linkage groups, hence you must be a gene hunter using trial and error and analysis of your data to eliminate loci for unlinked probes and find linked probes that will help you pinpoint the locus for the mutant gene!
To use this function, click on the Genetic Calculator tab at the top of the screen, then click on the popup arrow next to the box labeled "Trait & Probe Autosomal, Linked" at the top of this window. This popup menu allows you to design genotypes for different individuals depending on whether you think that the trait you are trying to map is an autosomal or sex-linked trait, and whether or not this trait is linked or unlinked to the probe for a given marker. Because you know from studying the full pedigrees that ALS is inherited as an autosomal dominant condition, select the "Trait & Probe Autosomal, Linked" to design genotypes that would show the ALS trait and asr marker as linked on an autosome.
Notice that on the far left of this box you have four popup menu boxes that you can use to select the trait and marker combination for each homologue of a pair in a female parent. Click on one of the upper two boxes, these allow you to choose the genotype for the trait. Note that the dominant allele for ALS is shown in capital letters and the recessive allele for ALS is shown in lower case letters. Click on one of the lower two boxes, these allow you to choose the genotype for the probe (marker). Note that the genotype for a homologue with a restriction site is shown as "asr+", and the genotype for a homologue lacking a restriction site is shown as "asr-."
It is important that you determine the correct linkage combination for the parent who is a double heterozygote (A/a, +/-) for the trait and restriction site. Do this for the female parent. For the first homologue select a linkage combination with "ALS" and "asr+" on the first homologue. Select the opposite homologue with the linkage configuration of "als" and "asr-."
Repeat this process for a male parent who is a double homozygote for the trait and restriction site (a/a, +/+). For this parent, the linkage combination is the same on both homologues.
Once you have selected the genotypes for each parent, click on the Calculate Punnett Squares button. You should now see Punnett squares that show nonrecombinant and recombinant genotypes that would result from a cross between the parents with the trait and restriction site arrangements that you just set up. You are now ready to return to the Large Family pedigree function to look for a pedigree with parents that show the genotypes for the trait and marker combination that you set up with the Genetic Calculator. Once you have done this, use the results of the Punnett squares to help you look for the genotypes that you will count as recombinants and which you will count as nonrecombinants. Hint: To help you as you search the Large Family pedigrees you may want to export the results of your Punnett squares from the Genetic Calculator and print a copy of these results to have in front of you as you look at the large family pedigrees to count recombinants and nonrecombinants.
After counting offspring for one pedigree, choose another useful pedigree to look for recombination. Note that as you tally recombinants and nonrecombinants, a running tally for the total number of offspring that you have counted is shown at the bottom of the screen along with recombination frequency and LOD score. You will need to count offspring from several pedigrees to accumulate data that will help you determine if the trait and probe are linked. Remember that you are looking for a LOD score of 3 or greater, and a recombination frequency of less than 50% as good evidence that the trait and probe are linked. If you are seeing evidence for linkage then you are ready to map the trait to a chromosome.
Conversely, a recombination frequency of 50% indicates that the probe and trait are probably unlinked. This can usually be determined after analyzing just a few pedigrees. If this is the case, choose another probe and try again with another probe! You should find that recombination data using the asr probe indicates that the asr marker is not linked to the ALS gene. Click on the Chromosome View and note that the asr probe is located on chromosome 1, hence you can probably rule out this chromosome as the location of the ALS gene, and you may want to rule out using the other probes on this chromosome in your next search. You may find that you will have to search for recombination with many probes before you find one that is linked to your trait.
To look for linkage with another probe, return to the Large Family function. Click on the New Search button. This time, search the database for pedigrees using the "juva" probe. Be sure to answer each of the three questions to choose database criteria that will favor the pedigrees that you will need to study recombination. Once you have done this, count recombinant and nonrecombinant offspring again. This time you should see evidence for linkage of the juva probe and ALS gene. Follow the next procedure to map the ALS gene to the correct chromosome.
Notice the other markers on chromosome 21, "ddy" and "utc3." Return to the Large Family function and repeat a database search using probes for each of these markers to better pinpoint the location of the ALS gene. You should find that the ALS gene maps to a position just above the juva marker. See your instructor for the exact location of the ALS gene.
To print your recombination data, return to the Large Family view. You can export a single pedigree that you have labeled by clicking on the Export Pedigree and printing from the GIF image window that appears. You can also export all of the data (total number of recombinants, nonrecombinants, recombination frequency, and LOD scores) for each probe that you used by clicking on the New Search button, then clicking on the Export Whole List button. With this function you will open a window with your data that functions as a lab notebook. You can add any comments that you'd like to your data and print a copy of this data as evidence of your gene hunting progress.
Now that you have experienced how PedigreeLab functions to map the location of the ALS gene, you are on your own to be a gene hunter for some of the other mutations in PedigreeLab!
Follow the guidelines in the ALS assignment to help you identify the location and approximate locus for each of the following mutations:
Use the Genetic Calculator to help you select the correct Large Family pedigrees and decide what genotypes to look for when counting recombinant and nonrecombinant offspring. Once you have selected the correct pedigree, count the number of recombinant and nonrecombinant offspring. Do this until the recombination and LOD data indicate whether the trait and probe are linked or unlinked. If the trait and probe are linked, use the Chromosome View function to assign a position for the mutant gene on the correct chromosome. If the trait and probe are unlinked, then pick another probe to test for recombination and repeat the process until you find a probe that is linked to the trait.
The Human Genome Project is a worldwide initiative to develop genetic maps for the 22 autosomes and two sex chromosomes in humans. One long-term goal of the Genome Project is to identify and determine the nucleotide sequence for an estimated 100,000 genes that are thought to be present in the approximately 3 billion nucleotides of DNA that comprise the human genome. The Genome Project began in 1990 and was targeting as a 15-year project with an estimated cost of $3 billion. Although this project originated in the United States, labs around the world are contributing to the Genome Project. Many new technologies for identifying and sequencing genes have been developed as a result of the project. Because of this, the progress of the Genome Project is actually slightly ahead of schedule and just under budget. One technique being used by Genome scientists involves using RFLP analysis and other similar techniques to develop extensive genetic maps of chromosomes in humans and other animal and plant species such as the nematode Caenorhabditis elegans, Drosophila melanogaster, the common house mouse, Mus Musculus, and the flowering plant Arabidopsis thaliana. Some labs involved in the Genome Project are working on mapping assigned segments of certain chromosomes. Other groups are involved in checking the accuracy of maps completed to date.
The following exercises are designed to have you investigate the accuracy of genetic mapping as simulated in PedigreeLab, and enable you to map an entire chromosome. Work together in a group of four students to complete the following assignments.
Once you have agreed on a chromosome location, compare the accuracy of the locus that you identified for each mutation. How accurate were the maps when you compared them? How many map units did the two maps differ by? Explain possible reasons for any differences in loci assigned on each chromosome mapped.
How would you begin? Should you assign each student a mutant gene that you have not yet mapped and have each person study the inheritance for that gene? Or should you work as a group and look for inheritance patterns to first narrow your search for genes that appear to be X-linked? Decide on an approach to use then work together to develop a map of the X that will show all of the X-linked mutations present in PedigreeLab. Because PedigreeLab will show different regions of the X-chromosome depending on the probes you are using, PedigreeLab will not show one complete full length-map of the X. Your group will have to piece together the different maps of the X shown in PedigreeLab to draw one complete map.
Once you have done this, compare your map with another group of students in your class that has worked on the X.
How accurate were the two sets of maps? Were there any differences in genes mapped to the X when the maps were compared? If there were differences, work together with the other group to resolve these differences until you can agree on an accurate and complete map of the X chromosome. Print your map and show it to your instructor for comparison to the correct map.
Figure 1: Diagrammatic Representation of
Restriction Fragment Length Polymorphisms (RFLPs) for a Recessive Trait.
Shown is an example of two homologues from an individual who is heterozygous (Dd) for a recessive gene that is linked to a genetic marker. Arrows indicate the restriction enzyme recognition sequence that would be cut by restriction digestion of each chromosome with the enzyme EcoRI. Note two sites (1, 2) are present on the homologue with the dominant allele (D), while only one site (2) is present on the homologue with the recessive allele (d). The absence of restriction enzyme cutting site number 1 in the recessive allele due to a single nucleotide mutation is indicated with an "X." The location of a radioactive probe that is complementary to the marker sequence is also shown. The pattern of restriction fragment length polymorphisms that would be generated for these homologues is visualized after gel electrophoresis of the digested DNA fragments, transfer of the DNA fragments to a filter, and hybridization of the fragments to the radioactive probe, followed by exposing the filter to X-ray film to produce an autoradiogram. In the autoradiogram, lane 1 shows the migration of DNA size standards of known length that are used to determine the nucleotide length of each RFLP. Lane 2, RFLPs for a heterozygote (+/-) where the genotype is designated according to the presence (+) or absence (-) of a restriction site; cutting with EcoRI yields two fragments of 7 kb and 4 kb in size. For comparison, lanes 3 and 4 show RFLPs for individuals who are homozygous for the restriction site, +/+ or -/-.