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Project Summary

Difficulty	7  –  8
Time required	Short (several days)
Prerequisites	You should have a basic understanding of genetic principles.
Material Availability	Readily available
Cost	Very Low (under $20)
Safety	No issues

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Abstract

Objective

In this science fair project you will use the patterns of fur color in tortoiseshell cats to determine if X chromosomes are inactivated in a random or in an allele-dependent manner.

Introduction

Tortoiseshell cats aren't a special breed of cat. Rather, they are a cat of any breed that has an unusual pattern of fur coloration consisting of patches of red or orange, and black or brown fur. To be considered a tortoiseshell cat, the animal must have two of these fur colors—either black or brown and red or orange. Cats who also have white patches are called calico cats. Interestingly, almost all tortoiseshell cats are female! Why? The answer has to do with how fur color is inherited in cats.

Figure 1. Notice the hallmark black and orange patches on this female tortoiseshell cat (Kurynny, 2006).

Like many physical characteristics, feline fur color is determined by genetics. The genetics of cats' coat colors is complex, but there are three main genes (sequences of DNA) that determine fur color: the browning gene, the piebald gene, and the orange gene. Each gene has several alleles. Alleles are different versions of the same gene, which result in different physical outcomes, called phenotypes. Most of the time an animal inherits two copies of the gene, one from each parent. Since these can be different alleles, the combination of alleles is called the genotype. For example, the browning gene in cats has three alleles: B, b, and b^l. If a cat inherits the B allele from its mother and the b allele from its father then its genotype (allele combination) is Bb. This particular combination results in a phenotype (physical result) of black fur. Depending on the genotype a cat inherits, he or she will have a black, brown, or light brown (sometimes called cinnamon) coat of fur. The table below shows the three fur color genes, the possible genotypes for each gene, and the resulting phenotype.

Fur Color Genes	Alleles, Grouped into Genotypes	Phenotype
Browning gene	BB, Bb, or Bbl	Black fur
	Bb or bbl	Brown fur
	blbl	Light brown (cinnamon) fur
Piebald gene	SS	Many white patches of fur
	Ss	Some white patches of fur
	ss	Few or no white patches of fur
Orange	OO(female), or O(male)	All black/brown fur, turns orange/red
	Oo(female)	Some black/brown fur, turns orange/red
	oo(female) o(male)	No orange/red fur

The second fur color gene is the piebald gene, sometimes referred to as the white spotting gene. This gene determines how likely it is that the cat will have white spots. The white spots can color over any other existing fur color. Cats with an SS genotype are likely to have either a large number of spots, or at least a few very large spots. At the other end of the spectrum, cats with the ss genotype have very few, or even no, white spots. Because tortoiseshell cats do not have white spots they usually have the ss genotype.

Both the piebald gene and the browning gene are autosomal, meaning they are located on chromosomes called autosomes. All chromosomes, except the X chromosome and the Y chromosome, are autosomes. The X and Y chromosomes are called sex chromosomes because they determine gender. A cat, or human for that matter, is female if it has two X chromosomes (one inherited from its mother and one from its father), and male if it has one X (inherited from its mother) and one Y chromosome (inherited from its father). Genes that appear on either the X or the Y chromosome are called sex-linked.

The third fur color gene, orange, is a sex-linked gene, located on the X chromosome. This means that female cats will have two copies (one for each X chromosome they inherit) of the orange gene, while males will have only one copy. Since males have only one copy of the gene, their genotype is either O or o. The O genotype is dominant over any of the browning gene genotypes. This means that all the fur that would normally have been black/brown due to the browning gene is orange/red instead! In contrast, the o genotype is recessive to the browning gene genotypes, so the fur remains black/brown. So, males with the O genotype will have all orange fur (and white spots, depending on their piebald genotype), while males with the o genotype will have no orange fur. This means that normal males can not be tortoiseshell cats because they have either the O allele and thus orange fur, or the o allele and black/brown fur, but they can't have both orange and black/brown.

Unlike males, female cats have two X chromosomes so they can have both alleles of the orange gene (the Oo genotype), one allele on each chromosome. However, both X chromosomes cannot be active at the same time in a cell. That's because the genes on chromosomes give the cat's body information about what proteins to make. If both copies were telling the cell what to do, the female cat would end up with twice as much X-chromosome protein as the male cats, and this would be unhealthy for the female cat. So, to equalize the protein output between genders, the female's body inactivates one X chromosome in each cell. This X-chromosome inactivation, also called X-inactivation, means that a female cat with the Oo genotype will have some cells that express the O allele (and thus grow orange fur), while other cells express the o allele and grow black/brown fur. Females with the OO genotype will have only orange fur, and females with the oo genotype will have only black/brown fur. So a tortoiseshell cat has to be a female with the Oo genotype (to get both orange and black/brown patches of fur), any genotype of the browning gene (a tortoiseshell cat can have any shade of black or brown fur), and the ss genotype (so that there are no white spots of fur). The diagram in Figure 2 illustrates how X-inactivation and genotype work together in a gender-dependent manner to give rise to tortoiseshell cats.

Figure 2. This diagram depicts the how the various orange gene genotypes result in different phenotypes in a cat that has the BB genotype for the browning gene (black fur) and the ss genotype for the piebald gene (no white spots). Notice that because female cats can inherit and express both alleles of the orange gene, they can be tortoiseshell cats, but normal males cannot.

Does the X-inactivation occur in a specific pattern? Is it predetermined which cells will express which allele of the orange gene? Or does X-inactivation occur randomly with respect to the orange gene alleles? In this science fair project you will discover the answers by comparing the fur color patterns of several tortoiseshell cats. If the pattern is consistent between cats, it is evidence that X-inactivation is pre-determined. If there is no consistency, then the data will suggest that X-inactivation is not driven by orange gene allele type and that it may be random.

Terms, Concepts and Questions to Start Background Research

• Tortoiseshell cat
• Calico cat
• Gene
• Browning gene
• Piebald gene
• Orange gene
• Allele
• Phenotype
• Genotype
• Autosomal
• Chromosome
• Autosome
• Sex chromosome
• Sex-linked
• Dominant
• Recessive
• X-chromosome inactivation
• Barr body
• Dosage compensation

Questions

• What is the relationship between genotype and phenotype?
• What are the genotypic and phenotypic differences between tortoiseshell and calico cats?
• On rare occasions, a male tortoiseshell or calico cat is born. What genetic abnormality makes this possible?
• What is X-inactivation? Why does it occur?

Bibliography

Biology textbooks are a good resource for reviewing genetic terms and concepts. For some basic information about genetics, you can also try these websites:

• The Dr. John T. Macdonald Foundation Center for Medical Genetics. (2007). Learn More About Genetics. Retrieved June 9, 2008 from http://medgen.med.miami.edu/x62.xml
• GlaxoSmithKline. (2006). Kids Genetics. Retrieved June 9, 2008 from http://www.genetics.gsk.com/kids/index_kids.htm

For specific information about X-chromosome inactivation, try this website:

• The University of Alabama at Birmingham. (2005). X Chromosome Inactivation/Genomic Imprinting. Retrieved June 9, 2008 from http://main.uab.edu/show.asp?durki=20044

Additional information about the genetics of cats' coat color can be found at:

• Krempels, D.M. (n.d.). The Genetics of Calico Cats. Retrieved June 9, 2008 from http://www.bio.miami.edu/dana/dox/calico.html

Materials and Equipment

• Tortoiseshell cats, minimum of five
  • If you do not have access to five tortoiseshell cats, you can use pictures of tortoiseshell cats from books or the Internet for some or all of your data. Make sure the pictures clearly show the cat's entire face.
• Lab notebook
• Graph paper
• Camera (optional)
• Ruler (optional)

Experimental Procedure

1. To start this project you will need either several tortoiseshell cats, or pictures of tortoiseshell cats. Make sure all the cats you are using are tortoiseshell cats and not calico cats.
2. Look at the face of each cat and mentally divide the face into 12 regions, based on the location of the cat's facial features, like its ears, eyes, nose, and mouth. Figure 3 below shows a diagram of the 12 regions.
   1. If you are using pictures of cats, you can physically divide the picture into the 12 regions using a ruler. Make sure the pictures you have can be drawn on. For instance, if they are from a book, you probably want to make copies first.
   2. If you are using actual cats for this experiment, you may find it easier to use a camera to take pictures of their faces and draw the regions directly onto the pictures.

   Figure 3. This diagram illustrates how to divide each cat's face into 12 regions for data collection.

3. In your lab notebook, make a data table, like the one below, with a row for each cat and a column for each region of the face. For each cat, record whether the cat's fur is black or orange for each region of the face.
   1. Since black and all shades of brown are created by the browning gene (the variations are different alleles of the same gene), record any shade of black or brown as "black" in your data table.
   2. Some regions of the face may have both black and orange fur. Write down whichever color is more abundant in your data table. If the two colors are equally represented, record the color as mixed.

   Cat #	Color of Face Region (black/orange/mixed)
   	1	2	3	4	5	6	7	8	9	10	11	12
   Cat #1
   Cat #2

4. Now that you've gathered the data, you need to summarize the results for each region of the face.
   1. In your lab notebook, make a second data table showing the sum total of cats surveyed with a particular fur color (either black, orange, or mixed) for each face region. See the data table below for an example of how to organize your data.
   2. Using this second data table, create a series of either bar graphs or pie charts showing what fraction of the cats had which fur color for each face region.

   Fur Color	# of Cats with a Particular Fur Color per Face Region
   	1	2	3	4	5	6	7	8	9	10	11	12
   Black
   Orange
   Mixed

5. Look at your bar graphs/pie charts. Which alleles of the orange gene are represented on the cats' faces? Does your data suggest that X-inactivation is predetermined by orange allele type, or random in respect to orange allele type?

Variations

• At what stage of embryonic development does X-inactivation occur? Is it at the same stage for every cat? Design an experiment to find out. Hint: The earlier the X-inactivation occurred, the greater the number of cells which would have the same active allele—this would, in turn, increase the size of the color patch.
• What is the population frequency of each allele of the orange gene? Look at the cats you know and use their gender and phenotype to deduce their genotype. Use this information to calculate population frequencies for each allele.
• If you know a cat breeder, or have access to the pedigrees kept by one, use the phenotype/genotype data for the parental generation to predict the offspring's fur patterns and colors. Check your predictions against the actual phenotypes of the offspring.

• For more science project ideas in this area of science, see Mammalian Biology Project Ideas.

Credits

Sandra Slutz, PhD, Science Buddies

Last edit date: 2008-07-27 09:00:00

Career Focus

If you like this project, you might enjoy exploring careers in Mammalian Biology.

	Veterinarian Veterinarians help prevent, diagnose and treat health problems in a wide variety of animals. Regardless of whether the animal is a family pet, a prize-winning race horse, a dairy cow, a circus lion, or seal in a zoo, its healthcare depends on veterinarians.			Veterinary Technologist and Technician Everyday heroes in the animal healthcare world are veterinary technicians and technologists. Just as nurses assist doctors, veterinary technicians and technologists are on the front lines, assisting veterinarians. As part of their duties, they perform initial physical exams, take samples, run tests in the lab, monitor patients' heart and respiratory rates, give shots, and assist in surgery and dental work. Their work helps relieve animal suffering and prevent future disease.
	Animal Trainer Do you have a favorite pet food commercial or animal movie in which the animals do something really cool or cute? Animal trainers are responsible for these amazing animal performances. Animal trainers get involved in much more than the fun world of entertainment, though; they are also involved in the serious business of training animals for search and rescue missions, bomb and drug detection, criminal capture, and service to help people with disabilities.

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