From Genes to Phenotypes

   At one level, geneticists tend to think of genes in isolation. In reality, genes don't act in isolation. The proteins and RNAs they encode contribute to specific cellular pathways that also receive input from the products of many other genes. Furthermore, expression of a single gene is dependent on many factors, including the specific genetic backgrounds of the organism and a range of environmental conditions, temperature, nutritional conditions, population density, and so on.

Gene action

is a term that covers a very complex set of events, and there is probably no case where we understand all the events that transpire from the level of expression of a single gene to the level of an organism's phenotype. Two important generalizations about the complexity of gene action:   1. There is a one-to-many relationship of genes to phenotypes.

2. There is a one-to-many relationship of phenotypes to genes.

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One-to-many relationship of genes to phenotypes

  This relationship is called

pleiotropy

. Pleiotropy is inferred from the observation that mutations selected for their effect on one specific character are often found to affect other characters of the organism. This might mean that there are related physiological pathways contributing to a similar phenotype in several tissues.

 For example, the white eye-color mutation in

Drosophila

results in lack of pigmentation not only in compound eyes but also in ocelli (simple eyes), sheaths of tissue surrounding the male gonad, and the Malpighian tubules (the fly's kidneys). In all these tissues, pigment formation requires the uptake of pigment precursors by the cells. The white allele causes a defect in this uptake, thereby blocking pigment formation in all these tissues.

Often, pleiotropy involves multiple events that are not obviously physiologically related.

 For example, the dominant

Drosophila

mutation

Dichaete

causes the wings to be held out laterally but also removes certain hairs on the back of the fly; furthermore, the mutation is inviable when homozygous.

This example shows a real limitation in the way dominant and recessive mutations are named.

The reality is that a single mutation can be both dominant and recessive, depending on which aspect of its pleiotropic phenotype is under consideration. In general, genetic terminology is not up to the task of representing this level of pleiotropy and complexity in one symbol, and there is a certain arbitrary or historical aspect as to how we name alleles.

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One-to-many relationship of phenotypes to genes

  This concept is based on the observation that many different genes can affect a single phenotype. This is easy to understand in terms of a character such as eye color, in which there are complex metabolic pathways with numerous enzymatic steps, each encoded by one or more gene products.

Genetic heterogeneity

is the term used to refer to a given condition that may be caused by different genes.

One goal of genetic analysis is to identify all the genes that affect a specific phenotype and to understand their genetic, cellular, developmental, and molecular roles. To do this, we need ways of sorting mutations and genes.

  We first will consider how we can use genetic analysis to determine if two mutants are caused by mutational hits in the same gene (that is,

they are alleles

) or in different genes.

Later, we will consider how genetic analysis can be used to make inferences about gene interactions in developmental and biochemical pathways.

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The complementation test

  The allelism test that finds widest application is the complementation test, which is illustrated in the following example.

 Consider a species of flower in which the wild-type color is blue. We have induced three white-petaled mutants and have obtained pure-breeding strains (all homozygous). We can call the mutant strains $, £, and ¥, using currency symbols to avoid prejudicing our thinking concerning dominance. In each case the results show that the mutant condition is determined by the recessive allele of a single gene. However, are they three alleles of one gene, or of two or three genes? The question can be answered by asking if the mutants

complement

each other.

Complementation is the production of a wild-type phenotype when two recessive mutant alleles are united in the same cell.

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Performing the complementation test

 In a diploid organism the complementation test is performed by intercrossing homozygous recessive mutants two at a time and observing whether or not the progeny have wild-type phenotype. If recessive mutations represent alleles of the same gene, then obviously they will not complement because they both represent lost gene function. Such alleles can be thought of generally as

a’

and

a"

, using primes to distinguish between two different mutant alleles of a gene whose wild type allele is

a +

. These alleles could have different mutant sites but would be functionally identical. The heterozygote

a’/a"

would be  However, two recessive mutations in different genes would have wild-type function provided by the respective wild-type alleles. Here we can name the genes

a1

and

a2

, after their mutant alleles. Heterozygotes would be

a1/+

;

+/a2

(unlinked genes) or

a1+/+a2

(linked genes), and we can diagram them as follows:

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Mutants that complement

 We now return to the flower example and intercross the mutant strains to test for complementation. Assume the results of intercrossing mutants $, £, and ¥ are as follows:   From this set of results we would conclude that mutants $ and £ must be caused by alleles of one gene (say

w1

) because they do not complement; but ¥ must be caused by a mutant allele of another gene (

w2

).

The molecular explanation of such results is often in terms of biochemical pathways in the cell. How does complementation work at the molecular level? Although it is conventional to say that it is mutants that complement, in fact the active agents in complementation are the proteins produced by the wild-type alleles.

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The biochemical explanation

 The normal blue color of the flower is caused by a blue pigment called anthocyanin. Pigments are chemicals that absorb certain parts of the visible spectrum; in the case of the harebell the anthocyanin absorbs all wavelengths except blue, which is reflected into the eye of the observer. However, this anthocyanin is made from chemical precursors that are not pigments; that is, they do not absorb light of any specific wavelength and simply reflect back the white light of the sun to the observer, giving a white appearance. The blue pigment is the end product of a series of biochemical conversions of nonpigments. Each step is catalyzed by a specific enzyme coded by a specific gene. We can accommodate the results with a pathway as follows:  A mutation in either of the genes in homozygous condition will lead to the accumulation of a precursor, which will simply make the plant white. Now, the mutant designations can be written as follows:

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Complementation

Three phenotypically identical white mutants, $, £, and ¥, are intercrossed to form heterozygotes whose phenotypes reveal whether or not the mutations complement each other. (Only two of the three possible crosses are shown here.) If two mutations are in different genes (such as £ and ¥), then complementation results in the completion of the biochemical pathway (the end product is a blue pigment in this example). If mutations are in the same gene (such as $ and £), no complementation occurs because the biochemical pathway is blocked at the step controlled by that gene, and the intermediates in the pathway are colorless (white).

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Interactions Between the Alleles of One Gene

Incomplete Dominance 

Four-o'clocks

are plants native to tropical America. Their name comes from the fact that their flowers open in the late afternoon. When a wild-type four-o'clock plant with red petals is crossed with a pure line with white petals, the F 1 an F 2 is produced by selfing the F 1 , the result is has pink petals. If  Because of the 1:2:1 ratio in the F 2 , we can deduce an inheritance pattern based on two alleles of a single gene. However, the heterozygotes (the F 1 and half the F 2 ) are intermediate in phenotype, suggesting an incomplete type of dominance. Inventing allele symbols, we can list the genotypes of the four-o'clocks in this experiment as

c +

/

c +

(red),

c/c

(white), and

c +

/

c

(pink).

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Incomplete dominance

 Incomplete dominance describes the general situation in which the phenotype of a heterozygote is intermediate between the two homozygotes on some quantitative scale of measurement.

 This figure gives terms for all the theoretical positions on the scale, but in practice it is difficult to determine exactly where on such a scale the heterozygote is located. At the molecular level, incomplete dominance is generally caused by a quantitative effect of the number of "doses" of a wild-type allele; two doses produce most functional transcript and therefore most functional protein product; one dose produces less transcript and product, whereas zero doses have no functional transcript or product. In cases of full dominance, in the wild-type/mutant heterozygote either half of the normal amount of transcript and product is adequate for normal cell function (the gene is haplo-sufficient), or the wild-type allele is "up regulated" to bring the concentration of transcript up to normal levels.

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Codominance

 The human ABO blood groups are determined by three alleles of one gene that show several types of interaction to produce the four blood types of the ABO system. The allelic series includes three major alleles,

i

,

I A

, and

I B

, but of course any person can have only two of the three alleles (or two copies of one of them). There are six different genotypes, the three homozygotes and three different types of heterozygotes:  In this allelic series, the alleles

I A

and

I B

each determine a unique antigen, which is deposited on the surface of the red blood cells. These are two forms of a single protein. However, the allele

i

genotypes

I A /i

and

I B /i

results in no antigenic protein of this type. In the , the alleles

I A

and

I B

are fully dominant to

i

. However, in the genotype

I A /I B

each of the alleles produces its own antigen, so they are said to be

codominant.

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Relativity of dominance relationships

 The human disease sickle-cell anemia gives interesting insight into dominance. The gene concerned affects the molecule hemoglobin, which transports oxygen and is the major constituent of red blood cells. The three genotypes have different phenotypes, as follows:  In regard to the presence or absence of anemia, the HbA allele is obviously dominant. In regard to blood cell shape, however, there is incomplete dominance. Finally, in regard to hemoglobin itself there is codominance, as the two hemoglobin molecules HbA and HbS can be visualized simultaneously by means of electrophoresis

Sickle-cell anemia illustrates that the terms dominance, incomplete dominance, and codominance are somewhat arbitrary. The type of dominance inferred depends on the phenotypic level at which the observations are being made, organismal, cellular, or molecular. Indeed the same caution can be applied to many of the categories that scientists use to classify structures and processes; these categories are devised by humans for convenience of analysis Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini

An anomalous segregation ratio

  Normal wild-type mice have coats with a rather dark overall pigmentation. A mutation called

yellow

(a lighter coat color) illustrates an interesting allelic interaction. If a

yellow

mouse is mated to a homozygous wild-type mouse, a 1:1 ratio of yellow to wild-type mice is always observed in the progeny. This observation suggests (1) that a single gene with two alleles determines these phenotypic alternatives, (2) that the

yellow

mouse was heterozygous for these alleles, and (3) that the allele for

yellow

is dominant to an allele for normal color.

However, if two

yellow

mice are crossed with each other, the result is always as follows:  Note two interesting features in these results. First, the 2:1 phenotypic ratio is a departure from the expectations for a monohybrid self-cross. Second, because no cross of

yellow

×

yellow

ever produced all

yellow

progeny, as there would be if either parent were a homozygote, it appears that it is impossible to obtain homozygous

yellow

mice

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Letal alleles

 The explanation for such results is that all

yellow

mice are heterozygous for one special allele. A cross between two heterozygotes would be expected to yield a monohybrid genotypic ratio of 1:2:1. However, if all the mice in one of the homozygous classes died before birth, the live births would then show a 2:1 ratio of heterozygotes to the surviving homozygotes. The allele

A Y

for

yellow

is dominant to the wild-type allele

A

with respect to its effect on color, but

A Y

acts as a recessive

lethal

allele with respect to a character we would call

viability.

Thus, a mouse with the homozygous genotype

A Y

/

A Y

dies before birth and is not observed among the progeny. All surviving

yellow

mice must be heterozygous

A Y

/

A

, so a cross between

yellow

mice will always yield the following results: The expected monohybrid ratio of 1:2:1 would be found among the zygotes, but it is altered to a 2:1 ratio in the progeny born because zygotes with a lethal

A Y /A Y

genotype do not survive to be counted. This hypothesis is supported by the removal of uteri from pregnant females of the

yellow

×

yellow

cross; one-fourth of the embryos are found to be dead.

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What goes wrong in lethal mutations?

  In many cases it is possible to trace the cascade of events that leads to death. A common situation is that the allele causes a deficiency in some essential chemical reaction. The human diseases PKU and cystic fibrosis are good examples of this kind of deficiency. In other cases there is a structural defect. Sickle-cell anemia is another example.

Whether an allele is lethal or not often depends on the environment in which the organism develops. Whereas certain alleles would be lethal in virtually any environment, others are viable in one environment but lethal in another. Human hereditary diseases provide examples. Cystic fibrosis is a disease that would be lethal without treatment, and individuals with PKU would not survive in a natural setting in which the special diet would be impossible. As another example, many of the alleles favored and selected by animal and plant breeders would almost certainly be eliminated in nature as a result of competition with the members of the natural population. Modern grain varieties provide good examples; only careful nurturing by the farmer has maintained such alleles for our benefit.

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Complex gene interactions in coat color

 The analysis of coat color in mammals is a beautiful example of how different genes cooperate in the determination of overall coat appearance. The mouse is a good mammal for genetic studies because it is small and thus easy to maintain in the laboratory, and because its reproductive cycle is short.

 It is the best-studied mammal with regard to the genetic determination of coat color. The genetic determination of coat color in other mammals closely parallels that of mice, and for this reason the mouse acts as a model system. We shall look at examples from other mammals as our discussion proceeds. At least five major genes interact to determine the coat color of mice: the genes are A, B, C, D, and S

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The A gene

 This gene determines the distribution of pigment in the hair. The wild type allele

A

produces a phenotype called

agouti.

Agouti is an overall grayish color with a brindled, or "salt-and-pepper," appearance. It is a common color of mammals in nature. The effect is caused by a band of yellow on the otherwise dark hair shaft. In the nonagouti phenotype (determined by the allele

a

), the yellow band is absent, so there is solid dark pigment throughout. The lethal allele

A Y

, discussed in an earlier section, is another allele of this gene; it makes the entire shaft yellow. Still another allele

a t

results in a "black-and-tan" effect, a yellow belly with dark pigmentation elsewhere.

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The B gene

 This gene determines the color of pigment. There are two major alleles,

B

coding for black pigment and

b

for brown. The allele

B

gives the normal agouti color in combination with

A

but gives solid black with

a/a

. The genotype

A

/- ;

b/b

gives a streaked brown color called

cinnamon,

and

a/a

;

b/b

gives solid brown.

 In horses, the breeding of domestic lines seems to have eliminated the

A

allele that determines the agouti phenotype, although certain wild relatives of the horse do have this allele. The color we have called

brown

in mice is called

chestnut

in horses, and this phenotype also is recessive to black.

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The C gene

   The wild-type allele

C

permits color expression, and the allele

c

prevents color expression. The

c/c

constitution is epistatic to the other color genes. The

c/c

animals are of course albinos. Albinos are common in many mammalian species and have also been reported among birds, snakes, and fish.

In most cases, the gene codes for the melanin-producing enzyme tyrosinase. In rabbits an allele of this gene, the

ch

(Himalayan) allele, determines that pigment will be deposited only at the body extremities. In mice the same allele also produces the phenotype called

Himalayan

, and in cats the same allele produces the phenotype called

Siamese

.

The allele

ch

can be considered a version of the

c

allele with heat sensitive expression. It is only at the colder body extremities that

ch

functional and can make pigment. In warm parts of the body it is expressed just like the albino allele

c

. This allele shows clearly how the expression of an allele depends on the environment.

is

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The D gene

 The D gene controls the intensity of pigment specified by the other coat color genes. The genotypes

D/D

and

D/d

permit full expression of color in mice, but

d/d

"dilutes" the color, making it look milky. The effect is due to an uneven distribution of pigment in the hair shaft. Dilute agouti, dilute cinnamon, dilute brown, and dilute black coats all are possible. A gene with such an effect is called a

modifier gene

.

In horses, the D allele shows incomplete dominance. The figure shows how dilution affects the appearance of chestnut and bay horses. Cases of dilution in the coats of house cats also are commonly seen.

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The S gene

 The

S

gene controls the presence or absence of spots by controlling the migration of clumps of melanocytes (pigment-producing cells) across the surface of the developing embryo. The genotype

S

/- results in no spots, and

s/s

produces a spotting pattern called

piebald

in both mice and horses. This pattern can be superimposed on any of the coat colors discussed earlier, with the exception of albino, of course. Piebald mutations are also known in humans.

We see that the normal coat appearance in wild mice is produced by a complex set of interacting genes determining pigment type, pigment distribution in the individual hairs, pigment distribution on the animal's body, and the presence or absence of pigment. Such interactions are deduced from modified ratios in dihybrid crosses. The figure illustrates some of the pigment patterns in mice.

Interacting genes such as these determine most characters in any organism

.

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Modifier genes

  Modifier gene action can be based on many different molecular mechanisms. One case involves regulatory genes that bind to the upstream region of the gene near the promoter and affect the level of transcription. Positive regulators increase ("up regulate") transcription rates, and negative regulators decrease ("down-regulate") transcription rates.

As an example, consider the regulation of a gene

G. G

is the normal allele coding for active protein, whereas

g

is a null allele (caused by a base-pair substitution) that codes for inactive protein. At an unlinked locus,

R

codes for a regulatory protein that causes high levels of transcription at the

G

locus, whereas

r

yields protein that allows only a basal level. If a dihybrid

G/g

;

R/r

is selfed, a 9:3:4 ratio of protein activity is produced, as follows:

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Penetrance and Expressivity



Penetrance

is defined as the percentage of individuals with a given genotype who exhibit the phenotype associated with that genotype. For example, an organism may have a particular genotype but may not express the corresponding phenotype because of modifiers, epistatic genes, or suppressors in the rest of the genome or because of a modifying effect of the environment. Alternatively, absence of a gene function may intrinsically have very subtle effects that are difficult to measure in a laboratory situation.

 Another term for describing the range of phenotypic expression is called

expressivity

. Expressivity measures the extent to which a given genotype is expressed at the phenotypic level. Different degrees of expression in different individuals may be due to variation of the allelic constitution of the rest of the genome or to environmental factors. This figure illustrates the distinction between penetrance and expressivity.

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