Transcript Genetica per Scienze Naturali aa 05

1. One-to-many relationship of phenotypes to genes
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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.
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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.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
2. The complementation test
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The allelism test that finds widest application is the complementation
test, which is illustrated in the following example.
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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.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
3. Performing the complementation test
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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 wildtype allele is a+. These alleles could have different mutant sites but would be
functionally identical. The heterozygote a’/a" would be
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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:
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
4. Mutants that complement
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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:
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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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Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
5. The biochemical explanation
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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:
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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:
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
6. 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).
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
7. Complementation in human genetic diseases
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Profound congenital hearing loss is often genetic, and when genetic it is
usually autosomal recessive. However, when two people with autosomal
recessive profound hearing loss marry, as they often do, the children usually
have normal hearing.
This is an example of complementation. The children will have normal
hearing whenever the parents carry mutations in different genes. Diseases
and developmental defects represent the failure of a pathway. It is easy to
see that many different genes would be needed to construct so exquisite a
machine as the cochlear hair cell, and a defect in any of those genes could
lead to deafness. Such locus heterogeneity is only to be expected in
conditions like deafness, blindness or mental retardation, where a rather
general pathway has failed; but even with more specific pathologies,
multiple loci are very frequent.
A striking example is Usher syndrome, an autosomal recessive combination
of hearing loss and retinitis pigmentosa, which can be caused by mutations
at eight or more unlinked loci
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
8. Complementation test for rare human genetic diseases
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Genetic complementation for rare and invalidating genetic diseases
cannot be observed in human pedigree, because affected people do not
marry
However, some mammalian somatic cells can be
cultured in a well-defined medium. In addition,
cultured cells can be fused to produce somatic
hybrids; although cell fusion occurs spontaneously
at very low rate, it can be increased in the presence
of certain viruses that have a lipoprotein envelope
similar to the plasma membrane of animal cells. A
mutant viral glycoprotein in the envelope promotes
cell fusion. Cell fusion is also promoted by
polyethylene glycol, which causes the plasma
membranes of adjacent cells to adhere to each other
and to fuse.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
9. An early demonstration of complementation in fused
human cells
As most fused animal cells undergo cell division, the nuclei eventually fuse, producing
viable cells with a single nucleus that contains chromosomes from both “parents.” It is
even possible to fuse cells from different species
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
9. Xeroderma pigmentosum
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Cell fusion is the basis for the complementation test in human; if a
genetic defect is assayable in cultured cells, complementation analysis
by cell fusion can be undertaken.
For example, the autosomal recessive disease xeroderma
pigmentosum (XP) involves defects in repair of UV-induced damage
in DNA. Patients are abnormally sensitive to sunlight, developing skin
cancer after relatively brief exposure.
Multiple basocellular carcinomas on
the face of an XP patient. Thick arrow
points to a recent lesion, and thin
arrow to a scar of an old lesion
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
10. First demonstration of complementation groups in XP
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By fusing fibroblasts from various patients with XP, seven main
complementation groups have been defined
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
11. Different sensitivity to UV radiation of cells from different
complementation groups
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Hypersensitivity to UV radiation of XP cells in culture. Here the cells
from a number of complementation groups are shown. There is a
variation between complementation groups, but all are more sensitive
to UV radiation than are normal cells. The difference in UV
photosensitivity between normal and diseased cells is evident from the
survival curves of cultured cells treated with UV light
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini