POLIMORFISMI MOLECOLARI

POLIMORFISMI MOLECOLARI
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Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
Polimorfismi genetici prima del 1960
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Per più di metà del 1900 la variazione genetica osservabile in
natura che poteva essere interpretata a livello di singolo locus era
molto limitata
Il repertorio includeva il gruppo AB0 e le varianti emoglobiniche
nell'uomo, le variazioni del colore del mantello nei vertebrati e
delle colorazioni nelle chiocciole e nelle farfalle, oltre alle rare
mutazioni morfologiche osservabili nell'uomo e in Drosophila
Although they provided individual model systems for the study of
evolution in action, it was not clear how general a picture of
genetic variation they represented.
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
Polimorfismi cromosomici in Drosophila
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There were also extensive studies by Dobzhansky and his school of
inversion polymorphism in Drosophila
There was universal agreement that genomes totally homozygous
for one or more chromosomes were on the average lower in
viability and fecundity than were random heterozygotes
The problem was that the observations could not be interpreted at
the gene level
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Was the inbreeding effect the consequence of a few nearly
recessive deleterious alleles carried by each genome as a
consequence of the constant rain of mutations, or was it the
consequence of homozygosity at very large numbers of loci that
were normally heterozygous, held in heterozygous state in
natural populations by some form of balancing selection?
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
Due scuole di pensiero
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Evolutionary geneticists were divided into opposing schools with
more or less uncompromising views of the truth
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Dobzhansky and his followers belonged to what he called the
“balance” school, holding that every individual in a sexually
reproducing population was heterozygous at most or all of its loci.
Dobzhansky opponents were derogatorily called by him the
“classical” school, whose most influential spokesman was H. J.
Muller, a school that believed nearly all loci to be essentially
homozygous, with rare deleterious mutations segregating to produce
a “genetic load”
Population genetics seemed doomed to a perpetual struggle
between alternative interpretations of great masses of inevitably
ambiguous data
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
L'avvento dell'elettroforesi
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In 1966 two laboratories, one in Chicago (Lewontin) and one in
London (Harris), independently published experimental results that
apparently solved the problem, initiating 20 years of intensive
investigation of protein variation in natural populations by
hundreds of laboratories
They applied electrophoresis of proteins to measure allozyme
variation in natural populations of Drosophila and humans, and
identified some 30% of the studied proteins highly variable among
individuals
There was a virtual explosion of electrophoretic investigations. A
comprehensive literature search made 18 years after the first
experiments were published, found studies of intraspecific
variation in 1111 species, with an average of 23 loci and 200
individuals per species examined
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
Varianti allozimiche: un esempio
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Electrophoresis of tissue extracts from 15 different green treefrogs (Hyla cinerea)
reveals 4 allelic versions of the enzyme aconitase (one of the enzymes of the citric
acid cycle). The 4 alleles can be distinguished by the speed with which their protein
product migrates: Fast (F), moderately fast (E), medium (M), and slow (S)
The results: Eight frogs (#2, 3, 4, 6, 7, 9, 12, and 14) are homozygous for allele M.
Frog #8 was homozygous for allele E. Three frogs (#1, 11, 15) are heterozygous for
the M and S alleles. Two (#5, 13) are heterozygous for M and E.Frog #10 is
heterozygous for M and F.
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
Generalità della tecnica elettroforetica
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Electrophoresis is a technique that could be learned easily by any
moderately competent person, that is relatively cheap as compared
with most physiological and biochemical methods, that gives
instant gratification by revealing before one’s eyes the heritable
variation in unambiguously scoreable characters, and most
important, can be applied to any organism whether or not the
organism could be genetically manipulated, artificially crossed, or
even cultivated in the laboratory or greenhouse
A typical species population for most organisms is polymorphic for
about 1/3 of its loci that code for enzymes and other soluble
proteins
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
The meaning of genetic variation
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From the publication of the first results of electrophoretic surveys
of variation in 1966, the problem of the explanation of the variation
became primary
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is the large amount of standing genetic variation in populations a
consequence of some form of variation-preserving natural selection,
such as overdominance or frequency dependent selection
or is the variation simply what one would expect from the random
accumulation of selectively neutral mutations reaching intermediate
frequencies by genetic drift in finite populations accompanied by
some small migration between populations?
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
Neutralism vs selectionism
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The struggle between these two views of genetic variation was
evident from the beginning of electrophoretic studies
Lewontin and Hubby, already in 1966, pointed out the immense
genetic load that would exist in a population with 10%
heterozygosity if it were maintained by simple overdominant
selection, even very weak selection. Various more complex
selective schemes were immediately proposed to meet the difficulty
On the other hand, a theory of selectively neutral evolution of
protein differences between species was proposed by Kimura
(1968) and King and Jukes (1 969), and it was Kimura’s view that
electrophoretic polymorphism was simply a stage in this neutral
evolution of species differences.
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
Indecidibilità fra opposte teorie
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The old struggle between those who saw natural selection as the
preserver of variation and those who saw it as essentially a purifying
process, was transferred to the domain of electrophoretic polymorphism
Although no one could now deny that there was indeed a great deal of
genetic variation in natural populations, the assumption that this
variation was unselected made the observations perfectly compatible
with a view that when selection did occur, it was purifying in nature.
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
Inizio dell'era del DNA
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In 1970, Hamilton Smith discovered the first restriction enzyme
that cuts DNA at specific sites
In 1978, Yuet Wai Kan and Andrée-Marie Dozy were studying
patients with sickle-cell anemia. They noticed, after they used a
restriction enzyme to cut the DNA of affected subjects, that most of
the patients had a DNA fragment containing the beta-hemoglobin
gene that was 13 kb long, whereas normal subjects often lacked
this particular DNA fragment
Because the fragment produced was different in size from that
normally seen, it was called a restriction-fragment-length
polymorphism (RFLP).
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
PCR-RFLP
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A restriction fragment length polymorphism is defined by the
existence of alternative alleles associated with restriction fragments
that differ in size from each other
RFLPs are visualized by digesting DNA from different individuals
with a restriction enzyme, followed by gel electrophoresis to
separate fragments according to size
Coupled with PCR, restriction site polymorphisms can now easily
and efficiently typed; it is commonly applied to mitochondrial
DNA analysis
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini
RFLP analysis: example of a nuclear gene
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A 244-bp fragment of the apoE gene
covering the codons for amino acids
112 and 158 was amplified by
polymerase chain reaction using the
primer pair F4 and F6
After amplification, 8 µl of the PCR
product were directly digested with
12 units of the restriction enzyme
HhaI (Promega, Madison, WI) for 4
hrs at 37 °C
Gene fragments were separated using
8% polyacrylamide nondenaturing
gel electrophoresis (3 hrs, 45 mA)
and detected by ethidium bromide
staining under ultraviolet illumination
(0.2 mg/l, 30 mins), using a known
DNA size marker
Genotyping of apoE using polymerase chain reaction
and restriction fragment length polymorphism (PCRRFLP). Lane M, marker ladder; lane 1 to 6, e2/e2,
e2/e3, e2/e4, e3/e3, e3/e4, and e4/e4 genotypes,
respectively.
Genetica delle popolazioni
a.a. 11-12 prof. S. Presciuttini