“The show so far”
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
There are “particles of inheritance” (units of segregation) …
… that pass “unchanged” through generations …
… and behave according to certain laws …
… and inside the nucleus, which is the part of the cell responsible
for heredity, there are chromosomes …
… which behave a certain way during meiosis …
… and the two sets of behaviors are remarkably concordant …
… because the particles (“genes”) actually physically reside on
the chromosomes …
… as one can prove by the study of eye color inheritance in fruit
flies …
… and furthermore, a given chromosome carries more than one
gene, as discoved when linkage was observed …
… and the arrangement of genes on a chromosome is a linear
one, with a specific, fixed genetic “distance” separating one gene
from another …
… that one can experimentally measure by crosses.
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This week:
what we learn about “the truth” from
“exceptions” to the story
1.
A particular gene: multiple alleles; complementation
criterion for allelism and implications for what a gene
is.
2.
A particular trait:
1.
2.
3.
Gene-gene interactions  expressivity  epistasis
Gene-environment interactions  penetrance  norm of
reaction
Gene and ?  epigenetics
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More on what a gene is:
allelic series
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Thalassemia (from the
Greek word for “the
Mediterranean sea”) –
a hemoglobinopathy
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Two perspectives
on the same thing
Complementation test: a way to define, what a gene is. (“A
gene is a unit of genetic complementation”)
If two recessive mutations that exhibit the same phenotype
FAIL to complement each other, then they are in the
same gene (note that this defines what a “gene” is – it’s
the entire entity, recessive mutations in which do not
complement each other).
If two recessive mutations that exhibit the same phenotype
DO complement each other, they are in different genes
(by definition).
What, exactly, is “complementation”?
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Complementation test
“Complementation is the production of a
wild-type phenotype when two haploid
genomes bearing different recessive
mutations are united in the same cell.”
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Edward Lewis
(NP 1995)
The cis-trans test
(aka complementation test)
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Orgo
cis-2-butene
trans-2-butene
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The cis-trans test, 1949:
lozenge (M. Greene)
Two different recessive mutants, both with the same
phenotype (small eyes and fused facets).
What is the relationship of these two mutations to each
other?
Make two different fly lines and compare their phenotypes.
Cis:
Trans:
wt
wt
wt
lz(g)
lz(BS)
lz(g)
lz(BS)
wt
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Cis:
Trans:
wt
wt
wt
lz(g)
lz(BS)
lz(g)
lz(BS)
wt
This is a control experiment.
The flies will be wild-type
regardless of whether
BS and g are in the
same gene or not.
If flies are normal, then
mutations are in different genes.
If the phenotype is still mutant,
then BS and g must be in the
same gene!!!
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Why this is useful and how this relates
to the earlier definition of a gene
Mendel lacked the ability to “engineer
chromosomes” and perform crosses of such
sophistication.
Hence, Mendel’s gene is a unit of segregation: the
minimal entity that moves through crosses and
results in F2 phenotypes segregating according
to Mendel’s laws is a gene.
The complementation test offers a crisper
definition of what a “gene” is and – as we know
from molecular analysis – highlights the
molecular complexity of gene architecture.
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18.12
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A remarkably useful aspect of
complementation testing
All traits that organisms exhibit – from an ability of
convert sugar to ATP (glycolysis, Krebs cycle,
oxidative phosphorylation), to the ability to not
bleed to death from a simple cut (blood clotting
cascade) – require the function of multiple
proteins, each encoded by a separate gene.
Complementation testing provides a powerful tool
to take a large number of organisms exhibiting
the same phenotype of interest and “bin” them
according to the specific gene that they have a
mutation in.
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Sex determination in mammals is a genetic process:
XY
XX+Sry transgene
Koopman et al. (1991) Nature 351: 117.
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Shmoo
Al Capp (1948) – Li’l Abner
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Mating type determination in yeast has both
a genetic and an epigenetic component
A wild-type haploid yeast cell contains
THREE copies of mating typedetermining genes:
• Copy #1: the a1 and a2 genes
(silent).
• Copy #2: the a1 and a2 genes
(also silent).
• Copy #3: An additional copy of
genes in item 1, or of the genes in
item 2, but active.
Whichever genes are contained in
copy #3 determines the mating
type.
This means that a haploid yeast cell –
whatever its mating type – has two
identical genes in its genome, one
of which is active, and the other –
silent!
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Rine schematic
mate to a cells
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
Fig. 18.14
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The data
• Colonies screened: 675,000
• Colonies that mated to a: 295
• Major complementation groups: 4
silent information regulators:
SIR1, SIR2, SIR3, SIR4
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
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A trait that results from
the action of 3 genes:
A  B  C = trait
Have two yeast strains: 1
and 2.
Let’s MATE 1 and 2 and
ask, is this diploid wildtype or mutant.
Well, if the mutation in 1
is in gene A, and in 2 – in
gene B, then the diploid
will look like this:
Aa Bb CC = wild-type
If the mutation in 1 and in
2 is in gene B, then the
diploid will look like this:
AA bb CC = mutant
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Resveratrol: 2.2 mil Google hits
Baur et al. Nature 444: 337.
Lagouge et al. Cell 127: 1109.
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“Simple” Mendelian
inheritance in humans
The complexity of the truth
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(A) Autosomal dominant;
(B) autosomal recessive
(C) X-linked recessive
(D) X-linked dominant
(E) Y-linked.
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Suzanne Vega
Solitude Standing
Songs in Red and Gray
Mitsuko Uchida
Mozart Piano Sonatas
Note: NOT concerti
Tracy Chapman
Where You Live
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Gene for starting businesses
“If you belong to a certain extended family in Seattle, you're
probably an entrepreneur. It seems to be about the only
career many of the members ever considered. ''It's in our
blood'' said Brian Jacobsen, president of Madison Park
Greetings, a stationery and gifts company. Mr. Jacobsen's
brother, mother, grandfather, two uncles, two cousins and
an aunt all started and ran their own companies and say
they cannot imagine any other livelihood.
Why are so many people in the same clan hooked? Some
of them have a theory. They believe that somewhere in
their chromosomes lurks an actual entrepreneurial gene -that their bent for business really is in their blood.”
New York Times, Nov. 20, 2003 – p. C8
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New York Times,
Nov. 20, 2003 – p. C8
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The God Gene
“Modern science is turning up a possible
reason why the religious right is flourishing
and secular liberals aren’t: instinct. It turns
out that our DNA may predispose humans
towards religious faith. … Dean Hamer, a
prominent American geneticist, even
identifies a particular gene, VMAT2, that
he says may be involved. People with one
variant of this gene tend to be more
spiritual, he found.”
N. Kristof, New York Times, 2-12-05
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A useful litmus test to distinguish pure
conjecture (“handwaving”) from
statements that are evidence-based
PubMed (via Google).
Search:
Hamer [au] AND vmat2
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Cancelled health insurance?
“Kevin McCormick called today. There’s
another lawsuit from the Weller family.
This time it’s the son of the deceased, Tom
Weller. … Apparently, his health insurance
got cancelled.”
“Because?”
“His father has the BNB71 gene for heart
disease.”
© 2006 Michael Crichton – Next
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“Gene Variant Is Linked to Common
Type of Stroke” NYT 1/9/07
Japanese researchers have identified a gene variant that appears to
predispose a person to strokes, but it seems more prevalent in
Asians than in people of European or African descent.
In a paper to be published next month in the journal Nature Genetics,
researchers write that the presence of the variant raised the risk of
cerebral infarction, the most common type of stroke, by 40 percent.
Cerebral infarction occurs when blood supply to a part of the brain is
obstructed, resulting in death or serious damage to brain cells. The
obstruction can be caused by a blood clot, a buildup of fatty deposits
in blood vessels or cancerous cells.
The researchers studied 1,112 Japanese and found that the variant of
the gene PRKCH turned up more often in people who had had
strokes. The variant also appeared to be linked to an enzyme,
rendering it more active.
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A reminder
from the Boss himself
Well, you may think the world's black and white
And you're dirty or you're clean
You better watch out you don't slip
Through them spaces in between
Born to Run
Born in the USA
The Rising
Bruce Springsteen
“Cross MCB140
My Heart”
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Sickle-cell anemia – a brief history
“In the western literature, the first description of
sickle cell disease was by a Chicago physician,
James B. Herrick, who noted in 1910 that a
patient of his from the West Indies had an anemia
characterized by unusual red cells that were
sickle-shaped.”
By 1923, it was realized the condition is
hereditary.
In 1949, Neel realized that patients with SCA are
homozygous, and heterozygous carriers have a
much milder condition (sickle cell trait).
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Sickle cell anemia
NIH:
“Sickle cell anemia is the most
common inherited blood
disorder in the United States,
affecting about 72,000
Americans or 1 in 500 African
Americans. SCA is
characterized by episodes of
pain, chronic hemolytic anemia
and severe infections, usually
beginning in early childhood.”
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Pleiotropy
Steinberg M. N Engl J Med 1999;340:1021-1030
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Linus Pauling, 1949: HbS has different charge!!
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V. Ingram, Nature 1956
“On [the existing] evidence alone, it is not
possible to decide whether the difference
between the proteins, which is in any event
small, lies in the amino-acid sequences of
the polypeptide chains, or whether it lies in
the folding of these chains leading to the
masking of some amino-acid side chains.”
V. Ingram (1956) Nature 178: 792.
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The third most-famous experiment
in the history of molecular biology
•
•
•
Digest Hb A and Hb S with trypsin
(protease – cuts hemoglobin into ~30
peptides).
Separate resulting fragments by
electrophoresis, and then by
chromatography.
Trace the peptide map.
V. Ingram (1956)
Nature 178: 792.
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V. Ingram (1956)
Nature 178: 792.
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Correct
“One can now answer at least partly the
question put earlier, and say there there is a
difference in the amino-acid sequence in
one small part of one of the polypeptide
chains. This is particularly interesting in view
of the genetic evidence that the formation of
hemoglobin S is due to a mutation in a
single gene.”
V. Ingram (1956) Nature 178: 792.
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RFLP!!!
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Incomplete dominance
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3.2
Haploinsufficient:
1. Take gene (Q).
2. Q = wild-type.
3. Complete lack of Q (let’s call that allele q) = mutant. Normally, a gene
that’s absent makes for a recessive allele (which makes sense – the
wild-type copy will still work, so the wild-type allele will be dominant over
the “gene is gone” BB King allele).
4. The heterozygote, however (eg Qq), has a mutant phenotype.
You have to have two alleles’ worth of protein to have a normal phenotype!
“When a gene is haploid, that’s not sufficient”
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Penetrance and expressivity
“The terms penetrance and expressivity quantify the
modification of the influence on phenotype of a particular
genotype by varying environment and genetic background;
they measure respectively the percentage of cases in which
a particular phenotype is observed when the specific allele
of a gene of interest is present and the extent of that
phenotype.”
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Variable expressivity
The importance of genetic
background; epistasis
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“Treatment Directed at the Relief
of Symptoms – Painful Episodes”
“In a given year, about 60 percent of patients with
sickle cell anemia will have an episode of severe
pain. A small minority of patients have severe
pain almost constantly. These differences are
one manifestation of the heterogeneity of this
disease, which complicates the choice of
treatment. Episodes of pain are sometimes
triggered by infection, extreme temperatures, or
physical or emotional stress, but more often they
are unprovoked and begin with little warning.”
Steinberg M. N Engl J Med 1999;340:1021-1030
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Man’s best friend – from a scientific
perspective as well
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Recessive epistasis (9:3:4)
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Dominant epistasis (13:3)
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Complementary gene action (9:7)
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Fig. 3.18
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“… the stadium capacity is now
officially listed as 75,662”
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Steinberg Curr Opin Hematol 13: 131
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“An SCN9A channelopathy causes
congenital inability to experience pain”
Nature Dec. 14, 2006
“The index case for the
present study was a tenyear-old child, well known to
the medical service after
regularly performing 'street
theatre'. He placed knives
through his arms and
walked on burning coals,
but experienced no pain. He
died before being seen on
his fourteenth birthday, after
jumping off a house roof.”
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So – let’s think about this
The small fraction of African-Americans who are
relatively pain-free …
… could they be heterozygous for a loss-offunction mutation in SCN9A?
In other words, could this be recessive epistasis?
If yes, could this suggest that a small-molecule
inhibitor of that specific pain receptor could be a
more effective analgesic for SCA patients than
God-awful parenteral morphine!
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Calling Michael Crichton
“Gene for …”?!
“Patients who are homozygous for the sickle hemoglobin
mutation can present with remarkably different clinical
courses, varying from death in childhood, to recurrent
painful vasoocclusive crises and multiple organ damage in
adults, to being relatively well even until old age. Increasing
numbers of genetic loci have now been identified that can
modulate sickle cell disease phenotype, from nucleotide
motifs within the beta-globin gene cluster, to genes located
on different chromosomes. With recent success of the
human genome project, it is anticipated that many more
genetic modifiers of sickle cell disease will be discovered
that can lead to the development of more effective
therapeutic approaches. The multigenic origin of the
variable phenotype in sickle cell disease will serve as a
paradigm for the study of variation in phenotypes of all
single gene disorders in man.”
Curr Opin Pediatr. 2001 Feb;13(1):22-7.
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The ob mouse
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Variable penetrance
The importance of the
environment AND genetic
background
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60-80%
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Hereditary breast cancer caused by
mutations in BRCA1 is incompletely
penetrant
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Risk of breast cancer and physical exercise in
BRCA1/2 mutation carriers: an example of how the
norm of reaction illuminates the modification of a
“genetic tendency” by the environment
“Physical exercise and lack of
obesity in adolescence were
associated with significantly
delayed breast cancer onset.”
M.-C. King et al. Science 2003
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Norm of reaction
A plot of carefully measured phenotype
in large pool of genetically identical
individuals grown under a range of
environments.
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D. Rio (UCB)
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Three mutants that affect facet #
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Norm of reaction of
different genotypes of
the bar locus to
temperature.Note, in
general, that wild-type
flies have a “tendency”
to have more facets
than the two mutants.
WT flies, however, have
less facets as the
temperature increases,
so one cannot claim that
the WT genotype
predisposes to more
facets! Further, ultrabar
“tends” to have less
facets than infrabar,
except at 15, where it
has slightly more facets.
This means that the
genetic “predisposition”
of ultrabar cannot be
stated in one sentence.
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Achillea millefollium (yarrow)
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Take 7 yarrow plants,
grow cuttings from
each one at different
elevations.
Measure each “child”
at each elevation.
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A fact, and a problem
Fact: what we do is a function of what we
know (and many other things, of course).
Problem: our knowledge comes in shades of
gray, but actions tend to be black-andwhite.
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People with insufficient education in genetics AND statistics
and not enough time to look at the primary data
Data:
1.
Policymakers.
2.
Health insurance company
officials.
3.
Health care providers (i.e.,
physicians).
4.
Journalists who write about
science and medicine for major
newspapers.
5.
The patients themselves.
Policy:
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