Download CHAPTER 4 Gene Function

Peter J. Russell
A molecular Approach 2nd Edition
CHAPTER 4
Gene Control of Proteins
edited by Yue-Wen Wang Ph. D.
Dept. of Agronomy,台大農藝系
NTU
遺傳學 601 20000
Chapter 4 slide 1
Gene Control of Enzyme Structure
1. Genes encode proteins, including enzymes.
2. Genes work in sets to accomplish biochemical
pathways.
3. Genes often work in cooperation with other
genes.
4. These discoveries are the foundation of modern
molecular genetics.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 2
Garrod’s Hypothesis of Inborn Errors of
Metabolism
1. Alkaptonuria is a human trait characterized by urine blackening on exposure to air
and arthritis in later life.
2. Archibald Garrod and William Bateson (1902) concluded alkaptonuria is genetically
determined because:
a. Families with alkaptonuria often had several affected members.
b. Alkaptonuria is much more common in 1st cousin marriages than marriages with unrelated
partners.
3.Garrod showed that alkaptonuria results from homogentisic acid (HA) in the urine.
HA is absent from normal urine. Garrod reasoned that normal people metabolize
HA, but those with alkaptonuria do not because they lack the necessary enzyme. He
termed this an inborn error of metabolism (Figure 4.1).
4. The responsible mutation is recessive. The gene was later shown to be on
chromosome 3.
5. Garrod’s work was the 1st evidence of a specific relationship between genes and
enzymes. With the insight that a mutation can block a human metabolic pathway by
damaging an enzyme, causing a detectable buildup of that enzyme’s substrate, he
found a similar relationship in three other human diseases. His work, naturally, was
not appreciated by his contemporaries.
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Chapter 4 slide 3
The One Gene-One Enzyme Hypothesis
1. Genes act by regulating definite chemical events.
2. George Beadle and Edward Tatum (1942) showed
a direct relationship between genes and enzymes
in the haploid fungus Neurospora crassa. This led
to their one gene-one enzyme hypothesis, and a
share of the 1958 Nobel Prize in Physiology or
Medicine.
Animation: The One Gene-One Enzyme Hypothesis
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Chapter 4 slide 4
3. It is necessary to understand the life cycle of Neurospora crassa (orange
bread mold) to understand Beadle and Tatum’s work (Figure 4.2x).
a. Neurospora is a mycelial-form fungus with asexual spores. The spores are
called conidia. They are orange in color.
b. It is a haploid organism, so mutations are easily spotted.
c. Its life cycle is conveniently short.
d. Neurospora propagates asexually by dispersal of:
i. bits of mycelium.
ii. conidia.
e. It also propagates sexually by means of two mating types, A and a.
i. The two types are indistinguishable, except that A will not mate with A,
nor a with a.
ii. Only an A x a cross will result in gamete fusion, producing an A/a
diploid nucleus that quickly undergoes meiosis to produce four haploid
nuclei.
iii. After a round of mitosis, the ascus contains eight sexually produced
ascospores, each capable of forming a mycelium. Of the ascospores, four
are A and four are a.
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Chapter 4 slide 5
Fig. 4.2x Life cycle of the haploid, mycelial-form fungus Neurospora crassa
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 6
f. Wild-type Neurospora needs only simple minimal media with:
i. inorganic salts (including nitrogen source).
ii. an organic carbon source (such as glucose or sucrose).
iii. biotin (a vitamin).
g. To grow on minimal media, wild-type Neurospora synthesizes all
organic molecules it needs for growth. An auxotrophic mutant unable to
make a needed nutrient will only grow if that nutrient is provided as a
supplement in its medium.
4. Beadle and Tatum isolated auxotrophic mutants by mutating with X rays
and then crossing with a wild-type strain. The cross insured that effects
were due to inheritance, rather than direct damage from the radiation.
In their experiment:
a. One progeny spore per ascus was germinated in a complete medium so
that growth would occur regardless of nutritional mutations. Then
growth was transferred to minimal media, where auxotrophs won’t
grow.
b. Each mutant was then tested on an array of minimal media, each with a
different single supplement, to determine the type of nutritional
mutation (Figure 4.2).
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Chapter 4 slide 7
Fig. 4.2 Method devised by Beadle and Tatum to isolate auxotrophic mutations in
Neurospora
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 8
5. Beadle and Tatum assumed that many genes interact in
Neurospora cells. They reasoned that metabolism
proceeded by series of reactions, each catalyzed by an
enzyme, and organized into pathways. The analysis of
methionine biosynthesis is an example of the analytical
approach they used (Table 4.1):
a. Starting with a set of methionine auxotrophs, it was found that 4
genes are involved, met-2+, met-3+, met-5+, met-8+.
b. Checked each mutant on series of minimal media each
supplemented with a different chemical believed to be involved
in the pathway. Expected growth if providing a chemical used
after the metabolic block, so the earlier the mutated gene
functions in the pathway, the more supplements will support
growth.
c. Deduced the pathway of methionine synthesis, and correlated
mutations with enzymes used in the pathway.
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Chapter 4 slide 9
台大農藝系 遺傳學 601 20000
Chapter 4 slide 10
Fig. 4.3 Methionine biosynthetic pathway showing four genes in Neurospora crassa
that code for the enzymes that catalyze each reaction
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 11
6. Beadle and Tatum’s famous conclusion from this
type of experiment is that one gene encodes one
enzyme. Later work showed that some proteins
consist of more than one polypeptide, and that not
all proteins are enzymes. The principle is now
usually stated, “one gene-one
polypeptide.”
iActivity: Pathways to Inherited Enzyme
Deficiencies
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Chapter 4 slide 12
Genetically Based Enzyme Deficiencies in
Humans
1. Single gene mutations
are responsible for many
human genetic diseases.
Some mutations create a
simple phenotype, while
others are pleiotropic
(Table 4.2).
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Chapter 4 slide 13
Phenylketonuria
http://www.genetic-counseling.com.tw/datebase/PKU_10.21.htm
1. Phenylketonuria (PKU) is commonly caused by a mutation on chromosome 12 in the
phenylalanine hydrolase gene, preventing the conversion of phenylalanine into tyrosine
(Figure 4.1).
2. Phenylalanine is an essential amino acid, but excess is harmful, and so is normally converted to
tyrosine. Excess phenylalanine affects the CNS, causing mental retardation, slow growth and
early death.
3. PKU’s effect is pleiotropic. Some symptoms result from excess phenylalanine. Others result
from inability to make tyrosine; these include fair skin and blue eyes (even with brown-eye
genes) and low adrenaline levels.
4. Diet is used to manage PKU by providing just enough phenylalanine for protein synthesis, but
not enough that it accumulates. To be effective, the special diet must commence in the first
two months after birth, continue at least throughout childhood, and be resumed before
pregnancy in PKU women to avoid phenylalanine levels that would affect the fetus.
5. All U.S. newborns are screened for PKU using the Guthrie test:
a. A drop of blood on filter paper is placed on solid media containing β-2-thienylalanine and the
bacterium Bacillus subtilis.
b. Normally, β-2-thienylalanine inhibits growth of Bacillus subtilis.
c. Phenylalanine allows Bacillus subtilis to grow in the presence of β-2-thienylalanine, so bacterial
growth indicates high phenylalanine levels in the blood, and the possibility that the infant has PKU.
6. NutraSweet is aspartame, which breaks down to aspartic acid and phenylalanine, with serious
consequences for a phenylketonuric.
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Chapter 4 slide 14
Fig. 4.1 Phenylalanine-tyrosine metabolic pathways
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 15
Albinism
1. Classic albinism results from an autosomal
recessive mutation in the gene for tyrosinase.
Tyrosinase is used to convert tyrosine to DOPA in
the melanin pathway. Without melanin,
individuals have white skin and hair, and red eyes
due to lack of pigmentation in the iris.
2. Two other forms of albinism are known, resulting
from defects in other genes in the melanin
pathway. A cross between parents with different
forms of albinism can produce normal children.
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Chapter 4 slide 16
Lesch-Nyhan Syndrome
http://www.genetic-counseling.com.tw/datebase/Lesch_Nyhan_syndrome_10.30.htm
1. Lesch-Nyhan syndrome results from a recessive mutation on the X chromosome, in the gene for
hypoxanthine-guanine phosphoribosyl transferase (HGPRT). The fatal disease is found in males,
while heterozygous (carrier) females may show symptoms when lyonization of the normal X
chromosome leaves the X chromosome with the defective HGPRT gene in control of cells.
2. HGPRT is an enzyme essential to purine utilization. In Lesch-Nyhan syndrome this pathway is
highly impaired. Purines accumulate and are converted to uric acid.
3. Symptoms of Lesch-Nyhan syndrome:
a. Infants develop normally for several months. Orange uric acid crystals in diapers (of males) are only clue
of disease.
b. At 3–8 months, motor development delays lead to weak muscles.
c. Muscle tone is altered, producing uncontrollable movements and involuntary spasms.
d. At 2–3 years children show bizarre activity, such as compulsive self-mutilation that is difficult to control
and painful, as well as aggression toward others.
e. Lesch-Nyhan individuals score severely retarded on intelligence tests, possibly due to poor
communications skills.
f. Most Lesch-Nyhan individuals die before their 20s, typically from infection, kidney failure or uremia.
4. In the case of Lesch-Nyhan syndrome, a defect in a single enzyme, HGPRT, has very pleiotropic
effects, giving rise to uremia, kidney failure, mental deficiency and (so far inexplicably) selfmutilation.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 17
Tay-Sachs Disease
http://www.genetic-counseling.com.tw/datebase/Tay-Sachs%20disease-11.20.htm
1. Tay-Sachs is one of a group of diseases called lysosomal-storage diseases. Generally
caused by recessive mutations, these diseases result from mutations in genes
encoding lysosomal enzymes.
2. Tay-Sachs disease (aka infantile amaurotic idiocy) results from a recessive mutation
in the gene hexA, which encodes the enzyme N-acetylhexosaminidase A. The HexA
enzyme cleaves a terminal N-acetylgalactosamine group from a brain
ganglioside.(Fig. 4.5)
3. Infants homozygous recessive for this gene will have nonfunctional HexA enzyme.
Unprocessed ganglioside accumulates in brain cells, and causes various clinical
symptoms:
a. Infants have enhanced reaction to sharp sounds.
b. A cherry-colored spot surrounded by a white halo may be visible on the retina.
c. Rapid neurological degeneration begins about one year of age, as brain loses control of
normal functions due to accumulation of unprocessed ganglioside.
d. Progress is rapid, with blindness, hearing loss and serious feeding problems leading to
immobility by age 2.
e. Death often occurs at 3–4 years of age, often from respiratory infection.
4. The disease is incurable. Carriers and affected individuals can be detected by genetic
台大農藝系 遺傳學 601 20000
Chapter 4 slide 18
testing.
Fig. 4.5 The biochemical step for the conversion of the brain ganglioside GM2 to the
ganglioside GM3, catalyzed by the enzyme N-acetylhexosaminidase A (hex A)
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 19
Gene Control of Protein Structure
1. Genes also make proteins that are not enzymes.
Structural proteins, such as hemoglobin, are often
abundant, making them easier to isolate and
purify.
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Chapter 4 slide 20
Sickle-Cell Anemia
1. J. Herrick (1910) first described sickle-cell anemia, finding that red
blood cells (RBCs) change shape (form a sickle) under low O2 tension.
a. Sickled RBCs are fragile, hence the anemia.
b. They are less flexible than normal RBCs, and form blocks in capillaries,
resulting in tissue damage downstream.
c. Effects are pleiotropic, including damage to extremities, heart, lungs,
brain, kidneys, GI tract, muscles and joints. Results include heart
failure, pneumonia, paralysis, kidney failure, abdominal pain and
rheumatism.
d. Heterozygous individuals have sickle-cell trait, a much milder form of
the disease.
2. E.A. Beet and J.V. Neel independently proposed (1949) that sickle-cell
trait and disease were the result of a single mutant allele.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 21
Animation: Gene Control of Protein Structure and Function
3. Linus Pauling and coworkers (1949) used electrophoresis
(Figure 4.9) and showed:
a. Hemoglobin from individuals with sickle-cell anemia (Hb-S) has
altered mobility compared with normal hemoglobin (Hb-A).
b. Hemoglobin from individuals with the sickle-cell trait shows
equal amounts of Hb-A and Hb-S, indicating that heterozygotes
make both forms of hemoglobin.
c. Therefore, the sickle-cell mutation changes the form of its
corresponding protein, and protein structure is controlled by
genes.
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Chapter 4 slide 22
Fig. 4.9 Electrophoresis of hemoglobin variants
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 23
4. Hemoglobin is formed by four polypeptide
chains, two molecules of the α polypeptide and 2
of the β polypeptide, each associated with a heme
group (Figure 4.10).
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Chapter 4 slide 24
Fig. 4.10 The hemoglobin molecule
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 25
5. V.M. Ingram (1956) found that the 6th amino acid
of the β chain in sickle-cell hemoglobin is valine
(no electrical charge) rather than the negatively
charged glutamic acid in the β chain of normal
hemoglobin (Figure 4.11).
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Chapter 4 slide 26
Fig. 4.11 The first seven N-terminal amino acids in normal and sickled hemoglobin 
polypeptides
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 27
6. Outline of the genetics and gene products involved in
sickle-cell anemia and trait:
a. Wild-type β chain allele is βA, which is codominant with βS.
b. Hemoglobin of βA/βA individuals has normal β subunits, while
hemoglobin of those with the genotype βS/βS has β subunits that
sickle at low O2 tension.
c. Hemoglobin of βA/βS individuals is 1⁄2 normal, and 1⁄2 sickling
form. (The two β chains of an individual hemoglobin molecule
will be of the same type, rather than mixed.) These
heterozygotes may experience sickle-cell symptoms after a sharp
drop in the oxygen content of their environment.
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Chapter 4 slide 28
Other Hemoglobin Mutants
1. Screening of hemoglobin for altered
electrophoretic mobility has identified over 200
hemoglobin mutants, showing a variety of amino
acid substitutions in both the α and the β chains.
Each appears to derive from a single amino acid
change (Figure 4.12).
2. Most effects are not as severe as those seen in
sickle-cell anemia.
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Chapter 4 slide 29
Fig. 4.12a Examples of amino acid substitutions found in  polypeptides of various
human hemoglobin variants
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 30
Fig. 4.12b Examples of amino acid substitutions found in  polypeptides of various
human hemoglobin variants
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 31
Cystic Fibrosis (囊腫纖維症)
1. Cystic fibrosis (CF) affects the pancreas, lungs and digestive system,
and sometimes the vas deferens in males. The disease is characterized
by abnormally viscous secreted mucus, and lung complications are
managed by percussion and antibiotics to treat infections. Life
expectancy with current treatments is about 40 years.
2. The affected gene is on the long arm of chromosome 7, and encodes a
protein called cystic fibrosis transmembrane conductance regulator
(CFTR). Comparing DNA sequences of cloned gene from normal and
CF individuals shows that the CF mutation commonly is the deletion of
a specific 3-bp region, removing one amino acid from the protein
product.
3. The structure of the protein has been deduced from its sequence (Figure
4.13). CFTR has homology with a large family of active transport
membrane proteins.
4. Functional analysis shows that CFTR normally forms a chloride channel
in the cell membrane. The mutated gene results in an abnormal CFTR
protein, preventing chlorine ion transport and resulting in CF
symptoms.
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Chapter 4 slide 32
Fig. 4.13 Proposed structure for cystic fibrosis transmembrane conductance regulator
(CFTR)
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 33
Genetic Counseling
1. Genetic testing can detect many inherited enzyme and protein defects,
yielding information about whether an individual has a disease or is a carrier.
Chromosomal abnormalities can also be detected.
2. Genetic counseling is advice based on genetic analysis, focusing either on the
probability that an individual has a genetic defect, or the probability that
prospective parents will produce a child with a genetic defect. Genetic
counselors have the task of explaining diseases, probabilities and options to
affected individuals or parents.
3. Some aspects of human heredity are well understood, others not yet so well.
Effective genetic counseling requires up-to-the minute knowledge of genetic
research, and the ability to offer clients unbiased and nonprescriptive
information from two main sources:
a. Pedigree analysis is an important tool of genetic counseling, considering
phenotypes found in both families over several generations. This is
particularly useful for identifying suspected carriers.
b. Fetal analysis includes assays for enzyme activity or protein level, or
detection of changes in the DNA itself.
4. For most defective alleles, there is currently no way to change the resulting
phenotype, and so genetic counseling focuses primarily on informing clients
of risks and probabilities.
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Chapter 4 slide 34
Carrier Detection
1. A carrier is heterozygous for a recessive gene
mutation. In a cross between two carrier parents,
1⁄4 of the offspring are expected to develop the
disease, and 1⁄2 to also be carriers.
2. The carrier’s phenotype is normal, but if levels of
the affected protein are determined, they may be
well below those of a normal individual.
3. Detection of carriers by direct DNA testing is
discussed in Chapter 8.
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Chapter 4 slide 35
Fetal Analysis
1. Genetic counseling is also concerned with whether a fetus
is normal. A sample of fetal cells is needed for the
analysis. There are currently two methods of obtaining the
necessary sample:
a. Amniocentesis is removal of a sample of amniotic fluid using a
syringe needle inserted through the uterine wall (Figure 4.14).
The procedure is seldom done before the 12th week of
pregnancy, due to small amounts of amniotic fluid and risk to the
fetus.
b. Chorionic villus sampling can be done in the 8th–12th weeks of
pregnancy, by removal of chorionic villus tissue either through
the abdomen as in amniocentesis, or via the vagina (Figure 4.15)
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Chapter 4 slide 36
Fig. 4.14 Amniocentesis, a procedure used for prenatal diagnosis of genetic defects
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 37
Fig. 4.15 Chorionic villus sampling, a procedure used for early prenatal diagnosis of
genetic defects
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 38
2. Once fetal cells are obtained they are usually cultured in
the laboratory, although chorionic villus sampling may
provide enough tissue to assay directly. They are examined
for protein or enzyme alterations or deficiencies, DNA
changes and chromosomal abnormalities.
3. Amniocentesis is costly and cannot be performed until the
second trimester, removing early abortion as an option in
cases of severe genetic defects. Chorionic villus sampling
can be done earlier, but carries a higher risk of fetal death
and inaccurate diagnosis due to the presence of maternal
cells.
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Chapter 4 slide 39