台大農藝系遺傳學 601 20000 Chapter 4 slide 1 CHAPTER 4 Gene Control of Proteins Peter J....

台大農藝系遺傳學 601 20000 Chapter 4 slide 1

CHAPTER 4Gene Control of

Proteins

Peter J. Russell

edited by Yue-Wen Wang Ph. D.Dept. of Agronomy, NTU

A molecular Approach 2nd Edition


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.


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.


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


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.

台大農藝系遺傳學 601 20000 Chapter 4 slide 6Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 4.2x Life cycle of the haploid, mycelial-form fungus Neurospora crassa


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).


Fig. 4.2 Method devised by Beadle and Tatum to isolate auxotrophic mutations in

Neurospora


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.


Fig. 4.3 Methionine biosynthetic pathway showing four genes in Neurospora crassa

that code for the enzymes that catalyze each reaction


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


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).


Phenylketonuriahttp://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.

http://www.genetic-counseling.com.tw/datebase/Lesch_Nyhan_syndrome_10.30.htm


Fig. 4.1 Phenylalanine-tyrosine metabolic pathways


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.


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) self-mutilation.

http://www.genetic-counseling.com.tw/datebase/Lesch_Nyhan_syndrome_10.30.htm


Tay-Sachs Diseasehttp://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 testing.

http://www.genetic-counseling.com.tw/datebase/Tay-Sachs%20disease-11.20.htm


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)


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.


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.


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.


Fig. 4.9 Electrophoresis of hemoglobin variants


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).


Fig. 4.10 The hemoglobin molecule


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).


Fig. 4.11 The first seven N-terminal amino acids in normal and sickled hemoglobin

polypeptides


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.


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.


Fig. 4.12a Examples of amino acid substitutions found in polypeptides of various

human hemoglobin variants


Fig. 4.12b Examples of amino acid substitutions found in polypeptides of various

human hemoglobin variants


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.


Fig. 4.13 Proposed structure for cystic fibrosis transmembrane conductance regulator

(CFTR)


Genetic Counseling1. 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.


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.


Fetal Analysis1. 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)


Fig. 4.14 Amniocentesis, a procedure used for prenatal diagnosis of genetic defects


Fig. 4.15 Chorionic villus sampling, a procedure used for early prenatal diagnosis of

genetic defects


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.

台大農藝系遺傳學 601 20000 Chapter 4 slide 1 CHAPTER 4 Gene Control of Proteins Peter J....

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Transcript of 台大農藝系遺傳學 601 20000 Chapter 4 slide 1 CHAPTER 4 Gene Control of Proteins Peter J....

台大農藝系 遺傳學 601 20000 Chapter 4 slide 1 CHAPTER 4 Gene Control of Proteins Peter J....

Documents

Transcript of 台大農藝系 遺傳學 601 20000 Chapter 4 slide 1 CHAPTER 4 Gene Control of Proteins Peter J....

台大農藝系遺傳學 601 20000 Chapter 4 slide 1 CHAPTER 4 Gene Control of Proteins Peter J....

Transcript of 台大農藝系遺傳學 601 20000 Chapter 4 slide 1 CHAPTER 4 Gene Control of Proteins Peter J....