1. 1. ) one gene one enzyme hypothesis. 2
1.) one gene one enzyme hypothesis. 2.) Analysis of mutations in biochemical pathways
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2. The Central Dogma of Molecular Genetics
DNA
Replication – in the nucleus
RNA
Transcription- prod. In the nucleus- travels to cytoplasm
Protein
Translation- occurs in the cytoplasm

3. 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.

4. 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.

5. 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.

6. 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.

7. Fig. 1 Life cycle of the haploid, mycelial-form fungus Neurospora crassa

8. 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 2)

9. Fig. 2 Method devised by Beadle and Tatum to isolate auxotrophic mutations in Neurospora

10. 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.

12. Fig. 3 Methionine biosynthetic pathway showing four genes in Neurospora crassa that code for the enzymes that catalyze each reaction

13. “One Gene-One Enzyme Hypothesis” “One Gene-One Polypeptide Hypothesis”
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.”
“One Gene-One Enzyme Hypothesis”
“One Gene-One Polypeptide Hypothesis”

14. We cannot study the effects of more than one mutated gene at a time
Mutant analysis: first make sure each mutant we study has one and only one mutated gene
We cannot study the effects of more than one mutated gene at a time
Ensure heritable single gene mutants by performing specific mating bread mold experiments
Mate wild type (A) and mutant (a) together:
produces A/a ascus
A/a ascus undergoes meiosis and 1 round of mitosis
produces 4 A and 4 a “ascospores
Ascospores can be germinated into 8 babies
Examine the babies: if 4 babies are wild-type (A), and 4 are mutant (a), by definition there is a mutation in a SINGLE gene

15. Life cycle of Neurospora crassa

16. The linear meiosis of Neurospora

17. Out of the single-gene mutants, how do we pick the ones in the same pathway? Done by testing different growing conditions (media):

18. Wild-Type (non-mutated) Neurospora is prototrophic
Needs only minimal media with:
Inorganic salts (including Nitrogen source)
Organic Carbon source (sugar)
Biotin (a vitamin)

19. Minimal media Complete media Supports growth of wild-type organisms
Wild-type organisms can make whatever is not supplied using raw materials in minimal media
Requires functional genes to make functional protein enzymes
Complete media
Has everything plus the kitchen sink
Supports wild-type and auxotrophic mutants
Supplies anything mutants cannot make due to faulty genes/enzymes

20. Auxotrophic Mutant Mutants are produced by X-ray exposure
Already seen how potential mutants are mated to wild- type strain to ensure X-ray damage is heritable and in only one gene
And how progeny spores that can be germinated
Auxotrophic mutants can no longer grow on minimal medium
In order to grow, mutant needs
minimal media PLUS particular nutrient supplement to overcome the gene mutation
Or can grow on complete medium (not diagnostic)

21. Method to isolate auxotrophic mutations in N. crassa

22. How do we pick the correct auxotrophic mutants?
Consider the biosynthetic pathway:
A > B > C > D > E
Bread mold needs E to live
Wild type bread mold can make E from D from C from B from A in minimal media
But only if all genes/enzymes are OK
If any one step (>) in the pathway leading to E is blocked (due to a mutation in that enzyme gene), no E is made and the mutant dies on minimal media
So for this pathway, test mutants to see if they will live when given E
If adding E overcomes the mutation: yes the success
If not, it must be a mutant in a different pathway

23. E.g. Methionine (E) Biosynthesis
Involves 4 protein enzymes in a linear pathway
Encoded by 4 wild-type DNA genes: met-2+, met-3+, met-5+, and met-8+:
(met-5+) (met-3+) (met-2+) (met-8+)
enz enz enz enz 4
A B C D E
(substrate) (intermediates) (end
product
Methionine)

24. 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 :-)
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25. Phenylketonuria
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 given).
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.

26. Fig. Phenylalanine-tyrosine metabolic pathways

27. 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.

28. Lesch-Nyhan Syndrome
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.

29. Tay-Sachs Disease
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.given)
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.

30. Fig. The biochemical step for the conversion of the brain ganglioside GM2 to the ganglioside GM3, catalyzed by the enzyme N-acetylhexosaminidase A (hex A)

31. 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.

32. 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.

33. 3. Linus Pauling and coworkers (1949) used electrophoresis (Figure given ) 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.

34. Fig. 4.9 Electrophoresis of hemoglobin variants

35. 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).

36. Fig. 4.10 The hemoglobin molecule

37. 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).

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

39. 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.

40. Fig. 4.12a Examples of amino acid substitutions found in  polypeptides of various human hemoglobin variants

41. Fig. 4.12b Examples of amino acid substitutions found in  polypeptides of various human hemoglobin variants

42. Cystic Fibrosis
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.

43. Fig. 4.13 Proposed structure for cystic fibrosis transmembrane conductance regulator (CFTR)

44. 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.

45. 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.