CHAPTER 4 Gene Function
• There is a specific relationship between genes and enzymes, initially embodied in the one gene-one polypeptide hypothesis. • Since some enzymes consist of more than one polypeptide chain and each gene codifies for only one polypeptide chain, the hypothesis became one gene-one polypeptide. • Many human diseases are caused by enzyme deficiency (usually recessive traits). • There is a large amount of evidence that genes control the structure of all proteins.
A brief review of protein structure. • The building blocks of proteins are amino acids. • An amino acid is a molecule that has an carboxyl group and an amino group. R represents the radical or rest of the molecule.
Amino group
Carboxyl (acid) group
There are 20 different amino acids. These can be non-polar (hydrophobic), polar and electrically charged. Those that are electrically charged can be acidic (2 carboxyl groups) or basic (2 amino groups)
• Cells link amino acids together by dehydration synthesis • The bonds between amino acid monomers are called peptide bonds
A protein’s primary structure is its amino acid sequence. Secondary structure is polypeptide coiling or folding produced by hydrogen bonding. Tertiary structure is the overall shape of a polypeptide. Quaternary structure is the relationship among multiple polypeptides of a protein
Primary structure Amino acid Secondary structure
Hydrogen bond Alpha helix
Pleated sheet
Tertiary structure Polypeptide (single subunit of transthyretin)
Quaternary structure Transthyretin, with four identical polypeptide subunits
Garrod’s hypothesis of Inborn errors in Metabolism. In 1902 Archibald Garrod studied Alkaptonuria, an inherited condition that causes urine to turn black when exposed to air. Ochronosis, a buildup of dark pigment in connective tissues such as cartilage and skin, is also characteristic of the disorder. People with alkaptonuria typically develop arthritis in adulthood.
Garrod and William Bateson (1902) concluded alkaptonuria is genetically determined because: a. Families with alkaptonuria often have several affected members. b. Alkaptonuria is much more common in first cousin marriages than marriages with unrelated partners.
• 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). • Garrod’s work was the first evidence of a specific relationship between genes and enzymes. He also studied three other human diseases and provided further evidence. Its most important contribution was the insight that a mutation can block a human metabolic pathway by damaging an enzyme, causing a detectable buildup of that enzyme’s substrate.
*
* Phenyalanine
and Tyrosine are amino acids found in the dietary protein
*
Not found in normal urine. It turns black in contact with air.
How common is alkaptonuria? • The condition is rare, affecting 1 in 250,000 to 1 million people worldwide. Alkaptonuria is more common in certain areas of Slovakia (where it has an incidence of about 1 in 19,000 people) and in the Dominican Republic. What genes are related to alkaptonuria? • Mutations in the HGD gene prevent the synthesis of the enzyme that catalyzes the breakdown of homogentisic acid. How do people inherit alkaptonuria? • This condition is inherited in an autosomal recessive pattern. Most often, the parents of an individual with an autosomal recessive disorder are carriers (heterozygous) and do not show signs and symptoms of the disorder.
The one-gene-one enzyme hypothesis • In 1942 Beadle and Tatum leaded the beginning of biochemical genetics, a branch of genetics that combines both disciplines to explain the nature of metabolic pathways. • Beadle and Tatum used a haploid fungus, Neurospora crassa (the orange mold that grows on bread).
Neurospora as a Model organism. • The mold Neurospora has a life cycle well suited to its use as a model organism in genetics. First of all, the organism spends most of its life cycle as a haploid organism. This means it is possible to study the expression of genes without worrying about dominance or recessive alleles. • The fungus has alternate mating strains, here called type A and type a. Mating can only take place between different mating strains and the result is a diploid cell in a long sac. • Because of the ascus the results of segregation during metaphase 1 are kept in order.
• Another advantage of neurospora is that in addition to ascospores, the fungus also produces asexual spores that allow scientists to clone any interesting Neurospora genotypes. • Next, the life cycle of Neurospora, is quite rapid requiring about 2 weeks, allowing scientists to rapidly conduct experiments. • Wild-type Neurospora needs only simple minimal media with: – Inorganic salts (including a nitrogen source). – An organic carbon source (such as glucose or sucrose). – Biotin (a vitamin).
• To grow on minimal media, wild-type Neurospora synthesizes all organic molecules it needs for growth. However if a mutation is introduced that impedes the production of one of these molecules, it will not grow unless the molecule is added to the minimal media . Organisms that grow in minimal media are prototrophs. • Auxotrophs or auxotrophic mutant are the type of mutant that are unable to make a needed nutrient and will grow only if the growth media is supplemented with the nutrient. • A complete growth medium includes all 20 amino acids. Most nutritional mutants can survive in this media.
• Wild type conidia (asexual spores) were irradiated with x rays. After irradiation, they were germinated and cultured on maximal media so that all Neurospora carrying possible mutations resulting from the x rays would grow. • These irradiated Neurospora were crossed with wild type Neurospora to produce asci containing segregated products of meiosis. • The ascosposres were then isolated and grown on complete media. Many hundreds of tubes are used for this step. • Once the cultures were mature, asexual spores were isolated from each tube and grown on minimal medium. Failure of a specific spore to grow on minimum medium indicates the presence of a mutant unable to synthesize a required compound from the raw materials in the minimum medium.
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).
They started with a set of methionine auxotrophs, and found that four genes are involved, met-2+, met-3+, met-5+, met-8+ .
• They checked each of these mutants on a series of minimal media, each supplemented with a different chemical believed to be involved in the pathway. They 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. • They were able to deduce the pathway of methionine synthesis, and to correlate mutations with enzymes used in the pathway.
• Beadle and Tatum’s famous conclusion from this 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, as one gene–one polypeptide.
Genetically Based Enzyme Deficiencies in Humans • Single gene mutations are responsible for many human genetic diseases. Some mutations create a simple phenotype, while others are pleiotropic (affect many traits) (Table 4.2). The lack of the enzyme that transforms Phenylalanine into tyrosine produces PKU. The lack of the enzyme that transforms the tyrosine into DOPA, a precursor of melanine produces albinism.
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). Phenylalanine is an essential amino acid, but in excess it is harmful, and so it is normally converted to tyrosine. Excess phenylalanine affects the CNS, causing mental retardation, slow growth, and early death. 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.
Albino British Blackbird. Photographed at the Natural History Museum, South Kensington, London.
Adelie Penguin at Cape Bird Antarctica Name: Copito de Nieve / Floquet de Neu / Snowflake / Nfumu Ngui Special characteristics: he was the first known albino gorilla
Albinism •
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. This enzyme defficiency is also found in a large number of animals.
•
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.
Gene Control of Protein Structure • 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 is an example of how genes control protein structure. • J. Herrick (1910) first described sickle-cell anemia, finding that red blood cells (RBCs) change shape (form a sickle) under low O2 tension . Sickled RBCs are fragile and less flexible than normal, hence the anemia and capillaries blockage.
• 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.
• The genes bA and bS are codominant. Hemoglobin of bA/bA individuals has normal b subunits, while hemoglobin of those with the genotype bS/bS has b subunits that sickle at low O2 tension. Hemoglobin of bA/bS individuals is 1⁄2 normal, and 1⁄2 sickling form. These heterozygotes may experience sickle-cell symptoms after a sharp drop in the oxygen content of their environment.
• The genes bA and bS are codominant. Hemoglobin of bA/bA individuals has normal b subunits, while hemoglobin of those with the genotype bS/bS has b subunits that sickle at low O2 tension. Hemoglobin of bA/bS individuals is 1⁄2 normal, and 1⁄2 sickling form. These heterozygotes may experience sickle-cell symptoms after a sharp drop in the oxygen content of their environment.
