Chapter 4: Gene Function

1. Chapter 4: Gene Function • Genes code for enzymes, non-enzymatic proteins, and other functional molecules like RNA. • Beadle & Tatum’s “One Gene-One Enzyme Hypothesis” • Life cycle of Orange Bread Mold (Neurospora crassa) • Deduction of biochemical pathways using auxotrophic mutants • Human genetic disorders caused by mutations • enzyme deficiency disorders • protein disorders (non-enzymatic deficiencies) • Examples of adaptive evolution (on Monday!) • Some examples from my research

2. 1942: George Beadle and Edward Tatum • Discovered that genes act by regulating definite chemical events. • Studied relationships between genes and enzymes in the haploid fungus Neurospora crassa (orange bread mold). • One Gene-One Enzyme Hypothesis • Each gene controls synthesis/activity of a single enzyme. • “one gene-one polypeptide” 1958: George Beadle (Cal Tech) & Edward Tatum (Rockefeller Institute)

3. Life cycle of the haploid fungus Neurospora crassa (orange bread mold): • Neurospora reproduces in a web-like pattern with asexual spores called conidia, which are orange in color. • Neurospora is haploid, so mutations are spotted easily (unlike diploid organisms). • Life cycle is short, and grows on simple media (N, C, biotin). • Sythesizes all other substances from these basic nutrients. • Propagates vegetatively (asexually) by dispersal of mycelium and asexual spores (conidia), OR • Sexually, by means of two mating types, A and a (only A will mate with a and result in a gamete fusion and meiosis). • Meiosis leads to 4 haploid nuclei (2A, 2a) • After one round of mitosis, 8 ascospores (4A, 4a).

4. Life cycle of the haploid fungus Neurospora crassa (orange bread mold) Fig. 4.2:

5. Beadle’s and Tatum’s Experimental Method • Isolated mutant auxotropicNeurospora by irradiating conidia with X-rays and crossing with wild-type strain of the opposite mating type. auxotrophic mutant = unable to make a required nutrient • Mutant ascospores were germinated on complete media. • Progeny conidia (asexual spores) were tested on an array of minimal media, each with a different supplement to determine the type of nutritional mutation. e.g., amino acids and vitamins • Assuming that many genes interact in pathway for synthesis of amino acids and other cellular products, Beadle and Tatum then dissected the biochemical pathways. e.g., synthesis of the amino acid Methionine

6. Fig. 4.3:

7. Pathway can be deduced using the following logic: The mutant strain is blocked farther along in a pathway if fewer intermediate compounds permit the strain to grow. OR The mutant strain is blocked at earlier steps in the pathway if a larger number of intermediates enable the strain to grow.

8. Fig. 4.4

9. Beadle and Tatum proposed: “One Gene-One Enzyme Hypothesis” However, it quickly became apparent that… More than one gene can control each step in a pathway (enzymes can be composed of two or more polypeptide chains, each coded by a separate gene). Many biochemical pathways are branched. “One Gene-One Enzyme Hypothesis” “One Gene-One Polypeptide Hypothesis”

10. Post-modern formulation: One gene can actually code several different polypetides or RNA subunits, by the process of alternative splicing. Alternative splicing of different exon sequences in different combinations creates different types of related proteins. These may be tissue specific. Many different types of gene products are transcribed to RNA but not translated to protein (e.g., rRNA, tRNA, snRNA). Other types of RNA sequences also are important for development and regulation of gene expression (e.g., miRNAs, siRNA). Non-coding DNA sequences also function importantly in gene regulation (e.g., binding sites for transcription etc.). Ultimately, the # of genes in not so important. This # is remarkably conserved among species (e.g., Drosophila vs Human). How the genes are expressed is more essential.

11. Human genetic disorders with demonstrated enzyme deficiencies: • Effects may be simple or wide-reaching pleiotropic = wide-reaching • Three common examples: Phenylketonuria (PKA) Type I Albinism Tay-Sachs Disease • Learn more about genetic disorders at NIH’s website: Online Mendelian Inheritance in Man (OMIM) http://www.ncbi.nlm.nih.gov/omim/

12. Phenylketonuria (PKU, OMIM-261600) • Mutation in the gene for phenylalanine hydoxylase, which prevents the conversion of phenyalanine to the amino acid tyrosine (phenylalanine is essential to the human diet). • Homozygous recessive: ~1 in 12,000 Caucasian births on chromosome 12. • Children born with PKU accumulate excess phenyalanine, which is converted to phenylpyruvic acid. • Results are damage to the nervous system: severe retardation, slow growth rate, and early death (prior to birth fetus is protected by maternal enzymes). • Pleiotropic effects: tyrosine is required for the production of the hormones thyroxine and adrenaline and the synthesis of melanin. • Can be managed by controlling dietary intake of phenylalanine beginning at 1-2 months after birth to ~10 years of age. • Pregnant women with PKU should maintain their diet through pregnancy, or their children will experience mental retardation independent of their genotype. • U.S. requires all newborns to be screened for PKU; the Guthrie test • “NutraSweet” contains phenylalanine

13. Type I Albinism (OMIM-203100) • Mutation affects the gene that codes for tyrosinase, which converts tyrosine to DOPA (dihydroxylphenylalanine), a precursor to melanin. • People with albinism produce no melanin. • Homozygous recessive: ~1 in 33,000 Caucasian births and 1 in 28,000 African Americans in the U.S. • No known problems resulting from accumulation of other precursors in the pathway. • Two other forms of albinism are known (Type II, Type III)

14. Tay-Sachs Disease (OMIM-272800) • Lysosomal storage disease caused by mutation in a lysosomal enzyme. • Homozygous recessive, occurs on chromosome 15. • Relatively rare, but common (~1 in 3,600) in Central European origin Jews. • Caused by a mutation in the HexA gene, which codes for the enzyme N-acetylhexosaminidase. • Results in the accumulation of unprocessed gangliosides in brain cells. (ganglioside = complex glycolipid common in nerve membranes) • Infants experience acute reactions to sharp sounds, a spot on the retina, and rapid neurological degeneration beginning at one year of age. • Followed by paralysis, loss of hearing, inability to feed, and death at age 3-4. • No cure, but carriers (heterozygotes) can be detected by genetic screening. • High-frequency in Jewish population has led some to speculate that heterozygotes may have tuberculosis (TB) resistance, but not verified. • More likely that frequency of Tay-Sachs was driven to high levels by chance (genetic drift) following a population bottleneck.

15. Most enzymes are proteins, but not all proteins are enzymes: Examples of traits and diseases that result from mutations in genes coding for non-enzymatic proteins. • Genetics of ABO Blood Groups (trait) • Cystic fibrosis (disease) • Sickle-cell Anemia (disease)

16. Genetics of the Human ABO Blood Group • ABO blood groups discovered by Karl Landsteiner early 1900s. • Four phenotypes determined by three alleles, IA, IB, and i. • ABO locus produces RBC antigens by adding sugars to membrane glycolipids using enzymes called glycosyltransferases. • Most people produce a glycolipid called the H antigen. • Next, depending on allele types, -N-galactosamine is added to produce either an A antigen or B anitgen. • antigen = any molecule recognized as a foreign by an organism and stimulates the productions of antibodies. • antibodies = specific molecules that recognize and bind antigens. 1930: Karl Landsteiner (Rockefeller Institute)

18. ABO blood groups and their properties: Donor relationships: Type AB produce A & B antigens and can be transfused only to blood types AB (universal recipients). Type B produce B antigen and can donate to type B and AB. Type A produce A antigen and can donate to type A and AB. Type O produce no antigen and can donate to all blood types (universal donors).

19. Cystic fibrosis (OMIM-219700): • Most common genetic disease in people of European descent, occurs in ~1 in 2,000 (1 in 23 are carriers). • Affects the pancreas, lungs, digestive system, and sometimes the vas deferens in males, resulting in sterility. • Characterized by abnormally viscous mucus and frequent lung infections. Life expectancy is generally ~40 years. • Autosomal recessive deletion on chromosome 7, commonly 3 base pairs. • Heterozygous state may confer resistance to diseases involving a loss of body fluid (i.e., resistance to cholera or TB). • Three base pair deletion (deletion of a codon) results in the deletion of an amino acid at an ATP binding site in the CFTR protein = Cystic Fibrosis Transmembrane Conductance Regulator • Examples of common allelic variants: • PHE508 DELETION ASP110 HIS • ILE507 DELETION ARG117 HIS • GLN493 STOP CODON ARG347 PRO

20. Sickle-cell anemia (OMIM-141900): • First described by J. Herrick (1910): discovered that red blood cells (RBCs) change shape to form a sickle under low oxygen pressure. • Sickle-shaped RBCs are fragile and less flexible than normal RBCs, resulting in anemia, blocking capillaries, and damaging tissues. • Results from an amino acid substitution in the 6th amino acid of the  chain of the hemoglobin molecule (hemoglobins are composed of four polypeptides (2  and two  chain), each associated with a heme). • Effects include: anemia, damage to the extremities, heart, lung, brains, kidney, GI tract, muscles, and joints. • Heterozygotes produce both normal and sickle-shaped RBCs and show a sickle-cell trait, but it is a much milder form of the disease. • Sickle-cell trait also protects heterozygotes against malaria (when the malarial parasite Plasmodium falciparum infects a sickle-cell, the RBC and parasite are destroyed, resulting in a lower parasite count). • In tropical Africa, as many as 20-40% of people are heterozygotes.

21. Fig. 4.9

22. Fig. 4.8

23. Fig. 4.10 Glu = glutamic acid (acidic amino acid with a negative charge) Val = valine (nonpolar amino acid with no electrical charge)

24. What happens when the heterozygote confers resistance to a disease? Heterozygote superiority: Heterozygote has higher fitness than either homozygotes, and both alleles are maintained in the population because the heterozygote genotype is favored (e.g., sickle cell trait). Also known as: heterosis or overdominance Distribution of malaria and Hb-S allele.

25. Fig. 4.10a Examples of amino acid substitutions found in  polypeptides of various human hemoglobin variants Fig. 4.11