Textbook Assignment: Chapter 6, Pages 156-175
Major concepts:
Introduction: Chapter 6 of our textbook presents an integrated view of gene expression with emphasis on functional proteins as the determinants of phenotypes. The chapter begins with an analysis of the human beta-globin gene and the biological function of the protein it encodes, beta-globin. Pathologies caused by a beta-globin missense mutation and by genetically determined absence or reduced levels of synthesis of beta globin are also discussed. The rest of the chapter is devoted to biochemical pathways and various lines of evidence concerning the one-gene:one-enzyme concept (which we will refine later in the semester to one coding unit (cistron):one peptide chain). At the end of these notes, I have retained a brief section on the ability of prions, which consist of normal proteins with altered patterns of folding, to cause pathology and function as infectious agents with no apparent nucleic acid genomes.
Beta globin -- gene to functional protein: Figure 6.2 presents the genomic sequence for human beta globin, including promoter, introns, and downstream sequences well beyond the polyadenylation cut point. A comparison with figure 3.9 shows conservation from mouse to human of key promoter sites, as well as the first few nucleotides transcribed adjacent to the cap site. The text points out consensus sites and individualized variations from them in the promoter, the polyadenylation signal, and the splicing signals for the two introns contained within the gene. The processed mRNA codes for a peptide containing 147 amino acids. The N-terminal methionine is subsequently cleaved off, leaving a mature beta-globin of 146 amino acids. Two-beta globulins and two alpha-globins combine into a quaternary structure that contains a hydrophobic pocket that is occurped by a heme molecule. The heme molecule binds and carries oxygen. This process is aided by the hydrophobic nature of the pocket. In the capillaries, carbon dioxide binds to arginine molecules locted near the carboxyl ends of the alpha subunits. This alters the protein confirmation and reduces the affinity for oxygen, which is expelled. A reverse exchange occurs in the high oxygen, low carbon dioxide environment of the lungs.
Sickle-cell anemia: One of the early pieces of evidence that genes code for amino acid sequences came from the discovery in 1957 that the mutation responsible for sickle cell anemia caused a single amino acid substitution in human beta globin. Sickle-cell anemia is a hereditary disease that causes red blood cells to become distorted in shape under conditions of low oxygen tension. These studies showed that one amino acid in position 6 (from the amino terminus) of the mature protein had been changed by the mutation from a glutamic acid to a valine. We now know that a one nucleotide change in the coding sequence, from GAG to GTG replaces the codon specifying glutamic acid with the codon specifying valine. That substitution causes the hemoglobin to precipitate into fibrous aggregates (figure 6.5) that distort the shapes of red blood cells (figure 6.6) under low-oxygen conditions, resulting both in blockage of capillary circulation and breakage of the red blood cells. The aggregation appears to be caused by interaction of the hydrophobic side chain of valine with the hydrophobic heme pocket when it is not occupied by oxygen (Figure 6.4).
Heterozygote advantage: Near the end of the semester, we will explore the mechanism that has allowed sickle cell anemia, which is a severe genetic disease, to become quite common in populations living in areas with a high incidence of malaria. In brief summary, individuals who are heterozygous for sickle cell anemia (possessing one copy of the sickle cell gene and one normal beta globin gene) are resistant to malaria and thus have a substantial survival advantage over either type of homozygoous individuals. Those who are homozygous wild type tend to die of malaria and those who are homozygous for sickle cell anemia die early of that disease in the absence of medical intervention. As we will see in chapter 19 (pages 596-598), the selective advantage for the heterozygous individuals is large enough so that the sickle cell gene is maintained in the population at a relatively high level.
Beta-Thalassemia: Failure to make adequate amounts of beta-globin chains results in a clinical condition known as beta-thalassemia. The textbook describes two types. One is caused by a defective splice junction, which results in use of alternative cryptic splice junctions that cause frameshifts in the mRNA, and thus total loss of functional beta-globin. The second disrupts the normal polyadenylation signal, resulting in an mRNA lacking poly (A), which is less stable, and thus does not produce an adequate amount of beta-globin.
Pigment pathways: The textbook presents a brief summary of the multiple biochemical steps and branched pathways that are involved in the production of human hair and skin pigmentation. This is presented as an illustration of the way in which enzymes, which in turn are encoded by genes can cause phenotypic variation.
Early evidence for one gene-one enzyme concept from alkaptonuria: The first evidence that genes could control the synthesis of enzymes was obtained shortly after the rediscovery of Mendelian genetics when Archibold Garrod was able to show as early as 1902 that the human genetic disease alkaptonuria (also spelled alcaptonuria) resulted from absence of a single enzyme (figure 6.10). One of the major symptoms of alkaptonuria is that the urine turns black because of excretion of copious amounts of homogentisic acid, which is normally one of the intermediate steps in the breakdown of dietary tyrosine. Garrod was able to show that the accumulation of homogentisate was caused by absence of an enzyme that should have converted it to 4-maleylacetoacetate, the next step in the breakdown of tyrosine in normal individuals.
Phenylketonuria: Phenylketonuria is caused by hereditary absence of the enzyme phenylalanine hydroxylase (PAH), which normally converts phenylalanine to tyrosine as the first step in ridding the body of excess dietary phenylalanine. Mutations in the human PAH gene were used as examples in the discussion of types of point mutations in Chapter 5 (figure 5.2). In the absence of the enzyme, high levels of phenylalanine accumulate and a portion of it is converted to phenylpyruvic acid, which is excreted in the urine, giving the disease its name. The excess of phenylalanine and phenylpyruvic acid has severe toxic effects, particularly on the brain, and causes severe mental retardation and other symptoms in affected individuals. Most of the damage can be prevented if a newborn infant is placed immediately on a low phenylalanine diet. This is another example of a hereditary disease that is linked to the absence of a specific enzyme.
Drosophila eye pigment: Drosophila undergoes complete metamorphosis from a worm-like larva to an adult fly. The eyes (and many other adult structures) of Drosophila are derived from a small clusters of cells known as imaginal discs that develop within the larvae and become transformed to adult structures at metamorphosis. If an eye disc is transplanted into the abdomen of a larva, it will form an eye-like structure within the abdomen of the fly that emerges from metamorphosis. Beadle and Ephrussi used this approach to study environmental effects on eye color mutations. They found that two mutations, vermillion (v) and cinnabar (cn), which cause loss of a brown pigment and give the eye an abnormally bright red color, could be converted to wild-type when the imaginal discs were placed in the abdomens of wild type larvae and allowed to develop. They concluded correctly that metabolic intermediates that the mutant discs were unable to make for themselves were diffusing into the discs from the wild type host tissue and being used for synthesis of brown pigment.
Metabolic pathway leading to synthesis of brown pigment: When they tested vermillion discs in cinnebar larvae and vice versa, they found that vermillion discs were "cured" in a cinnebar larva, but cinnebar discs retained a mutant phenotype in a vermillion larva. The interpretation was that the wild-type allele at the vermillion locus (v+) controls the first step in a metabolic pathway and cn+ controls the second step. The cn larvae could provide the v discs with enough of the intermediate produced by the v+ enzyme so that the v discs, which had a normal cn+ enzyme could then make brown pigment. However, in the reverse situation, the v host could not make any of the "downstream" intermediates, including the one that is normally synthesized by the cn+ gene product, and thus could not supply the missing intermediate needed by the cn discs to make brown pigment. These experiments are discussed as boxed example 6.2 on pages 166-170 (see figures 6.11 and 6.12). Note that In theory, the cn discs could make the v+ product, which could then diffuse out into the host tissue and be converted the the cn+ product, which could then diffuse back in, but the dilution factor in the host would be too large for this to support appreciable brown pigment formation.) These studies served as a prelude to the studies on auxotrophic mutations of Neurospora described below.
Complementation: The example described above introduces the concept of biochemical complementation, in which two genetically defective tissues or organisms can potentially work together with each providing what the other lacks to achieve an end result that neither could alone. In the material that follows, we will see two mutant strains of the mold Neurospora working together to grow under conditions where neither can alone. We will explore the genetic implications of complementation more fully in a future lecture.
Auxotrophic mutations: For microorganisms that can be grown on defined (or semi-defined) culture media, it is possible to select for auxotrophic mutations that require nutrients that the wild type organisms can make for themselves. Wild type organisms that are able to multiply in a medium lacking such a nutrient are called prototrophs.
Studies on auxotrophic mutants by Beadle and Tatum: In studies that resulted in a Nobel prize, George Beadle and Edward Tatum collected a large number of auxotrophic mutations in the mold, Neurospora crassa , and then studied groups of related mutations in detail. In an early test, they demonstrated that a mutant that required the vitamin pantothenate, could not carry out the last step in the synthesis of pantothenate, namely joining two precursor molecules together to yield pantothenate. This was due to loss of the required enzyme.
Arginine biosynthesis: One set of studies on arginine auxotrophs revealed an apparent pathway of precursor --> ornithine --> citrulline --> arginine, with mutants blocked at each of the steps (boxed example 6.3). Each strain could be grown on any of the intermediates that were beyond the missing step.
Tryptophan biosynthesis: A more complex pathway, with a total of five enzymatic steps involved in the biosynthesis of tryptophan has also been studied. In brief summary, the pathway leading from chorismate (the first precursor in the series) to tryptophan in Neurospora (figure 6.15) is:
Tryptophan auxotrophs blocked at any one of the enzymatic steps can be grown on all intermediates beyond the block, but none before. If you would like to see a more extended discussion of this pathway from last year's notes, click here. I have not made that material a formal part of this year's notes because it has been given only limited coverage in our current textbook.
Gene-protein colinearilty: One of the earliest lines of experimental evidence supporting the concept that genetic information was in a linear array corresponding to the amino acid sequence of a protein was provided by studies on the A subunit of tryptophan synthetase from E. coli in the laboratory of Charles Yanofsky. These studies verified that the relative map position of each mutation analyzed corresponded to the relative position within the protein of the resulting amino acid substitution (figures 4.5 and 4.6, boxed example 4.1, pages 92-94).
The material that follows was moved to Lecture 6 on September 11, 1999. For this year, a duplicate copy is retained here to be certain that everyone sees it.
Protein structure: Section 4.9 on protein structure and function should be read as additional bakcground information that is essentially a review of material from MCDB 1150. Four levels of structural information are commonly recognized.
Post-translational modification: Proteins are subject to a variety of post-translational modifications, including frequent removal of N-terminal methionine, removal of other N- or C- terminal sequences, removal of internal sequences, removal of signal or targeting sequences, modification of specific amino acids (such as conversion of proline to hydroxyproline), phosphorylation of hydroxyl groups, addition of carbohydrate side chains (glycosylation), complexing with metals or other prosthetic groups, and a long list of other possibilities that are not discussed particularly well in the textbook. Some of these modifications are illustrated in a section on enzymes and how they function at the end of Chapter 4.
Prions: A boxed section at the end of chapter 13 of Klug and
Cameron, Concepts of Genetics, 5th Edition (the previous textbook for
this course, available from Norlin reserve) discusses
an unusual pathogenic unit called a prion (proteinaceous infective
agent). Although the prion theory remains controversial, a very
large amount of evidence has accumulated showing that prion proteins
are coded by the host, and subsequently modified to function as
pathogens. The modified proteins accumulate in aggregates that
cause degenerative diseases of the brain. The best available evidence
seems to indicate that a conformational modification of the normal
host protein gives it pathogenic properties plus the ability to
catalyze similar modification of additional normal proteins, such
that the pathology is infectious. The most current focus on prions
is the mad cow disease , which apparently
got its start when proteins derived from sheep infected with a
similar disease, scrapie, were used in cattle feed.
Ordinary sterilization techniques do not inactivate the prion
infectivity. Similar diseases are known in humans, including Kuru
and Creutzfeld-Jacob disease. There may
also have been some cases of animal to human transmission, although
these have not been positively verified. Stanley Prusiner was
awarded the Nobel Prize in 1998 for his work on prions.