This is old Lecture 24
Textbook Assignment: Chapter 13, Pages 364-388.
Major concepts:
Introduction: Chapter 13 focuses on proteins as the end product of genetic expression. The first half of the chapter is devoted to various lines of evidence concerning the one-gene:one-peptide-chain concept, which we have already previewed in our discussion of genetic complementation in Chapter 6. The second half of the chapter focuses on protein structure and function, and will only be covered in outline form as a part of our review of topics covered in MCDB 1150. There is also an interesting boxed section on the ability of proteins with altered structures (prions) to function as infectious agents with no apparent nucleic acid genomes. I have moved our discussion of this chapter forward from its textbook position to link it with the introduction to complementation provided in the previous lecture. In addition, it contains a lot of historical material that predated modern molecular biology, which we will be moving into starting with the next lecture.
Complementation: The concept of complementation was introduced in the previous lecture, with emphasis on complementation of mutations in the rIIA and rIIB cistrons in bacteriophage T4. This lecture examines the one gene - one protein relationship in greater detail, with emphasis on complementation among mutations that affect biosynthetic pathways. The critical requirement for complementation is that the two mutations must not destroy precisely the same genetic function. Complementation strongly suggests that the two complementing mutations have damaged the coding sequences for two different proteins (polypeptide chains). However, as discussed in the previous lecture, intracistronic complementation can also sometimes occur within the coding sequence for a single protein in cases where two or more molecules of that protein must interact to achieve a biological function.
Early evidence for one gene-one enzyme 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 (page365). One of the major symptoms of alkaptonuria is that the urine turns black because of excretion of copious amounts of homogentisate, 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 (see Fig. 13.1 in the textbook).
Phenylketonuria: Phenylketonuria is caused by hereditary absence of the enzyme phenylalanine hydroxylase, which normally converts phenylalanine to tyrosine as the first step in ridding the body of excess dietary phenylalanine (see figure 13.1). 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.
Evidence for 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 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. (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.
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. Each strain could be grown on any of the intermediates that was beyond the missing step.
Tryptophan biosynthesis (not in book): In one of their most revealing studies, Beadle and Tatum collected a group of mutations that required tryptophan for growth and then tested the ability of each of the mutations to grow on a series of known precursors for the biosynthesis of tryptophan. For tryptophan, they were able to obtain mutants blocked at five different steps leading from chorismate (the first precursor in the series) to tryptophan. The patterns of which intermediates would or would not support growth of each mutant strain agreed exactly with the known pathway for biosynthesis of tryptophan. The overall pathway of tryptophan biosynthesis in Neurospora is chorismate --> anthranilate --> phosphoribosyl anthranilate --> 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate --> indoleglycerol phosphate --> tryptophan. The enzymes involved in each step are shown below in italics. A crudely simulated arrow head (\:/) has been used to depict the reaction arrow for each catalytic step because of lack of an appropriate vertical arrow symbol in html.
Growth characteristics of mutants: The table that follows
shows the growth of seven mutant strains (M1 - M7) of Neurospora
on TRP and the its biosynthetic intermediates.
Complementation analysis of these seven mutations yields the results
shown in the table below (mut = mutant phenotype, requiring tryptophan
for growth; wt = wild-type phenotype, capable of growing without
added tryptophan of any of its biosynthetic intermediates):
Conclusions: All seven mutations fail to complement themselves, as expected. Mutations 3 and 5 fail to complement each other, indicating that both affect the same peptide chain in the enzyme, anthranilate phosphoribosyl transferase, which is needed for synthesis of P-ANTH. Mutations 4 and 7 do complement each other, despite the fact that both cause loss of tryptophan synthase activity. This is consistent with the fact that tryptophan synthase is known to be composed of two subunits designated alpha and beta. Thus, complementation analysis precisely verifies the biosynthetic pathway and allows identification of mutations that affect the same step as well as identification of enzymes that appear to be composed of two or more subunits.
Suicide substrates: Genetic studies on tryptophan biosynthetic pathways have also been done in Arabadopsis, a small flowering plant. In this case, a suicide substrate was used to select for mutant strains. The enzymes that convert anthranilate to tryptophan can also convert 5-methylanthranilate to 5-methyltryptophan, which is incoprorated into proteins like tryptophan, but results in non-functional proteins, such that the plant does not survive. 5-methyltryptophan also blocks de novo synthesis of tryptophan by feedback inhibition of anthranilate synthase, thus preventing biosynthetic tryptophan from competing with the 5-methyltryptophan. Plants with mutations blocking any steps in the pathway between anthranilate and tryptophan can thus grow in the presence of 5-methylanthranilate when also supplied with small amounts of tryptophan, whereas those that are not blocked will make enough 5-methyltryptophan to inhibit growth even in the presence of the small amount of added tryptophan. This provides a powerful way to select for tryptophan auxotrophs in Arabadopsis.
Sickle-cell anemia: As more diverse types of mutations were studied, it was shown that genes specified the amino acid sequences for all types of proteins and not just for enzymes. One of the early pieces of evidence for this was the change in the beta globin chain of adult hemoglobin (HbA) that occurs in sickle-cell anemia, 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. This was one of the early lines of direct evidence that mutation changes the amino acid composition of a protein. 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.
Heterozygote advantage: The textbook briefly describes the high incidence of sickle cell anemia in African American populations without explaining its origin. In areas with a high level of malaria, individuals who are heterozygous for sickle cell anemia 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. However, 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. If we have time, we may explore this further in the section on population genetics, although the textbook appears not to provide any additional information.
Sequential expression of hemoglobin genes: The hemoglobins
also offer an interesting study in sequential gene expression
during embryonic and fetal development. Early embryos have a hemoglobin
called Gower I, which consists of two zeta chains and two epsilon
chains. Around eight weeks of gestation, this is replaced by fetal
hemoglobin, consisting of two alpha and two gamma chains. Adult
hemoglobin, which appears soon after birth, consists primarily
of two alpha and two beta chains, with a minor component of two
alpha and two delta chains. (see table 13.1)
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 (figure 13.8).
Protein structure: The section on protein structure 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 the formation of collagen fibers and genetic modifications of collagen in various disease states.
Prions: A boxed section at the end of chapter 13 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 this year for his work on prions.