Genetica per Scienze Naturali a.a. 06-07 prof S. Presciuttini 1. An early hypothesis on the function...

Genetica per Scienze Naturalia.a. 06-07 prof S. Presciuttini

1. An early hypothesis on the function of genes

The first clues about the nature of primary gene function came The first clues about the nature of primary gene function came from studies of humans. Early in the twentieth century, Archibald from studies of humans. Early in the twentieth century, Archibald Garrod, a physician, made several observations about alkaptonuria Garrod, a physician, made several observations about alkaptonuria (a form of arthrytis) and proceeded to propose the hypothesis that (a form of arthrytis) and proceeded to propose the hypothesis that the information for producing specific enzymes in humans is the information for producing specific enzymes in humans is inherited. He observed that inherited diseases reflect a patient's inherited. He observed that inherited diseases reflect a patient's inability to make a particular enzyme, which he referred to as inability to make a particular enzyme, which he referred to as ""inborn errors of metabolisminborn errors of metabolism“.“.

Garrod predicted that individuals affected with alkaptonuria Garrod predicted that individuals affected with alkaptonuria would be would be deficientdeficient in one of the enzymes in a degradative, in one of the enzymes in a degradative, biochemical pathwaybiochemical pathway. . He had suggested that the specific enzyme was involved in the He had suggested that the specific enzyme was involved in the degradation of degradation of homogentisic acidhomogentisic acid, an intermediate in the breakdown , an intermediate in the breakdown pathway of phenylalanine and tyrosine. He came to this conclusion by pathway of phenylalanine and tyrosine. He came to this conclusion by feeding homogentisic acid to alkaptonuric patients and noting that the feeding homogentisic acid to alkaptonuric patients and noting that the chemical was excreted in the urine in quantitatively similar amounts to chemical was excreted in the urine in quantitatively similar amounts to what was administered.what was administered.


Highly colored polymer

2. Alkaptonuria as a Mendelian disease

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Alkaptonuria (AKU) was the first disease to be interpreted as a Alkaptonuria (AKU) was the first disease to be interpreted as a single gene trait. In 1902 Garrod reported the mode of single gene trait. In 1902 Garrod reported the mode of inheritance in AKU. He noted that the rare affected individuals inheritance in AKU. He noted that the rare affected individuals had normal parents and normal offspring and were frequently had normal parents and normal offspring and were frequently children of consanguineous marriages. children of consanguineous marriages.

Advised by Bateson, Garrod suggested that the Advised by Bateson, Garrod suggested that the peculiar form of heredity of AKU was best peculiar form of heredity of AKU was best explained by the Mendelian theories of explained by the Mendelian theories of inheritance of a recessive character.inheritance of a recessive character.

In AKU patients, homogentisic acid (HGA) is excreted in large In AKU patients, homogentisic acid (HGA) is excreted in large amounts into the urine, which darkens on standing. This staining amounts into the urine, which darkens on standing. This staining of the urine, which can be detected from early childhood, is the of the urine, which can be detected from early childhood, is the first and best known manifestation of the disease. Over the years, first and best known manifestation of the disease. Over the years, benzoquinone acetic acid (an oxidation product of HGA) is benzoquinone acetic acid (an oxidation product of HGA) is deposited either directly or as a polymer into connective tissues, deposited either directly or as a polymer into connective tissues, causing their pigmentation (ochronosis) and eventually leading causing their pigmentation (ochronosis) and eventually leading to serious arthropathy to serious arthropathy

Genetica per Scienze Naturalia.a. 06-07 prof S. Presciuttini3. The OMIM home page

Genetica per Scienze Naturalia.a. 06-07 prof S. Presciuttini4. “Alkaptonuria” in OMIM


5. Identifying the gene for AKU

The biochemical evidence of the defect in AKU was provided by La Du The biochemical evidence of the defect in AKU was provided by La Du in in 19581958, exactly fifty years after Garrod’s hypothesis. He demonstrated , exactly fifty years after Garrod’s hypothesis. He demonstrated the absence of enzyme activity for the absence of enzyme activity for homogentisate 1,2 dioxygenasehomogentisate 1,2 dioxygenase (HGO) in a liver homogenate prepared from an AKU patient and (HGO) in a liver homogenate prepared from an AKU patient and established that the defect was limited to HGO, suggesting that in established that the defect was limited to HGO, suggesting that in affected individuals there is a failure to synthesize active enzyme.affected individuals there is a failure to synthesize active enzyme.

The gene responsible for AKU was located in man to 3q2 in The gene responsible for AKU was located in man to 3q2 in 19931993 by by linkage analysislinkage analysis. However, The decisive contribution to the . However, The decisive contribution to the characterization of the human characterization of the human HGOHGO gene came from work with the gene came from work with the ascomycete fungus ascomycete fungus Aspergillus nidulansAspergillus nidulans. A gene encoding an HGO . A gene encoding an HGO enzyme in this organism, denominated hmgA, was cloned and enzyme in this organism, denominated hmgA, was cloned and characterized in characterized in 19951995. The deduced amino acid sequence of its encoded . The deduced amino acid sequence of its encoded protein product was used to identify human DNA clones putatively protein product was used to identify human DNA clones putatively corresponding to the HGO gene.corresponding to the HGO gene.


6. The HG0 gene With this ESTclone the human HGO gene was characterized and With this ESTclone the human HGO gene was characterized and demonstrated that it is the AKU gene by showing that the AKU patients demonstrated that it is the AKU gene by showing that the AKU patients in two Spanish pedigrees were homozygous, or compound heterozygous, in two Spanish pedigrees were homozygous, or compound heterozygous, for the loss-of-function mutations P230S and V300G.for the loss-of-function mutations P230S and V300G.

The HGO human gene is now completely sequenced and a remarkable The HGO human gene is now completely sequenced and a remarkable number of AKU mutations have been already identified in patients from number of AKU mutations have been already identified in patients from many different countries. In 2000, the crystal structure of the human many different countries. In 2000, the crystal structure of the human HGO enzyme has been determined, which has provided a framework for HGO enzyme has been determined, which has provided a framework for understanding the structural basis for the AKU mutations.understanding the structural basis for the AKU mutations.


7. The Beadle and Tatum experiment

Garrod's hypothesis was ahead of its time. Experiments that clarified Garrod's hypothesis was ahead of its time. Experiments that clarified the actual function of genes came from research in the 1940s on the actual function of genes came from research in the 1940s on NeurosporaNeurospora by George Beadle and Edward Tatum, who later by George Beadle and Edward Tatum, who later received a Nobel Prize for their work.received a Nobel Prize for their work.

Beadle and Tatum analyzed mutants of Beadle and Tatum analyzed mutants of Neurospora crassaNeurospora crassa, a fungus , a fungus with a with a haploidhaploid genome. They first irradiated genome. They first irradiated Neurospora Neurospora cells to cells to produce mutations and then tested cultures from produce mutations and then tested cultures from ascosporesascospores for for interesting mutant phenotypes. They detected numerous interesting mutant phenotypes. They detected numerous auxotrophsauxotrophs strains (that cannot grow on a minimal medium unless the medium strains (that cannot grow on a minimal medium unless the medium is supplemented with one or more specific nutrients). In each case, is supplemented with one or more specific nutrients). In each case, the mutation that generated the auxotrophic requirement was the mutation that generated the auxotrophic requirement was inherited as a single-gene mutation: each gave a 1:1 ratio when inherited as a single-gene mutation: each gave a 1:1 ratio when crossed with a wild type.crossed with a wild type.


8.The life cycle of a fungus

The fungus The fungus NeurosporaNeurospora spends most of its life cycle as a multicellular haploid organism, in spends most of its life cycle as a multicellular haploid organism, in which the cells are joined end to end to form hyphae, or threads of cells. The hyphae grow which the cells are joined end to end to form hyphae, or threads of cells. The hyphae grow through the substrate and send up aerial branches that bud off haploid cells known as conidia through the substrate and send up aerial branches that bud off haploid cells known as conidia (asexual spores). Conidia can detach and disperse to form new colonies (asexual spores). Conidia can detach and disperse to form new colonies

The fungus has alternate mating strains, The fungus has alternate mating strains, called type A and type a. Mating can only called type A and type a. Mating can only take place between different mating strains take place between different mating strains and the result is a diploid cell in a long sac and the result is a diploid cell in a long sac (ascus). The diploid cell undergoes meiosis (ascus). The diploid cell undergoes meiosis producing four haploid cells.producing four haploid cells.

In the ascus, the results of segregation during In the ascus, the results of segregation during metaphase 1 are kept in order. These haploid metaphase 1 are kept in order. These haploid cells undergo one cycle of mitosis in the cells undergo one cycle of mitosis in the ascus leading to 8 spores (called ascospores) ascus leading to 8 spores (called ascospores) in order in the ascus.in order in the ascus.


One set of mutant strains required arginine to grow on a minimal medium. These strains provided the focus for much of Beadle and Tatum's further work.They found that the mutations mapped into three different locations on separate chromosomes, even though the same supplement (arginine) satisfied the growth requirement for each mutant.

9. Auxotroph mutants


10. Inferring a metabolic pathwayBeadle and Tatum discovered that the auxotrophs for each of the three loci differed in their response to the chemical compounds ornithine and citrulline, which are related to arginine.

One mutant strain grew when supplied with ornithine, citrulline, or arginine in addition to the minimal medium, another grew on either arginine or citrulline but not on ornithine, and the third grew only when arginine was supplied. On the basis of the properties of the arg mutants, Beadle and Tatum and their colleagues proposed a biochemical model for such conversions in Neurospora


11. The one-gene one-enzyme hypothesis

Beadle and Tatum concluded that a mutation at a particular gene affects the Beadle and Tatum concluded that a mutation at a particular gene affects the functioning of a single enzyme. The defective enzyme, then, creates a block in some functioning of a single enzyme. The defective enzyme, then, creates a block in some biosynthetic pathway. biosynthetic pathway.

This entire model was inferred from the properties of the mutant classes detected This entire model was inferred from the properties of the mutant classes detected through genetic analysis. Only later were the existence of the biosynthetic pathway through genetic analysis. Only later were the existence of the biosynthetic pathway and the presence of defective enzymes demonstrated through independent and the presence of defective enzymes demonstrated through independent biochemical evidence.biochemical evidence.

This model, which has become known as the This model, which has become known as the one-gene one-enzymeone-gene one-enzyme hypothesis, hypothesis, was the source of the first exciting insight into the functions of genes: genes was the source of the first exciting insight into the functions of genes: genes somehow were responsible for the function of enzymes, and each gene apparently somehow were responsible for the function of enzymes, and each gene apparently controlled one specific enzyme.controlled one specific enzyme.

Other researchers obtained similar results for other biosynthetic pathways, and the Other researchers obtained similar results for other biosynthetic pathways, and the hypothesis soon achieved general acceptance. The one-gene one-enzyme hypothesis hypothesis soon achieved general acceptance. The one-gene one-enzyme hypothesis became one of the great unifying concepts in biology, because it provided a bridge became one of the great unifying concepts in biology, because it provided a bridge that brought together the concepts and research techniques of genetics and that brought together the concepts and research techniques of genetics and biochemistry.biochemistry.

Genetica per Scienze Naturali a.a. 06-07 prof S. Presciuttini 1. An early hypothesis on the function...

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