Genetica per Scienze Naturali a.a prof S. Presciuttini 1. An early hypothesis on the function of genes The first clues about the nature of primary gene function came from studies of humans. Early in the twentieth century, Archibald Garrod, a physician, made several observations about alkaptonuria (a form of arthrytis) and proceeded to propose the hypothesis that the information for producing specific enzymes in humans is inherited. He observed that inherited diseases reflect a patient's inability to make a particular enzyme, which he referred to as "inborn errors of metabolism“. Garrod predicted that individuals affected with alkaptonuria would be deficient in one of the enzymes in a degradative, biochemical pathway. He had suggested that the specific enzyme was involved in the degradation of homogentisic acid, an intermediate in the breakdown pathway of phenylalanine and tyrosine. He came to this conclusion by feeding homogentisic acid to alkaptonuric patients and noting that the chemical was excreted in the urine in quantitatively similar amounts to what was administered.

Genetica per Scienze Naturali a.a prof S. Presciuttini Highly colored polymer 2. Alkaptonuria as a Mendelian disease Air Alkaptonuria (AKU) was the first disease to be interpreted as a single gene trait. In 1902 Garrod reported the mode of inheritance in AKU. He noted that the rare affected individuals had normal parents and normal offspring and were frequently children of consanguineous marriages. Advised by Bateson, Garrod suggested that the peculiar form of heredity of AKU was best explained by the Mendelian theories of inheritance of a recessive character. In AKU patients, homogentisic acid (HGA) is excreted in large amounts into the urine, which darkens on standing. This staining of the urine, which can be detected from early childhood, is the first and best known manifestation of the disease. Over the years, benzoquinone acetic acid (an oxidation product of HGA) is deposited either directly or as a polymer into connective tissues, causing their pigmentation (ochronosis) and eventually leading to serious arthropathy

Genetica per Scienze Naturali a.a prof S. Presciuttini 3. The OMIM home page

Genetica per Scienze Naturali a.a prof S. Presciuttini 4. “Alkaptonuria” in OMIM

Genetica per Scienze Naturali a.a prof S. Presciuttini 5. Identifying the gene for AKU The biochemical evidence of the defect in AKU was provided by La Du in 1958, exactly fifty years after Garrod’s hypothesis. He demonstrated the absence of enzyme activity for homogentisate 1,2 dioxygenase (HGO) in a liver homogenate prepared from an AKU patient and established that the defect was limited to HGO, suggesting that in affected individuals there is a failure to synthesize active enzyme. The gene responsible for AKU was located in man to 3q2 in 1993 by linkage analysis. However, The decisive contribution to the characterization of the human HGO gene came from work with the ascomycete fungus Aspergillus nidulans. A gene encoding an HGO enzyme in this organism, denominated hmgA, was cloned and characterized in The deduced amino acid sequence of its encoded protein product was used to identify human DNA clones putatively corresponding to the HGO gene.

Genetica per Scienze Naturali a.a prof S. Presciuttini 6. The HG0 gene With this ESTclone the human HGO gene was characterized and demonstrated that it is the AKU gene by showing that the AKU patients in two Spanish pedigrees were homozygous, or compound heterozygous, for the loss-of-function mutations P230S and V300G. The HGO human gene is now completely sequenced and a remarkable number of AKU mutations have been already identified in patients from many different countries. In 2000, the crystal structure of the human HGO enzyme has been determined, which has provided a framework for understanding the structural basis for the AKU mutations.

Genetica per Scienze Naturali a.a prof S. Presciuttini 7. The Beadle and Tatum experiment Garrod's hypothesis was ahead of its time. Experiments that clarified the actual function of genes came from research in the 1940s on Neurospora by George Beadle and Edward Tatum, who later received a Nobel Prize for their work. Garrod's hypothesis was ahead of its time. Experiments that clarified the actual function of genes came from research in the 1940s on Neurospora by George Beadle and Edward Tatum, who later received a Nobel Prize for their work. Beadle and Tatum analyzed mutants of Neurospora crassa, a fungus with a haploid genome. They first irradiated Neurospora cells to produce mutations and then tested cultures from ascospores for interesting mutant phenotypes. They detected numerous auxotrophs strains (that cannot grow on a minimal medium unless the medium is supplemented with one or more specific nutrients). In each case, the mutation that generated the auxotrophic requirement was inherited as a single-gene mutation: each gave a 1:1 ratio when crossed with a wild type. Beadle and Tatum analyzed mutants of Neurospora crassa, a fungus with a haploid genome. They first irradiated Neurospora cells to produce mutations and then tested cultures from ascospores for interesting mutant phenotypes. They detected numerous auxotrophs strains (that cannot grow on a minimal medium unless the medium is supplemented with one or more specific nutrients). In each case, the mutation that generated the auxotrophic requirement was inherited as a single-gene mutation: each gave a 1:1 ratio when crossed with a wild type.

Genetica per Scienze Naturali a.a prof S. Presciuttini 8.The life cycle of a fungus The fungus Neurospora 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 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 The fungus has alternate mating strains, 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 (ascus). The diploid cell undergoes meiosis producing four haploid cells. In the ascus, the results of segregation during metaphase 1 are kept in order. These haploid cells undergo one cycle of mitosis in the ascus leading to 8 spores (called ascospores) in order in the ascus.

Genetica per Scienze Naturali a.a prof S. Presciuttini 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

Genetica per Scienze Naturali a.a prof S. Presciuttini 10. Inferring a metabolic pathway Beadle 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

Genetica per Scienze Naturali a.a prof S. Presciuttini 11. The one-gene one-enzyme hypothesis 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 biosynthetic pathway. 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 biosynthetic pathway. 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 and the presence of defective enzymes demonstrated through independent biochemical evidence. 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 and the presence of defective enzymes demonstrated through independent biochemical evidence. This model, which has become known as the one-gene one-enzyme hypothesis, 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 controlled one specific enzyme. This model, which has become known as the one-gene one-enzyme hypothesis, 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 controlled one specific enzyme. Other researchers obtained similar results for other biosynthetic pathways, and the 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 that brought together the concepts and research techniques of genetics and biochemistry. Other researchers obtained similar results for other biosynthetic pathways, and the 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 that brought together the concepts and research techniques of genetics and biochemistry.