Use of Neurospora in Understanding Genetical Principle!
George Wells Beadle (1903-1989) grew up on a 40-acre farm near the small town of Wahoo, Nebraska (USA).
Beadle (Fig. 2.7) might have become a farmer himself had it not been for the influence of his high school science teacher, Bess MacDonald, who persuaded him to enroll at die University of Nebraska College of Agriculture. Beadle entered Cornell in 1927 and joined Rollins Adams Emerson’s laboratory to work on the cytogenetics of maize.
Over the next 5 years he published 14 papers dealing with his investigations on maize, all initiated while he was a graduate student at Cornell. With the completion of his graduate work in 1931, Beadle headed off to the California Institute of Technology to work with future Nobel laureate Thomas Hunt Morgan. There he became interested in Drosophila and began doing research on genetic recombination.
In 1934, Boris Ephrussi, a Rockefeller Foundation Fellow from Paris, came to Morgan’s laboratory at Caltech to study Drosophila genetics. Beadle and Ephrussi teamed up and began examining eye pigment development in Drosophila after devising a method for larval embryonic bud transplantation. These studies were performed in Ephrussi’s laboratory in Paris. From these experiments, they proposed that eye color changes in mutant strains of Drosophila could be caused by inactivation of specific proteins, acting in a single biosynthetic pathway.
This suggested that development could be broken down into a series of gene-controlled biochemical reactions and laid the foundation for the one gene-one enzyme theory that Beadle would eventually propose and make famous. The idea that specific proteins were produced by specific genes was first mentioned in 1909 by Sir Archibald Garrod, an English physician. Garrod proposed that alkaptonuria, an inherited condition in humans in which the urine is black due to the presence of homogentisic acid, was associated with a recessive gene.
In 1937 Beadle accepted an appointment as Professor of Biological Sciences at Stanford University and invited Tatum to join him as a research associate. For his experimental organism, Beadle chose the red bread mold Neurospora crassa, whose life cycle had been characterized, making it an ideal organism for genetic study. He and Tatum knew from the studies of others that Neurospora could grow on a minimal medium composed of a sugar, salts, and the one vitamin, biotin.
Then they used X-rays to attempt to produce Neurospora mutants that had lost the ability to grow on their minimal medium. The 299th mutagenized culture they tested proved to be the lucky one. It did not grow in their minimal medium, but it did survive and grow when vitamin B6 was added.
To prove that a single gene had been mutated, Beadle and Tatum performed a genetic cross between the mutant strain and a wild type strain and tested cultures derived from the eight single spores that were the progeny of a single meiosis. Their tests showed that cultures from four progeny spores required vitamin B6 whereas the other four did not, confirming that a single gene had been mutated. Before this finding, mutants requiring amino acids, purines, and pyrimidine’s were also found, and the science of biochemical genetics was born.
However, when Beadle first presented this theory few scientists accepted the concept that one gene specifies the sequence of one enzyme. The one gene- one enzyme theory was eventually verified and accepted, when subsequent investigations by others established that genetic material was DNA and that DNA had a double helical structure, and determined how genetic material is replicated and how it functions in protein synthesis. Tatum later applied their methods to produce bacterial mutants.
Using these mutants, Tatum’s graduate student, Joshua Lederberg, demonstrated genetic recombination in Escherichia coli, thereby founding the field of bacterial genetics. In 1958, Beadle, Tatum, and Lederberg shared the Nobel Prize in Physiology or Medicine for their pioneering studies with Neurospora and E. coli.
Investigation of the Neurospora genome began in the mid-1920s, when C. L. Shear and B. O. Dodge named it and described its life cycle (Fig. 2.8). They also showed that the two mating types of Neurospora crassa segregate so as to give 1:1 ratios in randomly isolated ascospores and 4:4 ratios in individual unordered asci. Dodge found that morphological variants also showed Mendelian segregation. In the 1930s, Carl Lindegren used morphological mutants, centromeres, and mating type to construct the first genetic maps, which consisted of six loci in linkage group I and four in linkage group II.
Markers suitable for mapping became abundant in the 1940s, when mutagens were used effectively to obtain auxotroph’s and an array of other mutant types and techniques were devised for mutant enrichment. By 1949, six linkage groups had been established and the seventh was soon added. From eight loci mapped in 1937, the number had increased to about 50 in 1949, 75 in 1954, and 500 in 1982. Now, at the millennium, the number of mapped loci exceeds 1,000.Physical knowledge of the genome increased in parallel with the growth of genetic knowledge. Barbara McClintock showed in 1945 that the seven tiny Neurospora chromosomes can be identified individually using light microscopy. She and J. R. Singleton went on to describe chromosome morphology and behavior during meiosis and mitosis in the ascus.
Genetic evidence of chromosome rearrangements was confirmed cytologically. Genetically mapped rearrangements soon enabled linkage groups to be assigned to each of the seven cytologically distinguishable chromosomes. The discovery of insertional and quasiterminal rearrangements made it possible to map genes by duplication coverage.
The complete meiotic karyotype was reconstructed in three Dimensions using the synaptonemal complex with its associated recombination nodules. Electrophoretic separation of whole-chromosome DNAs provided estimates of the DNA content of individual chromosomes and yielded a value of 43 mega-bases for the entire haploid genome about three times that of yeast and roughly one-third that of Drosophila or Arabidopsis.
When the first Neurospora compendium was prepared in 1982, a gene could not be recognized until a phenotypically recognizable variant had been obtained, either as a mutant in the laboratory or as a novel allele from the wild. Molecular genetics has changed all that.
Genes can now be recognized on the basis of nucleotide sequence and mapped using restriction fragment length polymorphisms, all before any phenotype is known. Numerous genes in the present compilation have been identified using the cloned wild-type allele.
To determine the phenotype of such a gene, a null allele or a mutant allele must be obtained by gene replacement or by repeat-induced point mutation (RIP). Genes without known mutant phenotypes may soon outnumber those for which the mutant phenotype has been determined.
The pink bread mold Neurospora crassa can grow on medium containing only:
(1) Inorganic salts,
(2) A simple sugar, and
(3) One vitamin, biotin.
Neurospora growth medium containing only these components is called “minimal medium.” George Beadle and Edward Tatum reasoned that Neurospora must be capable of synthesizing all the other essential metabolites, such as the purines, pyrimidine’s, amino acids, and other vitamins, de novo.
Furthermore, they reasoned that the biosynthesis of these growth factors must be under genetic control. If so, mutations in genes whose products are involved in the biosynthesis of essential metabolites would be expected to produce mutant strains with additional growth-factor requirements. Beadle and Tatum tested this prediction by irradiating asexual spores (conidia) of wild-type Neurospora with X-rays or ultraviolet light, and screening the clones produced by the mutagenized spores for new growth-factor requirements.
In order to select strains with a mutation in only one gene, they studied only mutant strains that yielded a 1:1 mutant to wild-type progeny ratio when crossed with wild type. They identified mutants that grew on medium supplemented with all the amino acids, purines, pyrimidine’s, and vitamins (called “complete medium”), but could not grow on minimal medium. They analyzed the ability of these mutants to grow on medium supplemented with just amino acids, or just vitamins, and so on.
For example, Beadle and Tatum identified mutant strains that grew in the presence of vitamins but could not grow in medium supplemented with amino acids or other growth factors. They next investigated the ability of these vitamin-requiring strains to grow on media supplemented with each of the vitamins separately. In this way, Beadle and Tatum demonstrated that each mutation resulted in a requirement for one growth factor.
By correlating their genetic analyses with biochemical studies of the mutant strains, they demonstrated in several cases that one mutation resulted in the loss of one enzyme activity. The one gene-one enzyme concept thus became a central part of molecular genetics.
It is evident from the above account that Neurospora is a very suitable fungi producing 8 ascospores after meiosis in a very short time. There is direct relationship between gene and its product and phenotypic product can easily be separated as ascospores.
It is easy to create mutations by X-rays. Therefore, this material is very suitable to study genetic changes by mutations and changes in resulting proteins. At the same time new molecular techniques are helpful to determine altered sequences and to correlate them with characters.