Mutation & its detection

Full information about mutation for Agriculture students

1. Submitted by:- Name:- Bhumi A. Gameti An assignment on MUTATION AND IT’S DETECTION

2. MUTATION The term 'mutation' refers to sudden heritable changes in the genome , excluding those resulting from incorporation of genetic material from other organisms. In the molecular term mutation is defined as the permanent and relatively rare change in the number or a sequence of nucleotides. Mutation was first discovered by Wright in 1791 in male lamb which had short legs. Later on mutation was reported by Hugo de Vries in 1900 in Oenothera, Morgan in Drosophila (white eye mutant). The term mutation was coined by Hugo de Vries .

3. Terminology Muton : The smallest unit of gene capable of undergoing mutation and it is represented by a nucleotide. Mutator gene: A gene which causes another gene or genes to undergo spontaneous mutation. Antimutator genes :- Decrease the frequency of spontaneous mutation of other genes of same genome. Mutable genes: Genes which show very high rates of mutation as compared to other genes. Mutant: An organism or cell showing a mutant phenotype due to mutant allele of a gene. Mutagen: A physical or chemical agent which induces mutation. Hot spots: Highly mutable sites within a gene. Gene mutations or point mutations: The changes which alter the chemical structure of a gene at molecular level.

4. Characteristics of Mutations 1. Mutations are mostly recessive and very rarely dominant. 2. Most mutations have harmful effects and very few (less than 0.1 %) are beneficial. 3. Mutations may be due to a change in a gene, a group of genes or in entire chromosome. 4. If gene mutations are not lethal, the mutant individuals may survive. 5. If mutation occur at both loci simultaneously, the mutants can be identified in M 1 generation. However, if it is restricted to one locus only, (dominant to recessive) the effect can be seen only in M 2 generation.

5. Characteristics of Mutations 7. Many of the mutants show sterility . 8. Most mutants are of negative selection value. 9. Mutations are random i.e. they can occur in any tissue or cell of an organism. 10. Mutations are recurrent i.e. the same mutation may occur again and again. 11. Induced mutations commonly show pleiotropy often due mutation in closely linked genes.

6. TYPES OF MUTATION

7. Based on source Spontaneous Mutations Occurs spontaneously. 1. Due to error during DNA replication. 2. Due to mutagenic effect of natural environment E.g. UV rays from sunlight

8. Based on source Induced Mutations Mutation that originates in response to mutagenic treatment

9. Based on direction Forward mutation Reverse mutation Any change from wild type into mutant allele A change from mutant allele to wild type. Based on site Nuclear mutation Cytoplasmic mutation Mutation in nuclear gene. Mutation in cytoplasmic gene.

10. Based on tissue Germinal Mutations A gene is altered in a germ cell. Because germ cells give rise to gametes, some gametes will carry the mutation and it will be passed on to the next generation. Typically germinal mutations are not expressed in the individual containing the mutation.

11. Based on tissue Somatic Mutations Mutations in somatic cells. To maintain this mutation, the individual containing the mutation must be cloned. Two example of somatic clones are…. navel oranges are seedless oranges cloned by cutting and grafting Red delicious apples. high in dietary fiber and contain vitamin C

12. Based on character Morphological Biochemical Alters Morphological character. Also known as visible mutation. i.e. Dwarf peas, short legged sheeps , curly wings in Drosophila Alters production of biochemicals by the organism.

13. Based on visibility Macro-mutations Micro-mutations Mutations with large no. of changes in phenotypes. Easily detected Found in qualitative characters. Mutations with invisible phenotypic changes. Not easily detected. (special techniques requires) Observed in quantitative characters.

14. Based on survival Lethal mutation:- Kill all the individuals Sub lethal mutation:- Kill most of the individual Sub vital mutation:- Kill some of the individual Vital mutation:- Do not affect the survival Super vital mutation:- Increase the survival

15. Based on cytology Chromosomal mutation:- Changes in either chromosome number or structure. Gene mutation:- A lterations in base sequences of concerned genes. Cytoplasmic mutation:- Changes in chloroplast DNA ( cpDNA ) and mitochondrial DNA ( mtDNA ).

16. Based on the effect on the functions Loss of function mutation Reduce function Gain of function mutation Change in function

17. Mutants The product of mutation. Morphological mutants detected by outward appearance of an individual. i.e. shape, size and colour. Lethal mutants cause mortal or lethal effect on the organism. Conditional lethal mutants that expresses characteristics of the wild type when grown under certain conditions Biochemical mutants For bacteria, biochemical mutants need to be grown on a media supplemented with a specific nutrient. Such mutants are called auxotrophs.

18. Mutagens “ Agents which greatly enhance the frequency of mutation.” Physical mutagens Chemical mutagens “Include various types of radiations” X-rays Gamma rays Alpha particles Beta particles Fast and thermal neutrons UV rays Alklyting agents Base analogues Acridine Dyes Other Mutagens Nitrous acid Hydroxylamine

19. Materials used for treating with mutagens Seeds Pollen Vegetative Buds Whole Plants Bulbils Tubers Suckers

20. Significance of mutations in Plant Breeding: 1. When a desirable character is linked with an undesirable character. 2. To know source of resistance gene in the available germplasm 3. To create variability 4. To develop male sterile lines 5. To create variations in vegetatively propagated plants

21. DETECTION OF MUTATIONS:- Scoring of some types of mutations in certain organisms is relatively easy. For example, mutations for antibiotic resistance in bacteria are simply detected by plating the bacterial cells on a medium containing a lethal concentration of the concerned antibiotic (selective medium). The colonies that develop on such a medium will be produced by cells resistant to the antibiotic. But detection of morphological mutations in eukaryotes requires examination of each individual of the population for the mutant phenotype. This is not only tedious requiring much time, but is also a source of errors.

22. Detection of Mutations in Prokaryotes

23. 1. Detection of Auxotrophic Mutations Auxotrophic mutants are unable to synthesize some biochemical essential for their growth and development that wild type cells are capable of producing. Therefore, such mutants cannot be detected using the simple approach described before; these mutants are detected by replica-plating. The replica-plating technique was developed by Joshua and Esther Lederberg in 1952 for direct selection of bacterial mutants.

24. In this technique, the cells are first plated onto the complete medium (inoculation) to obtain distinct colonies; this is called the master plate . A block of wood or cork of a size suitable for the master plate is covered with velvet cloth. This block is sterilized, and then lowered into the master plate till the velvet touches all the colonies. Generate the mutants by treating a culture with a mutagen.

25. Now the block is withdrawn and gently lowered onto a plate containing the selection medium so that the bacterial cells sticking onto the velvet are transferred onto the medium; such a plate is known or replica plate . For detection of auxotrophic mutants, the selection medium is the minimal medium in which only wild type cells can grow.

26. A reference point is marked both on the master plate and on the replica plate. This makes it possible to locate in the master plate any colony of the replica plate. A single master plate can be used to produce several replica plates. The colonies that develop on the selection medium plate are due to wild type cells. In contrast, those colonies of master plate that fail to grow on the minimal medium are auxotrophic mutants.

27. The mutant colonies can be isolated from the master plate and used for further investigations, e.g., confirmation of their auxotrophic nature, identification of the deficient biochemical, etc. This approach can be used for detection of other types of mutants, e.g., antibiotic resistance, etc.

28. 2. The Ames Test This test was developed by Bruce Ames (1974) and coworkers and is based on histidine-requiring ( his - ) auxotrophic mutants of Salmonella typhimurium . The routine Ames test addresses to both these needs as follows. The his - cells are plated onto a medium that contains traces of histidine, which is enough to allow a few cell divisions, but inadequate for visible colony formation.

29. Salmonella typhimurium Bacteria which is his -ve Bacteria + Minimal medium (contains traces of histidine) Chemical Agent Completely Non-Mutagenic Non Mutagenic Mutagenic Experiment should be repeat

30. Ames test is routinely used to investigate the mutagenicity of various chemicals. But, some of the chemicals may become mutagenic only when they are acted upon by liver enzymes. For example, nitrates themselves are neither mutagenic nor carcinogenic. But in eukaryotic cells, nitrates are converted to introsamines, which are highly mutagenic and carcinogenic. The test chemical is incubated with rat liver extract containing the liver enzymes, i.e., the microsomal fraction. This allows modification of the chemical in the same way as it would be in the liver of animals.

31. In order to increase the efficiency of the test, the his -ve strains used in the test are defective in DNA repair, and have increased permeability to chemicals. It has been observed that more than 90% of the chemicals that are mutagenic are also carcinogenic.

32. Mutation Detection in Drosophila In Drosophila, several genetic stocks have been constructed for the detection of lethal and visible mutations in the X-chromosome and in autosomes. The two genetic stocks most commonly used for mutation detection in X-chromosome are, CIB and Attached-X stocks.

33. 1. CIB Stock of Drosophila This method involves use of a ClB stock which carries… ( i ) an inversion in heterozygous state to work as crossover suppressor (C), (ii) a recessive lethal ( l ) on X-chromosome in heterozygous state, and (iii) a dominant marker, Barred ( B )for the barred eye (narrow eye). One of the two X-chromosomes in a female fly carried all these three features and the other X-chromosome was normal . Male flies irradiated for induction of mutations were crossed to ClB females. Male progeny receiving ClB X-chromosome will die.

34. The ClB female flies obtained in progeny can be detected by barred phenotype. These are crossed to normal males. In the next generation 50% of males receiving ClB X-chromosome will die. The other 50% males will receive X-chromosome, which may or may not carry the induced mutation. In case lethal mutation was induced no males will be observed. On the other hand, if no lethal mutation was induced, 50% males will survive. Thus, the ClB method was the most efficient method for detecting sex linked lethal mutations.

35. 2. Attached-X Chromosome Technique This technique is based on attached-X (X-XY) females. It is designed to study visible sex-linked mutations in Drosophila. Mutagen-treated males are mated with attached-X females. The X-XX (super female) and YY progeny produced from such crosses do not survive. Only the X-XY (female) and XY (male) progeny are recovered. All the male (XY) progeny receive their Y chromosome from the attached-X female parent, while their X chromosome is contributed by the mutagen-treated male parent.

36. The frequency (%) of a visible mutation in the X chromosome of the mutagen-treated males If a visible mutation was induced in the X chromosome of the sperm (produced by the mutagen-treated male), it will be expressed in the male progeny. Therefore, all the male progeny obtained from the cross are scored for visible mutations. The frequency of a visible mutation is expressed as the ratio between the number of males showing a mutation and the total number of males in the progeny.

37. Russell's Test for Dominant Lethals The dominant lethal assay in mice was initially used (in 1954) by Russell and coworkers and by others to detect radiation-induced mutations. Subsequently, it was extended to study the mutagenicity of chemicals. Therefore this assay system is often called Russell's test or dominant lethal test system (DLTS).

38. Detection of Mutations in Plants Techniques for detection of mutations are relatively poorly developed for plant species. The following three approaches are generally adopted for this purpose. In some species, e.g., maize, strains homozygous for several recessive genes as well as those for dominant alleles of these genes are available. In such a case, seeds or plants of a strain homozygous for several dominant genes are treated with a mutagen. These plants (M 1 , the generation treated with mutagen) are crossed with the strain having the recessive alleles of the same genes in homozygous state (tester strain). The treated plants are generally used as female parent due to partial male sterility in mutagen-treated plants. In the progeny of this cross, plants exhibiting recessive forms of the concerned characters are counted.

39. The frequency of mutation for a gene is estimated as follows. The plants showing the recessive form of the trait will receive one recessive allele from the tester parent having the recessive traits. But the other recessive allele would have been produced due to mutation in the mutagen-treated parent as this parent had the dominant allele of the gene.

40. 2. In most plant species, however, such strains are not readily available. In such species, seeds of a variety or strain are treated with a mutagen and grown to obtain the M 1 generation. The M 1 plants are selfed to avoid out crossing due to partial male sterility in M 1 , plants. The seeds thus obtained represent the M 2 generation. M 2 plants are grown and plants having mutant features are scored. The frequency of a given mutation is estimated as per cent ratio between the number of plants exhibiting a mutation in M 2 and the total number of plants in M 2 .

41. 3. The above procedures are applicable when mutations are to be detected in qualitative traits (macro mutations). In crop improvement, mutations in quantitative traits (micro mutations) are equally, often more, important. For detection of micro mutations, M 2 generation is grown as described above. All M 2 plants showing a visible macro mutation and/or partial pollen sterility are rejected. Seeds from all normal-looking fully fertile M 2 plants are harvested separately. They are planted in individual plant progeny rows in M 3 (preferably in a replicated trial). Data are recorded on quantitative traits. Means and variances of M 3 progenies are compared with those of the parental variety to ascertain if an M 3 progeny significantly from the parental variety for a quantitative trait. A significant deviated deviation from the parent variety will indicate the occurrence of micro mutations for the concerned trait.

42. REFERENCES B. D. Singh, 1990. Fundamentals of Genetics , Kalyani Publishers, Page no.: 290-308 Phundan Singh, 2004. Genetics , Kalyani Publishers, Page no.: 208-216 THANK YOU…