Mutation in Pharmaceutical Biotechnology

Mutations are fundamental to pharmaceutical biotechnology, influencing everything from drug development to biopharmaceutical production. Defined as permanent changes in the DNA sequence, mutations can be point mutations, insertions, deletions, or larger chromosomal alterations, each potentially altering protein function and leading to significant biological consequences. They are crucial in identifying drug targets, particularly in diseases like cancer, where mutations in oncogenes or tumor suppressor genes drive progression. Additionally, mutations serve as important biomarkers for personalized medicine, guiding treatment choices based on an individual's genetic profile. In the realm of drug production, genetic modifications enhance the efficacy of therapeutic proteins and vaccines through engineered organisms. However, the emergence of drug resistance due to mutations poses a significant challenge, particularly in infectious diseases. Emerging technologies such as CRISPR/Cas9 and directed evolution leverage mutations to create innovative treatments while raising ethical and safety considerations. As the field advances, a nuanced understanding of mutations will remain essential for overcoming obstacles and driving breakthroughs in medical therapeutics.

1. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 1 NANDHA COLLEGE OF PHARMACY ERODE-52 NAME: DAKSHINESH P COURSE: B.PHARM SEMESTER: VI 17-02-2022

2. PHARMACEUTICAL BIOTECHNOLOGY MUTATION PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 2 17-02-2022

3. OUTLINE Definition Causes Classification of mutation Mutants Isolation of mutants PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 3 17-02-2022

4. DEFINITION: A mutation is any sudden change in genetic material that may or may not affect phenotype i.e. the physical expression of a gene. Mutation can occur due to change in the nucleotide/base sequence of DNA, due to errors in DNA replication. Purines – Adenine and Guanine Pyrimidines – Cytosine and Thymine PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 4 17-02-2022

5. CAUSES: Error in DNA replication or transcription due to influence of environmental factors such as with examples, Radiation – UV radiation and X-Rays. Chemicals - Cigarette smoke, nitrate and nitrate preservatives, benzoyl peroxide. Infectious agents – Human Papillomavirus, Helicobacter pylori PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 5 17-02-2022

6. CLASSIFICATION OF MUTATION: Based on the survival of an individual Based on causes of mutation Based on tissue of origin Based on direction of mutation Based on type of trait affected Chromosomal mutation PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 6 17-02-2022

7. 1 . Based on the survival of an individual: lethal mutation – causes death of all mutants Sublethal mutation – causes death of 90% individuals Sub vital mutation – causes death less than 90% Vital mutation – do not affect the survival of mutants Super vital mutation – enhance the survival of mutants. 2. Based on causes of mutation: Spontaneous mutation – natural without any cause E.g. UV of sunlight causing mutation in bacteria Induced mutation - treatment with wither physical or chemical agents. E.g. X-Ray causing mutation in cereals. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 7 17-02-2022

8. 3. Based on tissue of origin: Germline mutation are the heritable alterations in the DNA which occurs in germ cell and it is included in every call of the body. These mutations are especially significant because they can be transferred to offspring and every cell in the offspring will have mutations. Somatic mutations are the modification in DNA that occurs after conception. It occurs in any other cells of the body except germ cell. These mutations may have little effect on the organism because they are restricted to just one cell and its daughter cells. Somatic mutations cannot be transmitted to next generation. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 8 17-02-2022

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10. 4. Based on direction of mutation: Forward mutation – normal allele to mutant allele Reverse mutation – mutant allele to normal/wild type allele 5. Type of trait affected: Visible – affect phenotypic character Biochemical – affect production of biochemicals and not show phenotypic character. 6. Chromosomal mutation: Modification in chromosomal number and structure. -Deletion, Inversion, Translocation, Nondisjunction, Duplication. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 10 17-02-2022

11. TYPE OF MUTATION: Types of mutations can arise due to modification of single pairs of nucleotides and from addition or deletion of one or two nucleotide pairs in the coding region of the genes. Mutation can be differentiated into two major types, Point mutation Frameshift mutation PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 11 17-02-2022

12. 1. Point Mutation: A point mutation is a single base substitution that will cause the replacement of a single base nucleotide with another nucleotide of the genetic material. DNA or RNA. The individual which is affected by a point mutation is called as point mutants. Transitions – In this case there is a replacement of purine base with another purine are substitution of pyrimidine with another pyrimidine. Transversions – In this case there is a replacement of purine with pyrimidine or vice versa. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 12 17-02-2022

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14. Outcomes of point mutation: Silent mutation : In silent mutation the mutated codon codes for the same amino acid. There will be replacement of single nucleotide but the new codes specify the same amino acid, resulting in unmutated protein PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 14 17-02-2022

15. ii. Missense mutation: In missense mutation, the mutated codon codes for the different amino acid. There will be single base pair substitution in DNA that will change a codon for one amino acid into a codon for another. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 15 17-02-2022

16. iii. Nonsense mutation : In nonsense mutation, there will be a replacement of single nucleotide that results in a stop codon replacing a normal amino acid codon. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 16 17-02-2022

17. 2. Frameshift Mutation: A frameshift mutation is insertion or deletion of one or more nucleotides that alters the reading frame of the base sequence. Deletions will remove nucleotides whereas will add nucleotides. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 17 17-02-2022

18. MUTANTS: Mutants are the individuals showing mutation. To detect particular mutant organism, must be avail of wild characters. In bacteria and other haploid microorganisms the detection system is simple. Other detection are complicated. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 18 17-02-2022

19. ISOLATION OF MUTANTS Replica Platting Technique Resistance Selection Method Substrate Utilization Method Carcinogenicity Test or Ames method PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 19 17-02-2022

20. Replica Plating Technique: In 1952, Joshua and Esther Lederberg develop replica platting technique. This technique is used to detect auxotrophic mutants which distinguish between mutants and wild type strains on the basis of ability to grow in the absence of an amino acid. This test is used to show the presence of antibiotic resistance in bacterial cultures prior to exposure of antibiotic. Steps involved are: The mutants are generated by treating a culture with a mutagen i.e. nitroguanidine. Then the plate is inoculated containing complete growth medium and incubated at proper temperature. Both wild type and mutant survivors will form colonies on the complete medium. This plate which contain complete medium is called as master plate. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 20 17-02-2022

21. A sterile velvet piece is prepared and is gently pressed on upper surface of master plate to pick up bacterial cells from each colony. As pressed on master plate, the velvet is again gently pressed on replica plates containing complete medium in one set and lacking only histidine in other set. So, the bacterial cells are transferred in a replica plates in the same position as in the master plate. Then plates are incubated and the replica plates are compared with master plate for bacterial colony not growing on replica plate. The Histidine auxotroph (His-) will not grow on replica plates which do not have leucine. Then, leucine auxotroph are isolated as cultured on complete medium. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 21 17-02-2022

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23. 2. Resistance Selection Method: This is another approach used for isolation of mutants. Mostly the wild type cells are not resistant either to antibiotics or bacteriophages. Thus, it is possible to grow the bacterium in the presence of agent. This method is applied for separation of mutants resistant to an chemical compounds that can adjust to agar, phage resistant mutants. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 23 17-02-2022

24. 3. Substrate Utilization Method: This method is involved in the selection of bacteria. Several bacteria use only a few primarily carbon sources. The cultures are plated on a medium which carry on alternate carbon sources. Any colony that grows on medium can utilize the substrate and are perhaps mutants, and then these can be isolated. Sugar utilization mutants are also separated by means of colour is sensitive to pH. This method comprises lactose sugar as carbon source and complete mixture of amin acids. Hence, both lactose wild type(Lac+) and lactose mutant (Lac-) cells can grow and form colonies on EMB agar plate. The Lac+ cells consumes lactose and secrete acids which will cause decrease in the pH of medium. It results in staining of colony to dark purple. Whereas, PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 24 17-02-2022

25. Lac- cells are not able to use lactose and utilize some of the amino acids as carbon source. After usage of amino acid, ammonia is produced that will increase the pH of medium and decolorise the dye resulting in white colony. (Lac+)-----utilise lactose-----Forms acid-----Decrease pH-----Staining of colony to dark purple colour. (Lac-)-----utilise amino acid-----Forms ammonia-----Increases pH-----decolorise the dye-----white colony. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 25 17-02-2022

26. 4. Carcinogenicity or Ames Test: In 1970s Ames test was developed by Bruce Ames, Professor of Biochemistry at UC-Berkeley, for evaluating mutagenicity of carcinogens i.e. chemical which is capable of causing cancer. In Ames test several special strains of Salmonella typhimurium are involved. Each strain contains a distinct mutation in the operon histidine biosynthesis. The Ames test follows the following steps: The culture of Salmonella histidine auxotroph (His-) are prepared. The bacterial cells and test substance i.e. mutagen are mixed in dilute molten top agar with small amount of histidine in one set, and control with complete medium plus large amount of histidine. The molten mix is poured on the top of minimal agar plates and incubated at 37 degree Celsius for 2 to 3 days. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 26 17-02-2022

27. Till histidine is depleted all the His- cells will grow in the presence of test mutagens. When histidine is completely depleted only the revertant i.e. the mutants which have retrieved the original wild type characters will grow on agar plate. The number of spontaneous revertant is low as compared to number of revertant induced by the test mutagen which is quite high. The higher the number of colonies, greater will be mutagenicity. Before plating, a mammalian liver extracts is added to the above molten top agar. The extract will transfer the carcinogen into electrophilic derivatives which will soon react with DNA molecule. It is a natural process which occurs in mammalian system when foreign substance are metabolized in liver. Bacteria do not have the metabolizing capacity as liver does, so the liver extract is added in this test just to promote the transformation. PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 27 17-02-2022

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29. THANK YOU PHARMACEUTICAL BIOTECHNOLOGY - DAKSHINESH P 29 17-02-2022