STRAIN DEVELOPMENT: STRAIN DEVELOPMENT Presented by Praful N. Giradkar M.Pharm I (Pharmaceutics ) Defination: Defination Improving the genetics of plant or animal cell so that it carries out a biotechnological process more effectively. What is the NEED ?: What is the NEED ? Essential when setting up a new process or maintaining the competitiveness of an existing one . Only few commercial fermentation processes use wild strains isolated directly from nature . Production of antibiotics, enzymes, amino acids, and other substances. Ideal Characteristics of Strain: Ideal Characteristics of Strain Depending on the system, it may be desirable to isolate strains : · R apid growth · Genetic stability · N on-toxic to humans · L arge cell size.. PowerPoint Presentation: Ability to metabolize inexpensive substrate. Do not show catabolite repression Permeability alterations. Shorter fermentation times. Do not produce undesirable pigments, R educed oxygen needs . W hich exhibit decreased foaming during fermentation. W ith tolerance to high concentrations of carbon or nitrogen sources. Methods of Strain Improvement: Methods of Strain Improvement Cell division (mitosis/meiosis) Mendelian genetics Mutation Protoplast fusion Recombinant DNA technology Cell Division Binary Fission, Mitosis & Meiosis: Cell Division Binary Fission, Mitosis & Meiosis Binary Fission: Binary Fission Most cells reproduce through some sort of Cell Division Prokaryotic cells divide through a simple form of division called Binary Fission 3 step process Single “naked” strand splits and forms a duplicate of itself. The two copies move to opposite sides of the cell Cell “pinches” into two new and identical cells called " daughter cells ". (Cell wall then forms if applicable) Mitosis: Mitosis Eukaryotes divide by a more complicated system called Mitosis This is because: They have a nucleus which must be broken up and then reformed They have their DNA “packaged” in the form of Chromosomes Chromosomes are composed of Chromatin Made of DNA Strands & Proteins Also contain Nucleosomes containing Histones - Proteins the DNA is wrapped around Name for the DNA/Protein complex is Chromatin They usually have more than 1 chromosome (Humans have 23 pairs) They have numerous organelles to equally share Overview of Mitosis: Overview of Mitosis Meiosis: Meiosis Similar in many ways to mitosis Several differences Involves 2 cell divisions Results in 4 cells with 1/2 the normal genetic information Overview of Meiosis: Overview of Meiosis Comparison of Mitosis & Meiosis: Comparison of Mitosis & Meiosis MITOSIS MEIOSIS Prophase Duplicated chromosome (two sister chromatids) Chromosome replication Chromosome replication Parent cell (before chromosome replication) Chiasma (site of crossing over) MEIOSIS I Prophase I Tetrad formed by synapsis of homologous chromosomes Metaphase Chromosomes positioned at the metaphase plate Tetrads positioned at the metaphase plate Metaphase I Anaphase I Telophase I Haploid n = 3 MEIOSIS II Daughter cells of meiosis I Homologues separate during anaphase I; sister chromatids remain together Daughter cells of meiosis II n n n n Sister chromatids separate during anaphase II Anaphase Telophase Sister chromatids separate during anaphase 2 n 2 n Daughter cells of mitosis 2 n = 6 Mendelian Genetics: Mendelian Genetics Gregor Johann Mendel PowerPoint Presentation: Mendelian inheritance (or Mendelian genetics or Mendelism or Monogenetic inheritance ) . S cientific theory of how hereditary characteristics are passed from parent organisms to their offspring. Theoretical framework was initially derived from the work of Gregor Johann Mendel published in 1865 and 1866 which was re-discovered in 1900; When Mendel's theories were integrated with the chromosome theory of inheritance by Thomas Hunt Morgan in 1915, they became the core of classical genetics . Breeding: Breeding Law of segregation: Law of segregation Mendel's law of segregation states that alleles of genes separate when gametes are formed. This apply to the segregation of alleles of one gene. Mendel's Law of Independent Assortment: Mendel's Law of Independent Assortment Mendel performed dihybrid crosses in plants that were true- breeding for two traits. Polygenetic Inheritance: Polygenetic Inheritance Qualitative variation usually indicates polygenic inheritance. This occurs when there is an additive effect from two or more genes. Pigmentation in humans is controlled by at least three (3) separately inherited genes. Mutation: Mutation Changes in DNA that affect genetic information. Mutation: Mutation Heritable change in the genetic material Permanent structural change of DNA Alteration can be passed on to daughter cells Mutations in reproductive cells can be passed to offspring Types of Mutation : Types of Mutation Mutations can be classified according to Cause Mechanism Types of Mutations according to Cause: Types of Mutations according to Cause Spontaneous Mutation Induced Mutation Spontaneous Mutation : Spontaneous Mutation The cause of a spontaneous mutation is unknown . Spontaneous mutations result from DNA replication errors The rate is approximately 1in10 6 replicated genes. Induced Mutation : Induced Mutation An induced mutation is brought about by a mutagen . A mutagen is a physical agent or a chemical agent that causes an alteration of the base sequence of the DNA molecule. Types of Mutagens : Types of Mutagens Physical mutagens heat ultraviolet radiation Chemical mutagens nitrous acid benzpyrenes aflatoxin base analogues Ultraviolet Radiation : Ultraviolet Radiation Ultraviolet radiation causes the formation of thymine dimers. Thymine dimers block DNA replication. Cell excises (cuts out) the thymine dimers using enzymes and repairs the damage. A mutation occurs when the repair is faulty . Nitrous Acid : Nitrous Acid Nitrous acid converts the adenine molecule so that it will pair with cytosine . When the DNA replicates, the newly synthesized stand will have a cytosine opposite the adenine on the conserved strand (instead of a thymine). Types of Mutations according to Mechanism : Types of Mutations according to Mechanism Missense Mutation: Missense Mutation A missense mutation results from a base substitution on the DNA molecule and leads to the insertion of a different amino acid into the protein during translation Nonsense Mutation : Nonsense Mutation A nonsense mutation results form a base substitution on the DNA molecule and leads to the “premature” termination of protein synthesis during translation Frame shift Mutation : Frame shift Mutation shifts the reading frame of the genetic message so that the protein may not be able to perform its function. Insertion THE FAT CAT ATE THE RAT THE FAT HCA TAT ETH ERA T Deletion THE FAT CAT ATE THE RAT TEF ATC ATA TET GER AT Chromosome Mutations : Chromosome Mutations Changes in number and structure of entire chromosomes Original Chromosome ABC * DEF Deletion AC * DEF Duplication ABBC * DEF Inversion AED * CBF Translocation ABC * JKL GHI * DEF Down Syndrome : Down Syndrome Chromosome 21 does not separate correctly. They have 47 chromosomes in stead of 46. Children with Down Syndrome develop slower, may have heart and stomach illnesses and vary greatly in their degree of inteligence . Cri-du-chat : Cri-du-chat Deletion of material on 5 th chromosome Characterized by the cat-like cry made by cri-du-chat babies Varied levels of metal handicaps Effects of Mutations : Effects of Mutations Most are neutral Eye color Birth marks Some are harmful Sickle Cell Anemia Down Syndrome Some are beneficial Sickle Cell Anemia to Malaria Immunity to HIV Protoplast fusion OR Somatic hybridization: Protoplast fusion OR Somatic hybridization PowerPoint Presentation: Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization PowerPoint Presentation: Somatic hybridization technique 1. isolation of protoplast 2. Fusion of the protoplasts of desired species/varieties 3. Identification and Selection of somatic hybrid cells 4. Culture of the hybrid cells 5. Regeneration of hybrid plants PowerPoint Presentation: Isolation of Protoplast ( Separation of protoplasts from plant tissue ) 1. Mechanical Method 2. Enzymatic Method PowerPoint Presentation: Mechanical Method Plant Tissue Cells Plasmolysis Microscope Observation of cells Cutting cell wall with knife Release of protoplasm Collection of protoplasm Enzymatic Method: Leaf sterilization, removal of epidermis Enzymatic Method Plasmolysed cells Plasmolysed cells Pectinase + cellulase Pectinase Protoplasm released Release of isolated cells Protoplasm released Isolated Protoplasm cellulase PowerPoint Presentation: Protoplast Fusion (Fusion of protoplasts of two different genomes) 1. Spontaneous Fusion 2. Induced Fusion Intraspecific Intergeneric Chemofusion Mechanical Fusion Electrofusion Spontaneous Fusion : Spontaneous Fusion Protoplast fuse spontaneously during isolation process mainly due to physical contact Intraspecific produce homokaryones Intergeneric have no importance Induced Fusion : Induced Fusion Chemofusion - fusion induced by chemicals Types of fusogens PEG NaNo 3 Ca 2+ ions Polyvinyl alcohal Induced Fusion : Induced Fusion Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette Electrofusion - Fusion induced by electrical stimulation Pearl chain of protoplasts is formed by low strength electric field (10kv m -1 ) Fusion of protoplasts of pearl chain is induced by the application of high strength electric field (100kv m -1 ) for few microseco Advantages of somatic hybridization : Advantages of somatic hybridization Production of novel interspecific and intergenic hybrid Pomato (Hybrid of potato and tomato) Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters Production of heterozygous lines in the single species which cannot be propagated by vegetative means Studies on the fate of plasma genes Production of unique hybrids of nucleus and cytoplasm. Limitations of Somatic hybridization : Limitations of Somatic hybridization Poor regeneration of hybrid plants Non-viability of fused products Not successful in all plants. Production of unfavorable hybrids Lack of an efficient method for selection of hybrids No confirmation of expression of particular trait in somatic hybrids Recombinant DNA technology : R ecombinant DNA technology PowerPoint Presentation: A series of procedures used to recombine DNA segments.. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate Recombination : Recombination Recombination is the process by which genes are rearranged on a chromosome or plasmid (extra chromosomal DNA). Occurs at the level of the chromosome or plasmid. the location of the gene is changed. The base sequence of a gene is unchanged. Mechanism of Recombination : Mechanism of Recombination Cross-over Transformation Conjugation Transduction Cross-Over : Cross-Over Cross-over -- the sense and anti-sense stands of the DNA molecule exchange pieces. Bases that were on the antisense strand are now on the sense strand, so these are now used for m-RNA synthesis . Transformation : Transformation Transformation -- process in which a sequence of genes are transferred from a donor bacterial cell to a closely related* recipient bacterial cell in the form of DNA in solution Conjugation : Conjugation Conjugation -- is the process in which plasmid genes are transferred from a donor bacterium to a closely related recipient bacterium. Transduction Transduction -- is the process in which bacterial genes are transferred from a donor bacterium to a closely related recipient bacterium by a bacteriophage. Bacteriophage -- a virus that infects a host bacterial cell. Applications rDNA : Applications rDNA Treatment of genetic diseases (gene therapy) e.g . SCID girl Production of medically useful biologicals (e.g. insulin) Vaccines production Large scale production of human proteins like interferon and growth hormones and blood clotting factor (VIII & IX). Stability of the Strain: Stability of the Strain An important consideration in strain improvement is the stability of the strain. An important aspect of this is the means of preservation and storage of stock cultures so that their carefully selected attributes are not lost. This may involve storage in liquid nitrogen or lyophilization . Strains transformed by plasmids must be maintained under continual selection to ensure that plasmid stability is retained. Instability may result from deletion and rearrangements of recombinant plasmids, which is referred to as structural instability, or complete loss of a plasmid, termed segregational stability. Some of these problems can be overcome by careful construction of the plasmid and the placement of essential genes within it. Segregational instability can also be overcome by constructing so-called suicidal strains that require specific markers on the plasmid for survival. Consequently, plasmid-free cells die and do not accumulate in the culture. These strains are constructed with a lethal marker in the chromosome and a repressor of this marker is located on the plasmid. Cells express the repressor as long as they possess the plasmid, but if it is lost the cells express the lethal gene. However, integration of a gene into the chromosome is normally the best solution, as it overcomes many of these instability problems. Applications of developed strain: Applications of developed strain Treatment of genetic diseases (gene therapy) Production of medically useful biologicals (e.g. insulin ) Vaccines production CONCLUSION: CONCLUSION References: References Aminetzach YT, Macpherson JM, Petrov DA (2005). "Pesticide resistance via transposition-mediated adaptive gene truncation in Drosophila". Science 309 (5735): 764 – Hastings , P J; Lupski , JR; Rosenberg, SM; Ira, G (2009). "Mechanisms of change in gene copy number" . Nature Reviews. Genetics 10 (8): 551 – http://technolog.it.umn.edu/technolog/issues/fall2004/genome.htm www.virtualsciencefair.org/ http://www.biology-online.org / Watson, James D. (2007). Recombinant DNA: Genes and Genomes: A Short Course . San Francisco: W.H. Freeman. ISBN 0-7167-2866-4 . Brown, Terry (2006). Gene Cloning and DNA Analysis: an Introduction . Cambridge, MA: Blackwell Pub. ISBN 1-4051-1121-6 .