[IMMUNO BLOTTING TECHNIQUES INTRODUCTION PRINCIPLE SOUTHERN BLOTTINGQ INTRODUCTION * Blotting techniques are very widely used analytical tools for the specific identification of desired DNA or RNA fragments from thousands of molecules. Blotting refers to the process of immobilization of sample nucleic acids or solid support (nitrocellulose or nylon membranes). The blotted nucleic acids are then used as targets in the hybridization experiments for their specific detection. Q PRINCIPLE. The blotting method involves the following four steps 1. Electrophoretic extraction of protein or nucleic acid. 2. Transfer and immobilisation on paper , fragments in the sample. 3. Binding of analytical probe to target molecule on paper. 4. Bound probe visualization. By using electrophoresis the molecules in the sample are separated and moved on a support medium or membrane. As a result, the protein or DNA fragments get immobilised providing a close copy of the original separation, and assisting subsequent biochemical analysis . Probes like antibodies or DNA that binds to the desired molecule are used to localise the immobilised protein or nucleic acid fragments after they are transferred to the support medium. Autoradiography is used in the final step for visualising the position of the probe bound to the immobilised target molecule.Q INTRODUCTION Enzyme-linked immunosorbent assay (ELISA) is a non-isotopic immunoassay. An enzyme is used as a label in ELISA in place of radioactive isotope employed in RIA. ELISA is as sensitive as or even more sensitive than RIA. In addition, there is no risk of radiation hazards (as is the case with RIA) in ELISA. Q PRINCIPLE. * ELISA is based on the immunochemical principles of antigen-antibody reaction . The antibody against the protein to be determined is fixed on an inert solid such as polystyrene. . The biological sample containing the protein to be estimated is applied on the antibody coated surface. . The protein antibody complex is then reacted with a second protein specific antibody to which an enzyme is covalently linked. These enzymes must be easily assayable and produce preferably coloured products. . After washing the unbound antibody linked enzyme, the enzyme bound to the second antibody complex is assayed. . The enzyme activity is determined by its action on a substrate to form a product (usually coloured). 44 44 ait -laat Laas | "Y Anbody Target protein 1 ereynetboed anboty Erayme reaction 0 ——> Antigen lovel| | * ELISA tests can be classified into three types depending upon the | different methods used for binding between antigen and antibodies, namely: 1. Indirect ELISA 2. Sandwich ELISA 3. Competitive ELISA 1. Indirect ELISA * Indirect ELISA detects the presence of an antibody in a sample. * The antigen is attached to the wells of the microtitre plate. A sample containing the antibodies is added to the antigen-coated wells for binding with the antigen. The free primary antibodies are washed away and the antigen- antibody complex is detected by adding a secondary antibody conjugated with an enzyme that can bind with the primary antibody. All the free secondary antibodies are washed away. A specific substrate is added which gives a coloured product. The absorbance of the coloured product is measured by spectrophotometry. Antigen Antibody — Enzyme-labelled antibody w A Substrate | | | A xv Advantages i) This method has an elevated level of sensitivity because more than one labelled antibody binds to each primary antibody. ii) Numerous labelled secondary antibodies are commercially available. Since the primary antibody is not labelled, its immunoreactivity is retained to the maximum. This method is versatile as many primary antibodies can be prepared in one species and the same labelled secondary antibody can be used for detection. This method is flexible as a single labelled secondary antibody can be used with many primary detection antibodies. This method is cost efficient as a less number of labelled antibodies are used. vii) With same primary antibody different imaging indicators can be used. v Disadvantages i) Cross-reactivity with secondary antibody may occur, causing non specific signal. ii) This method requires an additional incubation step. 2. Sandwich ELISA. Sandwich ELISA helps to detect the presence of antigen in a sample. The microtitre well is coated by the antibody. The sample containing the antigen is added to the well and washed to remove free antigens. Then an enzyme-linked secondary antibody, which binds to another epitope on the antigen is added. The well is washed to remove any free secondary antibodies. The enzyme-specific substrate is added to the plate to form a coloured product, which can be measured.Sandwich ELISA Vv. Bloctang Butter v Advantages i) This method has high specificity as the antigen is specifically captured and detected due to the use of two antibodies. ii) This method is suitable for complex samples as the antigen is not purified before measurement. iii) This method is flexible and sensitive as direct and indirect detection methods can be used. 3. Competitive ELISA * Competitive ELISA helps to detect antigen concentration in a sample. The microtitre wells are coated with the antigen. Antibodies are incubated in a solution having the antigen. The solution of the antigen-antibody complex is added to the microtitre wells. The well is then washed to remove any unbound antibodies. More the concentration of antigen in the sample, lesser the free antibodies available to interact with the antigen, which is coated in the well. The enzyme-linked secondary antibody is added to detect the | number of primary antibodies present in the well. The concentration is then determined by spectrophotometry.Y Advantages 1) 2) 3) This method is highly specific as two antibodies are used. This method is highly sensitive as both direct and indirect detection methods are used. This method is suitable for complex samples as the antigen is not purified before measurement. Q PROCEDURE 1. The steps involved in the procedure of ELISA are: Assay samples or standard solutions are added to the antibody- coated wells. and incubated for defined time period to capture the antigen molecules by the antibody After this binding reaction, the reaction mixture is discarded and excessive materials are removed from the wells by washing. A second antibody that identifies another epitope in antigen is | labelled with horseradish peroxidase (HPP) enzyme and then | added to the wells. |4. This enzyme-labelled second antibody binds to the antigen already | bound to the first antibody on the bottom of wells. This means that | HRP enzyme is also fixed on the bottom of wells. The amount of antigen captured and the fixed enzyme is directly proportional. 5. The enzyme activity is measured by adding a chromogenic substrate of the enzyme. In case of HRP, tetramethylbenzidine is often used. After incubation for a definite period, the chromogenis substrate is changed to a coloured product. 6. A standard or calibration curve is obtained by plotting the © concentration of standard solutions.4 ADVANTAGES OF ELISA Following are some of the advantages of the ELISA technique 1. Results fetched from ELISA gives an accurate diagnosis of a particular disease since two antibodies are used. Can be carried out for complex samples as the antigen is not required to get purified to detect. . It is highly responsive since direct and indirect analysis methods can be carried out. . Itis a rapid test, yields results quickly. . Possible detection for ELISA ranges from the quantitative, semi- quantitative, standard curve, qualitative, calibration curve models etc. . Easier to perform and uncomplicated process as compared to other assays which require the presence of radioactive materials. Q APPLICATION * The technique of ELISA has the following applications: it It can be used to detect the presence of antigen or antibody in a sample. . It can be used to determine serum antibody concentrations in a virus test 3. It can be used in food industry to detect potential food allergens. . Itcan be used in disease outbreaks to track the spreading of diseases HIV, bird flu, common, colds, cholera, STDs, etc.Q INTRODUCTION The method of western blot was discovered by George Stark at | Stanford, and the name western blot was laid down by W. Neal Burnette. This method , also known as immunoblot or protein blot, detects specific proteins in a sample tissue homogenate or extract. Western blotting is a qualitative and semi-quantitative technique for protein analysis. This technique is useful in cell and molecular biology. It aids in identification of desired protein from a mixture of proteins extracted from cells It also facilitates evaluation of the size and amount of protein. * The technique of western blotting requires SDS-PAGE. Q PRINCIPLE. * Western blotting is a fast and sensitive method of assay that detects and characterises proteins. It works on immunochromatography principle, where is separation of proteins as per their molecular weight occurs in polyacrylamide gel The separated proteins are transferred or electro-transferred on nitrocellulose membrane, and then detected by specific primary antibody and secondary enzyme labelled antibody and substrate Protein Separate RNA on SDS- Transfer Polymer polyacrymide gel * cena, antibodies are exposed to filmQO PROCEDURE 1, Tissue preparation . Gel electrophoresis . Transfer Immuno- botting . Detection 1. Tissue preparation * In the sample, ice cold PBS and lysis buffer (e.g., RIPA buffer that provides maximum proteins yield) are added. The lysis buffer is selected based on the localisation of the desired protein. Unlike isolated cytoplasmic proteins, the solubilisation of membrane bound proteins is achieved by stronger extraction detergents. Lysis buffer should possess protease inhibitors that avoid the degradation the desired protein. Lysis of cells is done by incubating on ice and their applying shear pressure through pipette. Later the cell mixture is centrifuged and the pellet is discarded. The supernatant obtained is the lysate of use. Lysising release cot proteins2. Gel electrophoresis * The separation is achieved by using a positive electrode that attracts a negatively charged protein. This is done by introducing the prepared protein sample into a polyacrylamide gel. The steps involved in gel electrophoresis are i) The gel is prepared by inserting it in the electrophoresis apparatus and filling with running buffer. The wells of the gel are washed with running buffer and the chambers are added with buffer. The samples are loaded into the wells. A pre-stained molecular weight ladder is loaded in one well to aid in monitoring protein separation during electrophoresis and verifying protein weight in the sample later during analysis. The electrophoresis unit is closed and a power supply is connected. The majority of units run for 45-60 minutes at 200 volts or until the loading buffer seeps to the bottom of gel. In this time period, the negatively charged sample proteins migrate towards the positive electrode through the polyacrylamide gel matrix. lgupantThe separated proteins from gel are transferred to a solid membrane or blot. The wet transfer method involves the following steps: The gel is removed from its cassette by cutting the top portion of the wells The top left corner is marked to indicate gel direction. iii. The gel is allowed to float in transfer buffer while preparing the | transfer sandwich using a cassette, sponges, filter paper, the gel, | and PVDF or nitrocellulose membrane. iv. The top left corner is marked on the blotting paper to indicate blot | direction. The membranes are incubated for 10 minutes in transfer buffer. i. A stack is prepared by placing sponge, filter paper, gel, membrane, filter paper, and sponge from the black negative cathode towards red positive anode. ii. The gel or membrane should not be touched with bare hands and clean tweezers or spatula should be used. iii. A clean roller should be employed at every layer to remove xi. bubbles they will interfere with efficient protein transfer. The cassette is locked and placed in the transfer apparatus containg cold transfer buffer positioned from positive to negative. The heat development can be avoided by transferring with a cold pack , the apparatus of operating in a cold room with the spinner bar at the bottom of the chamber. The chamber is closed and power supply is connected. xii. Transfer is done as per the manufacturer's instruction.Diectrode Electrophonically transfer of protein sample onto PVDF membrane 4. Immuno- botting * The immuno-blotting method involves the following steps: i) The membrane is detached from the cassette and rinsed thrice with water. ii) The blot areas which do not contain protein are blocked to avoid non-specific antibody binding and to minimise overall background signal. iii) The commonly used blocking buffers are 5% non-fat dry milk or BSA ina TBS-Tween solution. Milk solution is avoided when probing with phosphor-specific antibodies to prevent high background from its endogenous phosphoprotein, casein. iv) The membrane along with blocking solution is incubated for an hour at room temperature with slight agitation. v) The blocking solution is decanted and washed for 5 minutes with TBS-tween. vi) Before adding the primary antibody, it is diluted with a blocking buffer (in specified concentration) and incubated overnight with gentle shaking at 4°C temperature. vii) After incubation the primary antibody is decanted. The membrane is washed five times for five minutes each using large volumes of TBS tween with vigorous agitation to eradicate non- specific background signals.viii. Thereafter, the secondary antibody is diluted in blocking solution. The membrane is incubated for an hour at room temperature at the specified concentration. The membrane is decanted and washed five times for | five minutes each with large volumes of TBS tween | followed by vigorous agitation. 5. Detection * The detection method involves the following steps: i) Equal parts of ECL reagents are mixed in 1:1 ratio as per the manufacturer's instructions. ii) The membrane is incubated for 3-5 minutes with no agitation. iii) The ECL mixture is decanted after incubation, and the excess solution is wiped off from the membrane corner. iv) In order to avoid drying, the membrane is wrapped in a clear v) A roller is used to remove bubbles (if formed) or any excess solution. vi) Without further delay, the membrane is developed. vii) Manual adjustment of De exposure time is done by using film and camera systems to obtain a picture perfect western blot. viii) Quantification of relative band densities is done using the plastic-like sheet protector. | } commercially available software. ix) Molecular weight verification can also be achieved through comparison between the band sizes and the molecular weight ladder.SDS PAGE: separation ; ; ‘of protein sample ‘Sandwich preparation Ka Membrane Electrophonically transfer of protein sample onto PVDF membrane | ee a f— ££ Q APPLICATION Diagnosis of HIV by ELISA involves the western blotting technique. Western blotting technique is also used to detect some forms of lyme Diseases. Block the membrane with BSA or milk casein aoubody Secondary Protea antibody Incubate the membrane with primary antibody. specific Incubate the membrane the target protein ‘with HRP conjugated secondary antibody Xe film SOLD Autoradiography Western blotting technique is used in definitive test for BSE. Confirmatory test for Hepatitis B involves western blotting technique. Western blotting test is used in the analysis of Biomarkers such as hormones, growth factors and cytokines. This technique is also employed in The Gene expression studies.Q INTRODUCTION + E.M. Southern invented the technique of southern blotting. This method useful in molecular biology. In this method, hybridisation analysis is performed by transferring the DNA from a gel to a membrane. The DNA is cleaved using suitable restriction enzymes and made to run ona gel. Then it is treated sodium hydroxide that denatures the DNA to give a single strand. Q PRINCIPLE * Southern blotting is a hybridisation technique used for identifying particular of DNA from a mixture of other similar molecules. This technique relies on the principle of separating DNA fragments by gel electrophoresis and identifying them by labelled probe hybridisation. Electrophoresis ge! A \ Za “ oS ~< < - - Se 2 ye 3 - DNA transferred to filterQO PROCEDURE * In the method of southern blotting, the DNA is transferred from a gel to a membrane for hybridisation analysis. * Then the DNA is cut with suitable restriction enzymes and run ona gel. * On treating with NaOH, the DNA undergoes denaturation to yield a single strand. * The DNA is transferred from agarose gel to the membrane by capillary action. * The gel is placed ona filter paper saturated with buffer, and the nitrocellulose membrane is placed above the gel and covered with 2-3 layers of dry filter paper towel . * The buffer reaches the top papers by flowing through the gel and membrane. * While flowing it carries the DNA fragment with it, but DNA is retained on the paper as it cannot pass through the membrane. * This membrane with trapped DNA is exposed overnight to a solution containing the radio labelled cDNA probe. * The binding of probe to its complementary sequence detected by autoradiography. a Blotting Butter oO, wee _ TT = —— ortvion rites tr * p-probe _Bxpose to | Xeray Files” Nitrocellulose ornylon Filter Southern Blotting TechniqueQ) FACTORS AFFECTING SOUTHERN BLOTTING 1. Stringent conditions (stringency control) * Stringency refers to the specificity with which a particular DNA target sequence is detected by a probe. * Thus, with high stringent conditions only completely complementary DNA sequences will bind and hybridize. * However, low stringent conditions will allow hybridization of partially matched sequences. Therefore, stringency control is very _| essential for specific detection of DNA molecules in Southern | blotting 2. Membranes for blot transfer * In the early years, nitrocellulose was used for immobilization of DNA molecules during blot transfer. * The drawback with nitrocellulose is that it is fragile and has to be carefully handle. Q APPLICATION It is an invaluable method in gene analysis. Important for the confirmation of DNA cloning results. Useful for mapping restriction sites around a single copy gene sequence. Forensically applied to detect minute quantities of DNA. Highly useful for the determination of restriction fragment length polymorphism (RFLP) associated with pathological conditions. | DNA pieces from one species (e.g. human) can be used to detect DNA | molecules from related species (e.g. chimpanze, cow). This technique is referred to as Zoo-blotting.Q INTRODUCTION * Genetic organization refers to the linear order of DNA elements and their division into chromosomes. * Genetic organization can also refer to the 3D structure of chromosomes and the positioning of DNA sequences within the nucleus.OQ INTRODUCTION Eukaryotes are organisms whose cells contain a nucleus and other membrane-bound organelles. There is a wide range of eukaryotic organisms, including all animals, plants, fungi, and protists, as well as most algae. Eukaryotes may be either single-celled or multicellular. In eukaryotes, the major part of DNA is found in the chromosomes, which themselves are present within the nucleus. The eukaryotic cell membrane has pores and is continuous with the endoplasmic reticulum. The eukaryotes have varied number of chromosomes which is fixed for a biological species. During cell division the physical characteristics of chromosomes telomeres A eukaryotic chromosome has a single, very large, and linear double stranded DNA (ds-DNA molecule. For example, a diploid human cell that contains 46 chromosomes (22 pairs of autosomes and a pair of sex chromosomes) has a total of 6 x 10" base-pairs.* In individual human chromosomes, the length of DNA molecules ranges from 1.5-8.7cm. * These large DNA molecules are packed in chromosomes ranging from a few microns in length and breadth. * This is achieved by the formation of protein-DNA complex known as chromatin. * The proteins that bind to DNA are histones and non-histone proteins. Histones are basic in nature due to the presence of basic amino acids. Since they carry positive charges, they bind to the negatively charged DNA molecules forming characteristic structural units, ie. the nucleosomes. The long double stranded DNA molecule in each chromosome systematically surrounds the histones to form nucleosomes. * Nucleosome * The structure of nucleosome resembles beads connected to each other through linker DNA. A nucleosome bears a histone core having 8 sub-units (octamer) of 4 different histones, ie., H_A, H-B. H3, and H. with two molecules of each. This histone protein core is encircled by two turns of ds-DNA molecule, thus forming a nucleosome. The DNA molecule runs as a continuous thread from one nucleosome to another. The dominant part of DNA between the two nucleosomes is the linker Nucleosome width is 11mm, and it repeats itself at intervals of 200 nucleotide pairs of DNA. The linker between two nucleosomes may have varied length. The nucleosomes are basic structures that build chromatin. They are organised as closely packed 30nm chromatin fibres, which can be observed under high resolution electron microscope.Q INTRODUCTION * Prokaryote, also spelled procaryote, any organism that lacks a distinct ‘nucleus and other organelles due to the absence of internal membranes. * Bacteria are among the best-known prokaryotic organisms. Capsule Cell wall Plasma membrane. cytoplasm. Ribosomes. Plasmid Pill Bacterial Flagellum Nucleoid (circular DNA)QO) GENETIC ORGANIZATION IN PROKARYOTES The prokaryotic genome is structurally different from eukaryotic genome. On an average it has only about one-thousandth of DNA as in eukaryotic genome. In prokaryotes, mostly the genome has a DNA ring with few proteins linked to it. This genetic material ring is the prokaryotic chromosome. In eukaryotes, the chromosome lies within the nucleus, whereas in prokaryotes, the chromosome is in a nucleoid region. In prokaryotes and eukaryotes, fundamental similarity is observed in DNA replication, transcription, and translation however, some differences also exist. For example, ribosomes in prokaryotic cell are smaller than those in eukaryotic cells. Also the protein and RNA content of these ribosomes is different. These differences are important for some antibiotics as they bind to ribosomes and block protein synthesis. This blocking action takes place only in prokaryotes and not in eukaryotes. Hence these antibiotics kill bacteria without harming the human cells. Prokaryotes (like E. coli) lack a well-defined nucleus. Hence, the negatively charged DNA binds to each other through some positively charged proteins in the nucleoid region. The DNA in this region is arranged within the nucleus as large loops connected through proteins.Bacterial virus SVL? sRna J RNase cutting « Riboswitch 16S rRNA QO DIFFERENT BETWEEN PROKARYOTIC AND EUKARYOTIC CHROMOSOMES. Prokaryotic Chromosome Eukaryotic Chromosome The chromosome formation is not The genetic material is organised into present. chromosomes. Asingle chromosome is present in Two or more chromosomes are each cell. present in each cell. It is shorter. They are larger. It contains a covalently closed circular _ they contain a linear DNA with two DNA ends It codes for a few proteins, They code for numerous proteins. It occupies the center region of the They are enclosed in the nucleus cell and is not enclosed by the nucleus, It is in direct contact with the They are separated by the nuclear cytoplasm as the nucleus is absent membrane.It is associated with the mesosomes of the plasma membrane. DNA is not associated with histone proteins Nucleosomes are not formed It contains a single origin of replication (Ori). The DNA replication can occur at any stage of the life cycle. It has a negative charge that is nullified by Mg” ions. It is circular, thus telomere is absent. Centromere. kinetochore, secondary constriction and chromosomal arms are not formed. They are not associated with the | plasma membrane and stay away from it DNA is associated with histone proteins Nucleosomes are formed by the association of DNA with the histone proteins. They contain many origin or replications. The genetic material replicates at the S phase of cell cycle. They have a negative charge that is nullified by the positively charged histone proteins. Telomere is present in their tip. They contain centromere, kinetochore, and chromosomal arms.TRANSDUCTION CONJUGATION PLASMIDS TRANSPOSONSQ INTRODUCTION | * Microbial genetics is the study of genes and gene function in bacteria, archaea and other microorganisms. Microorganisms acquire genes and undergo recombination, in which the genetic material from two organisms combines to form a new | chromosome having a genotype different from that of the parent. This new arrangement of genes exhibits new physical or chemical properties. The microorganisms are known to undergo several recombination types, of which general recombination is the most common one. It involves a reciprocal exchange of DNA (between a pair of DNA sequences) on the microbial chromosome and is characterised by the exchanges occurring in bacterial transformation, bacterial recombination, and bacterial transduction. Chromosomal DNA F plasmid Chromosomal DN Recipient => DNA polymerase RelaxaSome Transferasome F plasmid F plasmid Pilus Old donor New donorQO INTRODUCTION * Transformation is a kind of genetic recombination which involves the passage of only gene carriers (i.e., the DNA molecules of donor cell) into the recipient cell through the liquid medium. * It was described by Frederick Griffith (an English bacteriologist) in 1928, Restriction enzyme Gene to be transferred Plasmid of cell Plasmid of EB bacterial cell \ Enzyme DNA ligase Ben CaliQO MECHANISM Transformation takes place in a few cells of the mixed population. A few donor cells break apart , resulting in an explosive release and fragments of DNA. A fragment of double stranded DNA gets attached to the recipient cell for entry into the recipient cell. During entry one strand of the fragment is dissolved by the enzyme, thus only the second strand is left , which passes into recipient cell | through cell wall and cell membrane. Within the recipient cell , a portion of single strand of double stranded DNA of recipient cell is displaced by the enzyme and then replaced by the DNA of donor cell. The displaced DNA is dissolved by another enzyme. Thus the recipient cell becomes transformed displaying the character of its own as well as of the newly incorporated DNA. Q INTRODUCTION * Transduction is a genetic recombination method which involves transfer of genetic material from to donor to the recipient cell | through a non-replicating bacteriophage. This process was discovered by Joshua Lederberg and Norton zinder in 1952 during their research with salmonella typhimurium. In transduction , a small fragment of bacterial DNA is incorporated into an attacking bacteriophage . When this bacteriophage infects a new bacterial cell , it transfers the genetic material into it, thus | genetic recombination take place.@® Bacterial DNA @® Viral ONA * The specialised transduction process occurs as follows: 1) The bacteriophage gets attached to a bacterial cell wall at the receptor site and its nucleic acid is transferred into the cytoplasm of the host cell. The phage does not cause lysis of the host bacterium. The phage nucleic acid codes for the synthesis of repressor | proteins in the bacterial cell. These proteins prevent the virus from producing the material required for its replication. In the bacterial cell, the viral DNA exists either as a fragment in the cytoplasm or attaches itself to the chromosome, known as prophage. the bacterial cell which carries the prophage is lysogenic and the phenomenon in which the phage DNA and bacterium exist together is termed lysogeny. The bacterial cell may remain lysogenic for many generations and during this period the viral DNA replicates a number of times with the bacterial chromosome. However, later the phage stops the synthesis of repressor proteins in the bacterial cell, followed by the synthesis of phage components,8. Thereafter, the phage DNA separates from the bacterial chromosome | and induces the synthesis of phage proteins. 9. During this separation, a number of genes of the bacterium get attached to it. 10. These attached genes replicate along with the phage DNA and later develop into phage particles, which come out from the bacterial cell by bursting. 11. When the new phage particle infects a new bacterial cell the attached bacterial genes along with the phage particle enter the chromosome of the new bacterium and cause recombination. | 12. Thus, the new bacterial cell contains genes of its own as well as from the | parent bacterial cell. Specialized transduction* Generalised transduction occurs more commonly than the specialized transduction , and involves the following steps 1) The prophage particle is present in the cytoplasm of the infected bacterial cell. 2) The phage DNA starts synthesising new phages. 3) The chromosome of bacterial cell gets fragmented. 4) Some fragments get attached to the DNA of some new phage particles , with the others remain with phage DNA. 5) The newly formed phage with fragment of bacterial chromosome in its DNA attacks a new bacterium. 6) Asa result, the gene of the parent bacterium is transferred to the bacterium and causes recombination. Transduced bacteriumQO) INTRODUCTION * Conjugation involves exchange of genetic material between two bacterial cell through a conjugation tube. * This process was first postulated in 1946 by Joshep Lederberg and Edward Tatum in Escherichia coli Later conjugation was also demonstrated in Salmonella. Vibrio, and Pseudomonas F plasmid Chromosomal DNA Donor (F* cell) Recipient (F° cell) @oO—-CS&) & Or & Original donor (F* cell) New donor (F* cell)In conjugation, two cells of opposite mating type temporarily attach with each other via sex pilus. There is a hole of 2.5m diameter in sex pilus, through which DNA can pass from the donor to the recipient cell. The F-factor or F-plasmid is a double stranded DNA loop found in the cytoplasm along with the nucleoid. After the conjugation tube is established, the F-factor prepares for replication by the rolling circular mechanism. The two strands of F-factor separate from each other and one of them passes to the F ~cell ( recipient cell ). On reaching the F ‘cell, the enzymes synthesise a complementary strand to form a double helix, which bends into a loop. Thus, the conversion process is completed. In F* cell (donor cell) also, a new DNA strand is formed to complement the left over DNA strand of the F-factor QO INTRODUCTION * Apart from bacterial chromosome (nucleoid), genetic elements are | also found to be present in the cytoplasm of bacterial cells. These | genetic elements, calles plasmids, replicate separately from the chromosome, In 1952, Lederberg demonstrated the existence of plasmids in bacteria cytoplasm while working on conjugation process in bacteria. —_ Bacterial DNA PlasmidsQO PROPERTIES * Given below are the properties of plasmids: 1) They are specific to one or a few particular bacteria. 2) They replicate separately from the bacterial chromosome. 3) They code for their own transfer. 4) They act as episomes and reversibly integrate into bacterial chromosome 5) They may pick-up and transfer certain genes of bacterial chromosome. 6) They may affect some of the characteristics of bacterial cell. 7) They differ from viruses as i. They do not cause damage to cells and are beneficial. ii. They do not have extracellular forms and exist inside the cells as free and circular DNA. Q REPLICATION Plasmids have their own replication origins, thus replicate independently. The enzymes involved in the replication of are normal cell enzymes. A very few genes (<30) are present in plasmids and they control the replication initiation process. They are also involved in the distribution of the replicated plasmids between daughter cells. The genetic information carried in plasmid genes is not essential to the host because the bacteria lacking them function normally. Plasmid DNA is small in size, thus its replication takes place rapidly Plasmids in gram-negative bacteria mostly replicate in a similar way as the bacterial chromosome that involves initiation at the replication origin site and bidirectional replication around the DNA circle giving a theta intermediate. Some plasmids of gram-negative bacteria, however, replicate by unidirectional method.Bacterial DNA Plasmids Integrated plasmid Plas: L integratio Cell replicatio TRANSPOSON: Q INTRODUCTION * A transposon is a DNA sequence that can move or insert itself at a new location in the genome. | This phenomenon of transposon movement to a new site in the genome is termed transposition. Transposons can encode transposase (a special protein) that catalyses the transposition process.ONA transposon (Manner type) B Transposase binding Cleavage Target capture and strand transfert target DNA Q TYPES * Transposons are categorised into the following types based on their | transposons mechanism: 1) Cutand paste transposons 2) Replicative transposons and 3) Retro elements1. Cut and paste transposons * These transposons transpose by cutting the transposable sequence from one position in the genome and inserting it into another position within the genome. * Inthe cut and paste transposition, two transposase sub-units are involved an each hinds to the specific sequences at the two ends of transposon. * Then this sub units of transposase protein come together and lead to the excision of transposon. * The so formed excised transposon-transposase complex integrated to the target recipient site Hence, in this way the transposon is cut from one site and pasted on the other site by a mechanism mediated by transposase protein. __ ee 1 1. Transposase binding sO nomen 2. Transposition complex formation a Lt 3. Excision <> + 4. Target site recognition <> yo WEEE2. Replicative transposons * These transposons transpose by replicating the transposable sequence, and inserting the so formed copy of DNA into the target site without altering the donor site. In this type of transposition, one copy of transposon is gained, and after transposition the donor as well as the recipient DNA molecule — exhibit one transposable sequence each. Target sequence3. Retro elements These elements transpose by DNA synthesis via reverse transcription by using elements RNA as the template. The transposable elements’ requiring reverse — transcriptase for their movement are the | retrotransposons. In this type of transposition, an RNA intermediate is involved, and the transposable DNA is transcribed to produce an RNA molecule. Then this RNA is used as a template for producing a complementary DNA by the activity of reverse transcriptase enzyme. The so formed single stranded DNA copy is made double stranded and inserted into the target DNA site. Formation of Ribonucleoprotein complexes LY Transcription Transcription SD) & Translation of Integration cotQ) SIGNIFICANCE * Transposons have the following significant values: 1) They have the ability to alter the structural and functional characteristics of genome by changing their position in the genome. 2) They can cause mutation by insertion, deletion, etc. 3) They can contribute in evolution as they can tremendously alter the | genetic organisation of organisms. | 4) They can be used as cloning vectors in gene cloning. For example, P | - elements are often used as vector for introducing transgenes into Drosophila. 5) They can also be used as genetic markers while mapping the genomes. 6) Transposon-mediated gene tagging can help in searching and isolation of a particular gene.Points to be covered in this topic INTRODUCTION TYPES OF REACTION APPLICATIONQ INTRODUCTION 2, Hydroxyiation * Microorganisms are capable of modifying a wide range of organic compounds enzymatically. * Bio-transformations are the processes in which microorganisms convert the organic compounds into structurally related products. * It can also be said that biotransformation involves microbial | conversion of a substrate into a product with a few number of enzymatic reactions. * Arule framed based on the results of earlier studies stated that if a | secondary hydroxyl group of a polyol is located between a primary and a secondary group, which is in the cis-position with respect to the oxidisable group, then the hydroxyl group is oxidised to give a ketone. * Example - conversion of naphthalene to salicylic acid. HO. HO . _Pseudomonas Aeruginess ° Naphthalene Salicylic acid * Hydroxylation of steroid isa monooxygenase reaction. * It has been established that the carbon atoms can be hydroxylated by different organisms. * The stereospecific microbial hydroxylation are practically available in all the carbon atoms of steroid molecule. * 11a-hydroxylation , 11f-hydroxylation , 16a-hydroxylation , and 21 hydroxylation are the industrially important hydroxylation reactions. * The increased demand for cortisone and hydrocortisone can be met by microbial hydroxylation of steroid intermediates at C-11.3. C- * The therapeutic properties of cortisone and cortisol were modified by introducing a double bond between C-1 and C-2, and producing | prednisone and prednisolone, respectively. 4. Reduction * The size and nature of the substituents in the substrates influence the stereospecific microbial reduction of ketonic substrates. * For example, highly stereospecific reduction of racemic decalone | and hexahydroindanone derivatives and of related di- and tri-cyclic ketones by Curvularia falcata. * Stereospecific reduction occurs when the ketone is flanked by large (L) and small (s) groups and gives alcohol of S-configuration. 5. Hydrolysis | * Alarge number of esters, glycosides, epoxides, laciones. P lactams, | and amides can be hydrolysed by microorganisms. For example tartaric acid is produced from maleic anhydride by Achromobacter tartarogenes. A 9 0 ° Achromobacter HO. HO OH Tartarogen ar CH ° Oo o | Maleic anhydride (Cis Epoxysuceinic acida . 8. Beicaa setation Asymmetric addition of water or ammonia to fumaric acid yields | optically active products, Therefore, aspartase-producing bacteria have been used for | manufacturing L-aspartic acid. Fumaric acid L-Aspartic acid Pre-treatment with detergents is recommended to suppress the formation of succinic acid, and this result into hydration to L-malic acid (with 70% yield). Amination is involved in the manufacture of many L-amino acids and guanosine-5'-monophosphate (5'-GMP) with the help of two mutants Brevibacterium ammoniagenes. Dehydration * Microbial dehydration of fatty acids. histidine, cis-terpne hydrate, L-phenylalanine, etc. are some commonly occurring reactions, 9. Deamination * Microbial deamination is either of the two types i, Hydrolytic: For example, conversion of L-tyrosineinto 4hydroxyphenylacetic acid ii. Oxidative: For example, conversion of isoguanine into xanthint and formycin A. which further converts into formycin B.10. Condensation * Condensation is the reaction of substituted benzaldehyde utilised in 1934 for synthesising natural (1R,2S)-ephedrine. * Acetaldehyde is reacted with benzaldehyde added to the fermenting yeast, to yield (R)-1-phenyl-1-hydroxy-2propanone. * Propanone undergoes reductive chemical condensation with methylamine to yield (IR, 2S)-ephedrine. ° “OA Of” Benzaldchyde Acetaldehyde = (Q)-1-Phenyl-I-hydroxy-2-propanone QR.2S)- Ephedrine “ A selected list of important biotransformation reaction aan Commonly used peerat Cet] pata ec ky Oxidation * Tryptophan * Bacillus subtitis Shydroxytrptophan * Corynebacterium Naphthalene —> Salicylic sP acid Reduction Benzaldehayde —> Benzyl Saccharomyces alcohol cerevisiae Nitropentachlorobenzol Streptomyces —> Pentachloroaniline aurec ace pydrolysis Anhydrotetracycline —> Streptomyces Tetracycline aureofaciens Mycobacteri Methyl laureate —> hat aaa phlei Menthol condensation Streptomycin > Streptomyces Streptomycin phosphate Brscus* The desired cells are cultivated in a suitable medium. * As the growth of the cells occurs a concentrated substrate is added to the culture. * Sometimes , addition of emulsifiers is required to solubilize substrates or products. + E.G. steroid biotransformation. 2. Non -growing cells * The non - growing cells are preferred for biotransformation reactions for the following reasons. v Very high concentration of substrate can be used. y Cells can be washed and used and thus there will be no contaminating substances. Y Conversion efficiency of substrate to product is high. Y Biotransformation can be optimized by creating suitable environmental conditions. Y Product isolation and its recovery are easy. 3. Immobilized cells * Biotransformation can be carried out continuously by employing immobilized cells. + Further, the same cells can be used again and again. * Several bioconversion reactions with single or multistage reactions are in fact carried out by using immobilized cells. * E.G. commercial production of] -alanine and malic acid 4. Immobilized enzymes * Cell-free enzyme systems in the form of immobilized enzymes are most commonly used in biotransformation , due to the following advantages. Y No occurrence of undesirable side reactions. | v The desired products are not degraded.v There is no transport barrier across the cell membrane for the substrate or product. v The isolation and recovery of the product is simpler and easier. Q APPLICATION 1. Bi f . f id * All the steroids possess the basic structure namely cyclopentanoperhydrophenanthrene. Steroids as hormones (glucocorticoids, mineralocorticoids, androgens, estrogens) perform a wide range of functions. * They are very useful therapeutically. For instance, cortisone, due to its anti-inflammatory action is used in the treatment of rheumatoid arthritis and skin diseases; derivatives of progesterone and estrogens are employed as contraceptives. v Examples Production of hormones like adrenocortical hormone. All carbon atoms present in the steroid molecule with 11a, 11, and16a-hydroxylases can be hydrolyzed by fungi giving the great economic worth Reduce the double bond in steroids such as androgens, estrogens, and - bile acids. Biosynthesis of androgens in Nectria haematococca.2. Bi f ion of antibioti * Production of new antibiotics or modifications in the existing ones for more effective treatment of the diseases is always on the priority of the pharmaceutical industry. * Further, antibiotics with wider antimicrobial spectrum, reduced toxicity, low allergic reactions and decreased resistance are highly advantageous. v Example TRY CEL Iho i] enhanced Benzopyrene Theophylline Carbamazepine Carbamazepine , clonazepam, itraconazole Chlorcyclizine Steroid hormones Ethchlorvynol Warfarin 3. Bi f ion for ti juction of bic acid * Ascorbic acid (vitamin C) can be commercially produced by a combination of chemical and microbial transformation processes. 4. Prod “ findi | * Indigo can be synthesized by microbial transformation. * This has been made possible by cloning a single Pseudomonas gene that encodes naphthalene dioxygenase in the creation of E. coli. 5. Environment * Biological processes are employed in the removal of contaminants an pollutants from the environment.Points to be covered in this topic INTRODUCTION TYPES OF MUTATION TYPES OF MUTANTS AGENTS OF MUTATIONQ INTRODUCTION + Mutation refers to a change in the DNA structure of a gene. * The substances (chemicals) which can induce mutations are collectively known as mutagens. * The changes that occur in DNA on mutation are reflected in replication, transcription and translation. QO TYPES OF MUTATION 1. Point mutations * The replacement of one base pair by another results in point mutation. They are of two sub-types. (a) Transitions : In this case, a purine (or a pyrimidine) is replaced by another. (b) Transversions : These are characterized by replacement of a purine by a pyrimidine or vice versa. —C-G-A—G— transition _—C—G—G—G— I | | ——+ 2. Frameshift mutations * These occur when one or more base pairs are inserted in or deleted from the DNA, respectively, causing insertion or deletion mutations. T. Il Tf pene ee ee ae ne » tt —G-C3. Neutral Mutation * In this type, the replacement of amino acid due to base substitution | does not cause any functional change in the protein. For example, the amino acid serine is converted to threonine, valine is converted to alanine etc. 4. Deletion mutation * In this type, the deletion can be short or long. * Bacterial genome generally bears long sequences that can be deleted with no loss of bacterial viability. * Recombination of directly repeated sequences in the bacterial genome causes deletion. 5. Duplication * In this type a piece of DNA is abnormally copied multiple times. * This unusual copying result in functional changes in the protein formed. 6. Inversion * Occasionally a region of DNA reversibly aligns into the opposite orientation. If the signals needed for expressing an adjacent gene are located in the inverted region, inversion will shift the associated gene on or off. Repeated sequences in a genome may present inversion as well. For example, variation in the flagellar antigens is seen in Salmonella typhimurium.7. Insertion * An insertion, as related to genomics, is a type of mutation that involves the 8 a page) segs addition of one or more nucleotides into a segment of DNA. * An insertion can involve the addition aggeoo cao oO of any number of nucleotides, from a single nucleotide to an entire piece of a chromosome. 8. Substitution M : * In this type, an exchange between two bases occurs. * This alters a codon encoding a different amino acid, thus producing a different protein. * At times the protein structure is not affected by substitutions; such mutations are named as silent mutations. 1 | | Starting sequence | TCTCGCAT AGAGCGIA 6 S (a) Substitution Transition: Purine for purine, pyrimidine for pyrimidine He TcecatMetracer _AGAGCGTABCATCCA ‘Transversion: Purine for pyrimidine, pyrimidine for purine or PEPER RE RNSnE eee 9. Repeat Expansion * In this type, the number of repetition of short DNA sequence is increased. * Thus, the formed protein functions differently than the original. * Nucleotide repeats are the short DNA sequences repeated times in a row. For example, a tri-nucleotide repeat comprises of 3 base- | pair sequences, and a tetra-nucleotide repeat comprises of 4 base- pair sequences.QO MUTANTS + A mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. QO TYPES OF MUTANTS" 1. Morphological Mutants * These mutants show alteration in their form (shape, size, and colour). * Some examples of morphological mutants are albino spores in Neurospora, curly wings in Drosophila, dwarf-sized peas, and sheep with short legs. 2. Lethal Mutants | * In these mutants, the new allele is recognised by its mortal or lethal | effect on the organism. | * Lethal mutant alleles lead to the death of individuals carrying them; | however, semi-lethal or sub-vital alleles let the individuals survive. Conditional Lethal Mutants * Under specific environmental conditions, some alleles generate a ' mutant phenotype called restrictive mutants. | * When the conditions are different, they produce a normal phenotype called permissive that can be grown under permissive conditions and later transferred to restrictive conditions for further evaluation. Bioct ical M * In these mutants, their biochemical function of the cell is lost. * The normal function of the cell is regained upon enriching the medium with suitable nutrients. * For example, adenine auxotrophs can be grown in adenine-rich medium, whereas wild type can grow in adenine deficient medium.QO AGENTS OF MUTATION * Physical or chemical agents that increase the frequency of mutations are termed mutagens. * Various physical and chemical mutagens are presented below: ohne oxy ouatd radiation Penetrating and non particulate Highly penetrating and non-particulate. Less penetrating, particulate, and positively charged. More penetrating than a rays, particulate, negatively charged. Highly penetrating, particulate , and neutral particles Low penetrating and non ionising Mode of action Induce mutations by forming free radicals and ions; cause addition, deletion, transition and transversion. Induce mutations by ejecting atoms from the tissues; cause addition, deletion, transition and transversion. Induce mutations by ionisation and excitation; cause chromosomal and gene mutations. Induce mutations by ionisation and excitation; cause chromosomal and gene mutations. Cause chromosomal breakage and gene mutation Cause chromosomal breakage and gene mutationEthyl methane sulphonate Ethyl ethane sulphonate 5 - bromo uracil 2- amino purine Acriflavin Proflavin Nitrous acid Hydroxylamine Sodium azide AT GC transitions GC AT transitions AT GC transitions AT GC transitions Deletion , addition and frame shifts AT GC transitions GC AT transitions