Seminar Paper Present (M.Sc; Biotechnology)

University of Yangon

Department of Zoology(BIOTECHNOLOGY)

Biotechnology

Biotechnology is defined as “the application of biological organism, system or process to manufacturing and service industries”. The “biotechnology” is sometimes use in a much narrower sense for the pursuit of manipulative genetics and molecular biology in hopefully practical direction. Biology is the controlled and deliberate application of sample biological agents-living or dead cells or cell components in technically useful operation either of production manufacture or as service operations. The word biotechnology can be interpreted in a very broad sense, the embrace all operation of all the operation applied biology from agriculture to cookery.

Biotechnology is a highly multidisciplinary applied science and has been defined in several way. The term biotechnology is the short form of biological technology. One of the most popular definitions of biotechnology is the application of scientific and engineering principles of the processing of materials by biological agents to provide good and services. Biotechnology is the development of products by exploiting biological process is substances. Another definition of biotechnology is the use of biological organisms or their constituents to the transformation of input in to commercial output. Biotechnology concerns the practical application of organisms or their components some definitions of biotechnology are as follows.

(1) Application of biotechnological organisms, systems or processes to manufacturing and service industries.

(2). The integrated use of biochemistry , microbiology and engineering science in order to achieve technology (industrial) application capabilities of microorganisms, cultured tissue cells and parts.

(3). A technology using biological pheromone for copying and manufacturing various kinds of useful substance.

(4) The science of the production processes base on the action of microorganisms and their active components and production process involving

Culture of Plant Protoplast

Introduction:-

Plant tissue culture is an abbreviation for plant protoplast, plant cell, plant tissue, plant organ and plant culture. General, the plant tissue culture is use form,

1. The production of pharmaceutical.

2. The preservation of valuable germplam.

3. The transport of plant material from one country to another.

4. The recovery of diaene-free-clones.

5. The rapid colonel multiplication of selected varieties and, and

6. The investigation of various aspect of development botany

In this paper culture of plant protoplast is described.The protoplast is the entire plant cell without its cellulose cell wall. When protoplasm is free from cell wall and it includes protoplasm and vacuole. Every plant cell possesses a definite cellulose cell wall and protoplast within the cell wall. Such the modification to the genome or cytoplasm requires that the cell wall is removed to give and isolated protoplast. Protoplast can be isolated from varieties of plant tissue. The basic principles of protoplast culture is the aseptic isolation of large number of intact living protoplast moving their cell wall and culture them on the suitable nutrient medium for their requisite- growth and development. In protoplast technology, from any two genotypically difference plant protoplasts are isolated from the somatic cell (diploid) are experimentally fuse to obtain parasexual hybrid protoplast. The hybrid protoplast contains hetroplasoic cytoplasm and fused parental nuclei. The fused protoplast is grown in vitro-with an aim to obtain hybrid plant. So in vitro fusion of plant protoplast derived either from somatic cell of plant is called somatic hybridization.

A major reason for rapidly expanding interest in protoplast culture is their potential used in plant cell genetic and specially in cell fusion and transfer of genetic information by DNA uptake organelle implantation. The protoplast culture are produced experimentally by the removal of all wall by either enzymatically or mechanical means from the artificially plasmolysed plant cell. Experimentally produced protoplasts are known as isolated protoplasts.

Principle of Protoplast culture

The basic principle of protoplast culture is the aseptic isolation of large number of intact living

protoplast removing their and culture them on a suitable nutrient medium for their requisite growth and development.

Isolation of protoplast

Isolated plant protoplast are cell from which the wall has been removed either mechanically or enzymatically . It is leaf and stem tissue from the auxiliary shoots provides a supply of surface, sterile meristematic tissue. It is difficult to regenerate plant from protoplasts isolated from leaf issue of monocosts. Cell wall of the explants tissue are removed by exposure lytic enzymes then after treatment such as electroporation, or fusion, the protoplast undergo cell division and generation. This tissue consists of cell without excessive secondary thickening of the cell wall so that protoplast can be release by only short-time exposure to potentially damaging lytic enzymes. Protoplast can be prepared from a variety of tissue but among them mesophyll tissue from a wide range of plant has been proved to be the most ideal source of plant materical for protoplast isolation. Leaves of Nacotiam tabacum is highly standardized material for easy entry in to the art of protoplast isolation and culture.

A considerably more efficient way of liberating the protoplasts to digest the cell wall away around them, using cell wall degrading enzymes such as cellulose, he-micellulase, pectinase or macerozyme etc. These enzymes are isolated from fungi and available commercially. Period of treatment and concentration of enzymes are the critical factors and both factors should be standardized for particular plant tissue. This method produces many damaged protoplast and the yield is always small. Techniques have been perfect for the removal of the cell wall by enzymatic digestion and it is the method which is now use predominantly. That treatment of cell which cell wall degrading enzymes may have an effect on the protoplast other than purely the removal of the cell wall. For the distance changes in the protoplast properties in relation to transminase activity were recorded when mechanically isolated protoplast were exposed to cell wall degrading enzymes for start time.

The essential step of the isolation of protoplast is the removal of the cell wall without damaging the cell or protoplast. Mechanical isolation involves breaking open each cell compartment to liberate the protoplast. This operation can be done carefully on small pieces of tissue under a microscope. The earliest upon mechanical removal of the cell wall.

Method of Protoplast Fusion

Protoplast fusion is a physical phenomenon. During fusion , two or more protoplast come in contact and adhere with on another either spontaneously or in presence of fusion including chemicals. Fusion of freely isolated protoplasts from different sources with the help of fusion including chemical agent is known as induced fusion. The isolated plant protoplasts can be induced to fuse by three ways,

(1) Chemical fusion

(2) Chemo – fusion

(3) Electro – fusion

Chemo-fusion

Several chemical have been used to induced protoplast fusion. Sodium nitrate(NaNO₃) polyethylene glycol (PEG), calcium iron(Ca²⁺), polyvinyl alcohol etc. The simplest and least expensive method to achieve protoplast fusion is by chemical method, of which the use of polymer polyethylene glycol (PEG) is the best known. The function of the PEG is the alter the membrane characteristic so that the protoplast become sticky and it the protoplasts are allowed to come into contact they will adhere together and the contents will fuse.

Electro-fusion

The first requirement in any experimental procedure that involves protoplast is to be able to isolate the protoplast of good “quality” that are variable and free from contamination debris. When protoplast that are in contact are subjected to suitable external electric field, which causes the formation of transient reversible pores in the plasma membranes they can fuse together. Protoplast have a negative surface change that helps repel neighbouring protoplasts. Protoplast can now be fused a large scale by electrical methods using charmer in which the cells are exposed to small electrical current. In diellectrophoresis protoplast in the medium of low conductivity (e.g a Manito solution of appropriate osmolarity) are placed between two electrodes, and a high frequency AC field (0.5 – 1.5 MHz) is applied. The value of critical voltage is in the order of 0.5 to 1.5v, and varies with membrane composition, cell type and origin.

Recently, mild electrical stimulation is being used to fuse protoplasts. This technique is known as electro-fusion of protoplasts. Two classes capillary microelectrodes are placed in contact with the protoplast. An electrical field of low strength (10Kvm^-1) gives rise to dielectrophoretic dipole generation within the protoplast suspension. This lead to pearl chain arrangement of protoplasts. The number of protoplast within the pearl chain depend upon the population density of the protoplast and the distance between the electrodes.

When the protoplast are in contact, the plasma membranes break and fuse forming a continuous membrane a round the pearl chains. In compare, the electrical method of fusion is much more production then the chemical method as up to 50% of protoplast may be as heterokaryons.

Figure;

The Composition of Culture Media and Culture of Protoplast

The media use for maintenance of tissue culture of the species or genotype being studied provides the basic nutrient for the culture of protoplasts.

Composition Medium for Protoplasts Culture

(Nagata and Takebe CNT) in 1971

Constituent Amount in mg/L	Constituent Amount in mg/L
Macronutrients NH4 NO3 825 KNO3 950 CaCL2, 2H2O 220 MgSO4,7H2O 1233 KH2PO4 680 Micro-nutrient KL 0.83 H3BO3 6.2 MnSO4,H2O 22.3 ZnSO4,4H2O 8.6 NO2NO4,5H2O 60.25 CuSO4,5H2O 0.025	Iron source FeSO4, 7H2O 27-28 Na, EDTA, 2H2O 373 Vitamin Meso-inositol 100 Thiamine HCL 1 Conbonhydrate source 1% Growth substances L-naplhalene acid (NAA) 3 6-Benzyleminopurine 1 Plansmolyticum 13% – 0% Mannitol pH 5.8

For solid medium 1.6% or 6% or 0.8% agar is added.

After isolation, the protoplast are very fragile and still need and osmoproteetant until cell wall are formed . The isolated protoplasts can be cultured either static liquid or agaritied medium. The media uses for the maintenance of tissue culture of the species or genotype being studied usually provides the basic requirement but than the auxin and cytokinin level may need adjustment . A typical medium would be MS medium 2.0 mg 1 NAA, 0.5 mgl-₁, BAP 3% source and 9% mannitol. The protoplast can be cultured either static liquid or agarified medium. The protoplast medium consists of mineral salts, vitamin, carbon sources and plant growth hormones as well as osmotic stabilizes and possibly organic nitrogen source, coconut milk and organic acids.

The initial stage of wall formation and division to form a callus is the first barrier to be overcome. All method of protoplasts culture is to protect protoplasts from the loss of soluble cell components by restricting the volume of surrounding medium. Once the callus is established and growing vigorously them it can be sub-cultured to normal regeneration medium. Isolated protoplast can be cultured in several ways of which agar embedding technique in small Petridis is commonly followed. In this technique protoplasts suspension is mixed with equal volume of melted 1.6% “Difeo” agarified medium (37*C) and the protoplast agar mixture are poured in to small petridishists. In petridishishers, embedding of protoplasts in solid agar medium is known as plating of protoplasts. In culture protoplast can reform a new cell wall around them . Then cell form, the protoplast subsequently enter cell division which is followed by the formation of callus and cell cultures. Such callus also retain the capacity for morphogenesis and plant regeneration.

Cell wall formation, Cell Division, Callus formation and Plant Regeneration

The viable protoplast in culture generate its on wall around them. Once the wall is formed, the protoplast becomes essentially a generated cell. The membrane of newly isolated protoplast contain protruding microtubules in that function in the orientation of newly synthesized cellulose microfibrits. The rate and regularity of cell wall regeneration depend on the plant species and the state of differentiation of the donor cell used for protoplast isolation calcaflvor white (CFW) is the most commonly used stain a detect the onset of cell wall regeneration. After the formation of cell wall, the walled cell expend and divide in to two cell. Several factors such as genotype if the donor plant, culture medium, hormones as well as physical factors are important for the division of protoplast and callus formation. The ultimate objective in protoplast culture is reconstruction of plant form the single protoplast. The strategy for plant generation has been to recover rapidly growing callus form protoplasts and to transfer the callus to species specific generation medium. Plant generation from protoplast derived callus tissue have been reported mainly form solanceous species. The list contain several species monot cot and dicot including carrot, alfalfa, clover, asparagus, cabbage, citrus etc.

Conclusion:-

All procedures must be conducted under aseptic condition. Aseptic isolation and in vitro culture of protoplast of higher plant is a novel technique in the field of plant tissue culture. The protoplast includes the plasma lemma and everything contained within i.e. the entire cell without it inherent cello sic cell wall plant protoplast can be isolated either mechanically or enzymatically for plasmolysed plant tissue or cells. Protoplast were first isolated using mechanical methods. By enzymatic method, protoplast are using cell wall degrading enzymes such as cellulose and pectinase. Experimental produced protoplast fusion is a physical phenomenon. Protoplast during isolation often fuse spontaneously and this phenomenon is called spontaneous fusion. Several chemical has been used to induce protoplast fusion. Sodium nitrate (NaNO₂), polyethylene glycol (PEG), (Calcium ions (Ca²⁺), polyvinyl alcohol etc. Recently, mild electrical stimulation is being used to fuse protoplasts. All method of protoplast culture is to protect the protoplasts from the loss of soluble cell component by restricting the volume of surrounding medium. In culture protoplasts can reform a new cell wall round them. Them cells from, the protoplast subsequently enter cell division which is followed by the formation of callus and cell culture. Such callus also retain the capacity for morphogenesis and pant regeneration. A sexual propagation is the potential for regeneration possessed by the vegetative ports of plants. A sexual propagation is important for growing cultures that produces no seeds and preserving clones. With some plant it may be more rapid and more economical than sexual propagation. Plant can be produced by vegetative and sexual reproduction although it is excluded sexual reproduction so that an organ of plant somatic tissue will be cultured to new generation plant. Plant inherent genetic information are controlled by plant tissue or, protoplast to the next generated plant. The fusion of nuclei in a binucleate heterokaryon result in the formation of a true hybrid protoplast. Protoplast fusion and somatic hybridization have opened up a new avenue in plant science. Protoplast fusion provides a method of combining the difference genomes of difference genera and species with potential of overcoming sexual incompatibility barrier between plants. Small species of plant can be two develop new plants in an artificial medium under aseptic condition, this process is micro-propagation or tissue culture. The advantages of micro-propagation are rapid, vegetative propagation of pathogen free, plants. This is specially useful for plant with very slow normal reproduction.

References books:

1. Plant tissue culture, Kalyan Kumar De, 1997.

2. Plant cell culture, Hamish A. Collin & Sue Edwards.

3. Plant Tissue culture and cell culture.(2^nd ed) Black well Scientific Publication, Oxford London.

4. Plant Biotechnology, Dr. U Win & Dr. Aye Pe, 2001.

Prokaryotes

Introduction

The kingdom monera contain all of prokaryotes organism i.e the cyanobacteria (Blue-green-algae) and the bacteria. There are two basic type of cell organization (1) prokaryotic cells and (2) Eukaryotic cells. Prokaryotic cell divided in to two division Cyanophytae, the cyanobacteria (Formerly known as blue-green-algae) and Schhizophytae, the bacteria. Eukaryotic cells found in animal, plant, fungi and unicellular organisms. And contain organelles that are bounded by membrane such as nuclei, mitochondria, plastids, lysosomes and so forth.

Prokaryotic cell

Prokaryotic cell found only inbacteria, don’t contain membrane-bone organelles. Prokaryotic cell differ from eukaryotic cell in three main ways (1) the structure and chemical composition of their cell wall, (2) the absence of the membranous-bound organelles (3) genetic materials.

1. The Structure and Chemical composition of their cell wall

It has no nuclear membrane, a prokaryotic cells has no distinct nucleus; they have ribosome and cell wall but ribosome are small and are usually made up of carbon hydrates and polypeptides chain rather than the cellulose. The prokaryotic cell wall is unique in that it is made up of mucocomplex substance, a polymer of glucose derivatives attach amino acids. In nature, prokaryotic cell produce a dense felt like melt of polysaccharides (Sugar-polymer) that enable them to stick surface soil particles, make possible rocks in stream beds on human teeth or gums for example.

In addition to plasma membrane and mesosmes, most photosynthetic prokaryotes also contain photosynthetic membranes but this one not enclosed in chloroplasts as they are in eukaryotic cell of plant.

Prokaryotic cells range in size from about 1 to 10 um: a few eukaryotic cells are smaller that about 7 nm is diameter, but most of much large. It may be that the size of prokaryotic cell is restricted by their lack of cellular organelles. The only organelles found consistently in all prokaryotes are ribosome, on which messager RNA transacted into proteins. However ribosome are smaller than the ribosome in cytoplasm of eukaryotes. Many biochemical reaction can take place only on the surface of the membrane, some prokaryotes lack of membranes except for their plasma membrane. However, it would be wrong to give the impression that no prokaryotes contain internal membranes. In some bacteria, the plasma membrane develop many loops and indentation that increase its surface area, forming a mesosome (Figure A). This is probably important to formation of a new cell wall during cell division, and to the separation of DNA prior to cell division. Furthermore photosynthesis prokaryotes may have internal membranous structure containing photosynthetic pigment and enzymes. There are no simple rules concerning the metabolism of prokaryotes. Some are aerobic, some are anaerobic and some are used nitrate (NO₂^-) or sulfate (SO₄^2-) instead of oxygen as electron acceptors in respiration.

2. Genetic Materials

Prokaryotic cells genetic material is contain in a large-single cell, circular molecule of double stranded chromoses chemically known as DNA in “a nuclear area” and RNA. Prokaryotic cell’s DNA lack the protein associated with eukaryotic DNA. Plasma membrane is present in prokaryotic cell as well as eukaryotic cells. The only membranous found in many prokaryotes is the mesosome, attach to plasma membrane and its function is a little understood. It plays a role in producing new cell material following cell division; that it is involved in replication of the DNA prior to cell division that is involved in breakdown of food molecules to provide energy. Prokaryote genetic material contain in an compound circular DNA molecule having only about one-thousand the DNA found in the even the smallest eukaryotic cell. The DNA is attached to the plasma membranes and cell containing at least two complete copies of the DNA divide into two equal or unequal halves, with at least one DNA molecule in each mitosis and meiosis are not occur in prokaryotic cells.

Figure (A) Diagram of heterotrophic bacterium undergoing cell division

Conclusion

The prokaryotic cells of bacterial also have cell wall, plasma membranes, cytoplasm and DNA differ from the eukaryotic cell of plants, animals, fungi, and protest in containing none of the major cytoplasmic organelles except ribosome. Their main internal membrane system is the mesosome. Prokaryotic exhibit a wide diversity of metabolic capabilities. Many bacteria are autographs, either photosynthetic or chemosynthetic. Nitrogen fixing bacteria and cyan bacteria convert gaseous nitrogen into a form that can be used by plants to make amino acids. Most bacteria heterotrophy is sap ropes or parasites. Some bacteria are part of the normal microbial flora of animal and some are pathogenic, causing disease by the production of toxins. Some bacteria are important in the production foods such as yogurts (yoghurts), cheese, and vinegar. Food poisoning is caused by ingesting serotoxins or pathogenic bacteria in food.

UNIVERSITY OF YANGON

M.Sc. (Biotechnology) 2^nd Year; Semester – I

Module No. BT- 631 (Bot)

Seminar on Gene Cloning

Date: June 18,2004

Presented By;

Mg Hliesa(M2 – BT – 1)

Contents:-

-Introduction

-Reproducing DNA

-Gene Splicing

-Plasmid and Phage

-Cloning a Gene

-Conclusion

Introduction

Recombinant DNA techniques enable researchers to isolate gene from one organism, manipulate the purified DNA in the laboratory and then transfer the genes into another process to the organism is called gene cloning. Gene cloning requires a host cell including prokaryotic and eukaryotic cell of chromosomal DNA. Gene cloning uses a vector or carrier molecule, there are two main type first plasmids which are circular DNA molecule over have been specially constructed and phages such as two types insertion vector and placement vectors. Now, to introduce May of the tools use in gene cloning, the aspect of reproduction is examined. For the present discussion, reproduction is defined as the duplicator of the information content of the cell followed daughter by segregation of this information two newly form daughter cells. Since information is stored in DNA, knowing how DNA duplicates is crucial to understanding reproduction. The next section discusses the machinery responsible for DNA duplication, the technical term for reproduction DNA. The gross aspects of information organization in DNA are easily described by means of analogies between DNA and motion picture film, film metaphors are used to begin describing gene cutting and splicing. Certain types of small DNA molecules are infections. This infection DNA molecules fall into two general types, the viruses and plasmids. It possible to cut DNA molecule from plasmids and bacteriophage ( Viruses that attack bacteria) in specific place, insert a piece of DNA from another source and still retain all the information necessary for infection by plasmids or phages. Thus, these infection DNA molecules are useful as tool to transfer DNA from one type of cell another. Gene cloning is much like baking a cake in that recipe are available for both processes. A few technical details have been added to complete your understanding of gene cloning. The original hemoglobin recipe is particularly instructive because it uses both plasmids and phages for cloning. The gene coding for hemoglobin fall into both categories. Hemoglobin is the blood protein responsible for moving oxygen form our lung to our body tissues. One of the medically important disorders caused by improper hemoglobin function is the serious disease called sick-cell anemia.

Reproducing DNA

DNA not only must be chemically stable, but it also must be copied accurately during replication. The two strand so each can act as a template for formation of a new strand. Nucleotide is aligned along each DNA strand according to the complementary base-pairing rule and they are joined to form a new DNA strand. Two DNA molecules arise from one, and they contain information. The DNA molecules move to different parts of the cell, cell division occur between the DNA molecules and two daughter cell arise haring identical DNAs. The two DNA strands begin to unwind; producing a replication fork, that move through the double stranded DNA molecules as replication occurs. Thus, two single strands are created, exposing the base. An enzyme complex called DNA polymerase binds to one of the single strands and moves into the direction of fork movement. As it moves along the single strand DNA polymerase mediate formation of base pair between free nucleotides and the linked DNA nucleotides. The polymerase moves down the chain one position a lings a next nucleotide, the links it the growing chain. Thus a new single stranded DNA chain is formed using the formation contain in the old one. Of course as a new chain forms, it is already base-paired with the old one; double-stranded DNA molecule containing one new and one old in produced.

Gene expression and DNA replication can now be used add important detail to the general strategy for cloning gene. DNA is first cut in specific places with a purified enzyme called a restriction endonuclease, many DNA species are produced, and they are spliced into cloning vehicles using another purified enzyme, DNA ligase. To be useful gene cloning must be removed from living cell. The mature of cellular components is called a cell extract. The enzyme of interest is separated from other cell components by a series of physical manipulation separation is possible because every type of enzyme is physically and chemically difference from every other type of molecule in the cell . For example, DNA polymerase is relatively small compound may cell.

Gene Splicing

Gene cloners move specific bit of genetic information from one DNA molecule to another by cutting and splicing procedures that utilize specific enzymes. Restrictions endonucleases are group of enzymes that correspond to the scissors in the analogies develop a above. They are recognize specific nucleotide sequence in DNA often four or six base pairs long and cut both strands of DNA within the recognition site. In some case the two DNA strands are not cut opposite each other rather-rather, the cut staggered. In the case, illustrate in figure. The cuts are offset by four nucleotides. When the cut DNA is gently warmed, the four base pairs will break apart, and the DNA molecule will separate into fragments.

Figure – Cleavage of DNA by restriction endonuclease

The motion picture metaphor is useful when considering the detail of cutting and splicing. The two films might represent many copies an animal DNA and a small infection DNA (the cloning vehicle), respectively in solution in the test tube. This step corresponds to the addition of identical restriction endonuclease molecule to the tube, to cut. The DNAs. If DNA ligase is added, the fragments will become permanently spliced. Restriction map, position of cutting sites for difference restriction nucleases are determined by comparing the sieges of DNA fragments produced. It is called restriction mapping. Restriction map show the location of each cutting (restriction) sites in relation to neighboring restriction sites. The difference short DNA sequence recognized by difference restriction map reflects their arrangement in the region.

Figure. General – scheme for forming Recombinant DNA molecules

Plasmids and Phages

Plasmids

Plasmids are small, circular double- stranded DNA molecule that occurs naturally in bacteria. Plasmid differs in length and the genes contain in their DNA. Some of smaller plasmids, which are popular in gene cloning, have about 5000 nucleotide pairs, enough DNA to code for about five average size proteins. E.coli contains slightly more than four million nucleotide pairs in its DNA. Many of the longer plasmids are difficult to handle and are transmissible are bacterial cell to another. Thus, they are generally not used in gene cloning. An important aspect of plasmid DNA molecules is that they often contain genes that make their host bacterial cell resistant to antibodies. For example in cloning procedure outlined earlier, DNA fragments are spliced into a plasmid DNA having a gene that confers resistance to penicillin. Under the proper experimental condition, the plasmid DNA enters the cell and multiplies along with the bacterial cell (transformation). The bacteria are next spread an agar plate containing penicillin and incubated for two days. Most of the bacteria are killed. However, the, the few cells that acquire a plasmid are penicillin resistant and they grow into colonies.

After a gene clones has spliced DNA fragments into cloning vehicles, transfer the vehicles into bacterial cell, separated the bacterial cells on agar plate grown the separated cell into colonies, and determined which colony contain the desired DNA and must retrieve the cloned DNA fragment by purifying the plasmid and phage containing it.

Operationally the process is much simpler than the explanation. A DNA preparation is mixed with the dye and a heavy salt in a test tube. At this stage one could rely on the force of gravity to generate the density gradient with the salt water, but it would take very long time for the salt molecules to accumulate in the bottom of the tube. It would also take a long time for the DNA molecules to settle to their own density. The time requires can shortened by putting the tube containing DNA, salt, and dye in centrifuge where forces in excess of 100000 times gravity can be obtained. After spinring the sample for a day or so, the centrifuge is turned off, and the tube is examined with ultraviolet light (black light). The emit a visible, fluorescent light. Two bonds can be observed in the tube. The upper one corresponds to the leaner DNA (bacteria DNA) and the lower one to the circular plasmid DNA. The circular plasmid DNA can be sucked out of the tube with a pipette, which will then contain pure plasmid DNA.

The clone gene can be cut out of the plasmid DNA by restriction endonucleases as described in gene splicing. This produce a small number of DNA fragments, the fragment can be separated from each other by gel electrophoresis.

Figure. Separation of Bacterial and DNAs by Dye Buoyant Density Centrifugation

Plasmid DNA, bacterial DNA, water (dye Cethidium bromide) and heavy salt (cesium chloride; CsCL) were mixed in plastic tube and centrifuged for two days at 35,000 rpm. Before centrifugation, mineral oil was added to fill the plastic tube to prevent it collapse from the force of the centrifugal field. After centrifugation, the tube was illuminate with ultra violet light, and bright orange bonds appeared in the tube, indicating the location of DNA molecules.

Bacteriophage

Plasmids are naked DNA molecules containing the organism of replication. Bacteriophage, commonly called phages, is more complicated. In addition to having an origin of replication, phage DNA contains gene coding for proteins that form the protective shell around the DNA. Both phages and plasmids can be used to separate and amplify specific DNA fragments but the two have very difference means of production. But difference cloning strategies are employed. The phage DNA is wrapped into a tight ball inside a head like structure made of proteins. A tail also made of proteins is attach to the head when such a phage particle comes into contact with a bacterial cell , the phage tail sticks to the cell wall and DNA is squirted out of the head, through the tail and into the bacterium. Many phages also have genes for their own DNA replication machinery. When this apparatus is in place, the phages can use nucleotides released from bacterial DNA are made, within minutes other genes on the phages are turned onto produce new head and tail proteins. The head proteins assemble into heads phages DNA is packaged inside them, a tail is attached to each head. The assembly of new phage occurs spontaneously, the total time from injection of DNA to production of new phage cans be less than 20 minutes. The bacterium becomes little more than a shell containing hundreds of new phage particles. As a final act, the phage produces an enzyme that destroys the bacterial cell wall releasing the phage particles to seek new hosts.

One phage produces hundreds of progeny particles. By the repeating the infection cycle just four times, a single phage particle can lead to the death of more than a billion bacterial cells. If a DNA fragment is splice into a phage DNA molecule without destroying important phage genes, the phage will produce the fragment along with its own DNA when it infects bacterial cell, phage particle can easily purified by density gradient centrifugation in a way similar to that described earlier for purification plasmid DNA . A tube containing the phage in a heavy salt solution is centrifuged until a density gradient is established and the phage sediments to its own density. When the tube is removed from the centrifuged and examined, the phage will appear as opalescent bonds that can be easily stickled out with a pipette. One of the phage use for cloning is called lambda. When lambda DNA is injected into a bacterial cell, it has two choices. The lambda DNA inserts into the bacterial chromosome. It becomes part of the bacterial DNA.

Fig. Purification of a Bacteriophage by centrifugation

Gene Cloning

The first step in the recipe is to find a source for hemoglobin genes. The initial studies were done with rabbit gene, with few exception cells in rabbit body contain identical DNA molecules. The DNA from virtually Hemoglobin genes and any type of body tissue can be ground up to obtain these genes. Hemoglobin genes can be obtained from a live or death donor, and they can be stored for an indefinite-time in a frozen state. Hemoglobin genes were initially separated from all the other genes in the DNA preparation by cloning into bacteriophage lambda DNA.

DNA was extracted from the phage particles. Since it is necessary to shorten the lambda DNA to make room for the rabbit DNA inside the virus particle, the lambda DNA was cut into several pieces by a restriction endonuclease. The two DNA fragments require by lambda to infect a bacterial cell were spliced together, and nonessential fragments were discarded. The rabbit DNA was broken into large pieces, and the two types of DNA were mixed. Ligase was added to splice the fragments, which were than coated with phage proteins. Thus, the recombinant DNA in many difference combinations was neatly packaged inside phage particles. In this form, they were easily transferred into bacterial cell by the mechanism normally used by the phage to inject it DNA. Often, the packaged phage DNA contained a piece of rabbit DNA.

Reverse transcriptase makes DNA from nucleotides, using the RNA as a template. The DNA called complementary DNA (cDNA), can be make highly radioactive if it components, the nucleotides, are radioactive.

In many examples of gene cloning, radioactive complementary DNA is suitable to locate plaque containing clone genes. The initial cloning studies with hemoglobin it was necessary to screen hundreds of thousands of phage plaques. Large amount of complementary DNA were needed. It was decided to first synthesized complementary DNA, convert the cDNA into double-stranded DNA using DNA polymerase, then clone it into plasmid to obtain large amounts. The cloned DNA would then be made radioactive. So the double stranded DNA copies of hemoglobin mRNA were spiced into plasmids, and the resulting the recombinant DNA molecules were transferred into E.coli cells. These colonies were tested for the present of hemoglobin nucleotide sequence using small amounts of radioactive hemoglobin messenger RNA. Some of the details of these procedures are presented below.

To insert DNA copies of hemoglobin mRNA into plasmid DNA, plasmid DNA was cut one with a restriction endonuclease to convert the circular DNA into a linear molecule. Long-single stranded tails were produces by treating each DNA with an enzyme called lambda exonuclease. Another enzyme was then used to put about 100 T’s onto the tails of the DNA copies an about 100A’s onto the ends of plasmid DNA. When the two DNAs were mixed, the A’s and T has formed base pairs, joining the NDA copies to the plasmids. The resulting circular DNA molecules were added to culture of E.coli cell, and the recombinant DNA molecules (cDNA plasmids) entered some of the cells. The plasmid DNA contained a gene conferring resistance to the antibiotic tetracycline. So when the E.coli cells were placed on agar plates containing the drug, only cells containing the plasmid grew into colonies.

Figure. Joining Hemoglobin Complementary DNA to plasmid DNA.

Conclusion

DNA is copied by a group of proteins traveling together a long double stranded DNA molecule, as the protein move along the DNA, they separate the two DNA strands and make a new strand adjacent to old once; one double-stranded molecule becomes two. Each DNA has one old and one new strand, and nucleotide in these strands are complementary. Occasionally errors occur while the new strands are being made and the nucleotide sequence in the new strand is not perfectly complementary to that in the old strand. If not corrected, these errors called mutations. The enzyme that connects the patches (DNA ligase) has been obtained in pure form and is now used by gene cloners to splice DNA fragments. Clones to make highly radioactive DNA in test tubes use other enzymes involved in DNA replication. This radioactive is utilized to find bacterial colonies that contain specific cloned genes. Gene cloners move specific bits of genetic information from one DNA molecules to another by cutting and splicing procedures that utilize specific enzymes. DNA is very long and contains a large number of sites at which cutting can occur. Consequently, cutting and splicing often result in many difference combinations of fragments being joined. Biochemical methods based on the principle of complementary based paring are used to detect and locate a specific combination of spliced fragments.

The enzymes that cut DNA are called restriction endonucleases and they recognize and cut at specific DNA sequences. Consequently, cutting DNA with these enzymes produces DNA fragments having discrete lengths. Enzymes can be obtained that recognize difference sites in a DNA molecule, making it possible to cut DNA at a wide variety of locations.

Plasmids and phages are submicroscopic agent that infect bacterial and use bacterial components to replicate himself or herself. They contain genetic information, which I most cases is stored in short DNA molecules. Molecular biologists have found plasmids and phages DNA molecules particularly easy to handle study. Genetic information from animals and other organisms having very long DNA molecule is difficult to study unless the DNA has been cut into small pieces and the pieces have been physically separated. Plasmids and phages assist us in separating DNA pieces. Since DNA fragments can be spliced into plasmid of phage DNA without impairing infectivity, these tiny infectious agents can be used as vehicle to place almost any DNA fragment reproduces as a part of the plasmid or phage DNA.

Gene cloning procedures involve splicing human DNA fragments into phage and plasmid DNA molecules. When phages are used, the recombinant DNA molecules one packaged inside phage particles so that they can infect bacteria and generate phage plaques. Thousands of phage plaques are then tested for the presence of a specific gene by nucleic acid hybridization using a highly radioactive probe.

Gene cloning strategy with plasmids often clone use the drug resistance properties of plasmids to help locate bacterial colonies into human genes have. In one type of strategy, plasmids having genes for resistance to two difference drugs are chosen. One-drug resistance gene contains a restriction endonuclease cleavage site where DNA fragment can be inserted, insertion of a fragment into this site on the plasmid destroys the drug resistance gene. Recombinant plasmid DNA molecule will counter the resistance to the second drug only. When bacterial cell are transformed with plasmids though to be recombinant, colonies are obtained that grow on agar containing the second drug. Each of these colonies then tested for growth on agar containing the first drug, those that fail to grow are saved and are tested for the presence of a specific gene by nucleic acid hybridization using a highly radioactive complementary DNA probe.

Reading Books

DNA and Gene cloning Karl Drilica,

University of Rochester (John Wiley & Sons)1976.

Genetic Engineering and Biotechnology,

Professor Dr. U Win, Pro-Rector, Yangon Eastern University (2004 – 2005).

Test book Module No. BT-632 (Chem.).

UNIVERSITY OF YANGON

Biotechnology Seminar Paper

On

CHEMICAL COMPOSITION OF EUKARYOTIC CHROMOSOME

Presented By,

Mg Hliesa

Roll No. M2 – BT-1

Date: July 1, 2004.

Introduction

Through biochemical methods, chromosome material (chromatin) isolated from eukaryotic nuclei has been shown to consist of a variety of macromolecules, chiefly protein and nucleic acids, which serve as structure components, as biocatalyst (enzymes), as hormone and as repositories for the genetic information characteristic species. Chromosomes have the complex of DNA and protein known as chromatin, a nucleoprotein complex. This material is composed of about equal amounts of long chained DNA and the smaller acid soluble (basic) histone proteins. The histones are intimately involved with maintaining the structural of the DNA double helix within the chromosome and some of they may function by the interacting with the acidic DNA phosphate groups. The RNA molecules are of various molecular weighs and serve purpose that include the transmission of information form DNA proteins. Histone protein composed argine (- amino – -guandino – -valeric acid) and lysine (a, – Diaminocproic acid).

Nucleoprotein

The nucleoproteins were so named because they constitute a large part of the nuclear material of the cell. As chromatin largely composed of nucleoproteins, which indicates that these compounds are involve in cell division and the transmission of hereditary factors. They are characterized by a non protein prosthetic group (nucleic acid). Which attach to one or more molecules of simple protein. The simple protein is usually a basic such as protimine or histone. These proteins are also found in cytoplasm, associated particularly with the ribosome where a ribonucleic acid is intimately concerned with synthesis of proteins. In the mechanism of nucleoprotein, formation is followed by alternation in cell growth and reproduction. When the purified the nucleoprotein is hydrolyzed with acid on by the use of enzymes, it is broken down into various components as shown in Fig. By the use of careful acid hydrolysis, the purine or pyrimidine nitrogen base may be removed from a nucleotide, leaving a sugar attached to phosphate

Nitrogen Base

The various purine and pyrimidine nitrogen base which occur in nucleotides of nucleic acid are divided by appropriate substitution on the ring structure of the parent substances, purine or pyrimidine the structure of this parent nitrogenous bases are as follows.

Purine Pyrmidine

There are three main pyrimidine bases, which have been isolated from nucleic acids, cytosine, thymine, and uracil. Cytosine (2- oxy – 6 – aminopyrimidine) is found in all nucleic acid accept the deoxyribonucleic acid of certain bacterial viruses. Vizoli bacteria phage of the Te – even series (T_2,T_4,T₆ etc), in its place there is five – hydroxymethylcytosine(HME). Thymie (2,6-dioxy-5methyl pyrimdine).Occur mainly is nucleic acids, which contain deoxyriboses as the characteristic carbohydrate, the so called deoxyribonucleic acid (DNA). However, minor amount have also been found in transfer RNA (tRNA). Uracil (2, 6 – dioxypyrmidine) on the other hand, is confirm to the ribonucleic acids (RNA)which contain ribose rather than deoxy sugar. Purine bases are adenine (6 – aminopurine) and guanine (2- amino acid – 6 – oxypurne).

A nucleotide, the structure unit of nucleic acid, is composed of a purine or pyrimidine base attach to sugar (Ribose or 2- deoxyribiose) by aglycosidic linkage; the sugar is tan combined with phosphoric acid. Purine or pyrimidine – sugar – phosphoric acid. Two general types of nucleotides are found in nucleic acid; one contains D- ribose; the other contains 2 – deoxyribonucleic acid. Removed of the phosphate moiety form a nucleotide produces nucleoside which composed of nitrogen base and sugar, either D- ribose or 2 – deoxyriboe.

Nucleic acids

Nucleic acids consist of long chains of nucleotides (polynucleotide) combined one, which one which another through phosphate diester linkage. There are two types of nucleic acid, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Particularly important is the role of DNA I directing the synthesis of messenger RNA (mRNA), referred to as “transcription” where by the genetic information store in DNA required to provide for the primary structure (i.e., the

amino acid sequences) of specific protein can be transmitted to the protein synthesing apparatus in the cytoplasm. Nucleic acid chain is a long, unbranched polymer composed of only four types of subunits. These are the D- ribo – nucleotide (or) 2 – deoxyribonucleotides containing the bases adenine (A), cytosine (C), guanine (G), and Uracil (U) or thymine (T). Biochemical experiments and conclusion derived from model building suggested that complementary base pairs (also called Watson – rick base pairs) form between A and T (or) U and between G and C. This building revealed that the numbers of effective hydrogen bounds that could be formed between G and C or between A and T or (U)

Simple Proteins

The major protein components of chromatin are the histones, small, basic (positively charged) protein that bind tightly to DNA. A chromosome contains five types of histone. H₁. (H₅), H₂ A, H₂B, H₃ and H_4. H_1, is homologous proteins and the other 4 types histones associated with