Prasanna

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Sexual Reproduction

In previous classes reproduction in lower plants like algae and bryophytes was discussed in detail. Sexual reproduction involves the production and fusion of male and female gametes. The former is called gametogenesis and the latter is the process of fertilization. Let us recall the sexual reproduction in algae and bryophytes.

They reproduce by the production of gametes which may be motile or non motile depending upon the species. The gametic fusion is of three types (Isogamy, Anisogamy and Oogamy). In algae external fertilization takes place whereas in higher plants internal fertilization occurs.

Flower

A flower is viewed in multidimensional perspectives from time immemorial. It is an inspirational tool for the poets. It is a decorative material for all the celebrations. In Tamil literature the fie lands are denoted by different flowers. The flags of some countries are embedded with flowers. Flowers are used in the preparation of perfumes.

For a Morphologist, a flower is a highly condensed shoot meant for reproduction. As you have already learned about the parts of a flower in Unit II of Class XI, let us recall the parts of a flower. A Flower possesses four whorls – Calyx, Corolla, Androecium and Gynoecium.

Androecium and Gynoecium are essential organs (Figure 1.3). The process or changes involved in sexual reproduction of higher plants include three stages. They are Prefertilization, Fertilization and Post fertilization changes. Let us discuss these events in detail.

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Vegetative Propagation – Definition, Types, Examples & Explanations

Natural methods

Natural vegetative reproduction is a form of asexual reproduction in which a bud grows and develops into a new plant. The buds may be formed in organs such as root, stem and leaf. At some stage, the new plant gets detached from the parent plant and starts to develop into a new plant.

Some of the organs involved in the vegetative reproduction also serve as the organs of storage and perennation. The unit of reproductive structure used in propagation is called reproductive propagules or diaspores. Some of the organs that help in vegetative reproduction are given in Figure 1.1.

A. Vegetative reproduction in root

The roots of some plants develop vegetative or adventitious buds on them. Example Murraya, Dalbergia and Millingtonia. Some tuberous adventitious roots apart from developing buds also store food. Example Ipomoea batatus and Dahlia. Roots possessing buds become detached from the parent plant and grow into independent plant under suitable condition.

B. Vegetative reproduction in stem

From the Unit 3 of class XI (Vegetative morphology) you are familiar with the structure of various underground stem and sub aerial stem modifiations. Thse include rhizome (Musa paradisiaca, Zingiber offinale and Curcuma longa); corm (Amorphophallus and Colocasia); tuber (Solanum tuberosum); bulb (Allium cepa and Lilium) runner (Centella asiatica); stolon (Mentha, and Fragaria); offet (Pistia, and
Eichhornia); sucker (Chrysanthemum) and bulbils (Dioscorea and Agave). The axillary buds from the nodes of rhizome and eyes of tuber give rise to new plants.

C. Vegetative reproduction in leaf

In some plants adventitious buds are developed on their leaves. When they are detached from the parent plant they grow into new individual plants. Examples: Bryophyllum, Scilla, and Begonia. In Bryophyllum, the leaf is succulent and notched on its margin.

Adventious buds develop at these notches and are called epiphyllous buds. Thy develop into new plants forming a root system and become independent plants when the leaf gets decayed. Scilla is a bulbous plant and grows in sandy soils. The foliage leaves are long and narrow and epiphyllous buds develop at their tips. Thse buds develop into new plants when they touch the soil.

Advantages of natural vegetative reproduction

• Only one parent is required for propagation.
• The new individual plants produced are genetically identical.
• In some plants, this enables to spread rapidly. Example: Spinifex
• Horticulturists and farmers utilize these organs of natural vegetative reproduction for cultivation and to harvest plants in large scale.

Disadvantage of natural vegetative reproduction

New plants produced have no genetic variation.

Artificial Methods

Apart from the above mentioned natural methods of vegetative reproduction, a number of methods are used in agriculture and horticulture to propagate plants from their parts. Such methods are said to be artifiial propagation.

Some of the artifiial propagation methods have been used by man for a long time and are called conventional methods. Now-a-days, technology is being used for propagation to produce large number of plants in a short period of time. Such methods are said to be modern methods.

A. Conventional methods

The common methods of conventional propagation are cutting, grafting and layering.

a. Cutting:

It is the method of producing a new plant by cutting the plant parts such as root, stem and leaf from the parent plant. The cut part is placed in a suitable medium for growth. It produces root and grows into a new plant.

Depending upon the part used it is called as root cutting (Malus), stem cutting (Hibiscus, Bougainvillea and Moringa) and leaf cutting (Begonia, Bryophyllum). Stem cutting is widely used for propagation.

b. Graftng:

In this, parts of two different plants are joined so that they continue to grow as one plant. Of the two plants, the plant which is in contact with the soil is called stock and the plant used for graftng is called scion (Figure 1.2 a). Examples are Citrus, Mango and Apple.

There are different types of graftng based on the method of uniting the scion and stock. Thy are bud graftng, approach graftng, tongue graftng, crown graftng and wedge graftng.

(i) Bud graftng:

A T – shaped incision is made in the stock and the bark is lifted. The scion bud with little wood is placed in the incision beneath the bark and properly bandaged with a tape.

(ii) Approach graftng:

In this method both the scion and stock remain rooted. The stock is grown in a pot and it is brought close to the scion. Both of them should have the same thickness. A small slice is cut from both and the cut surfaces are brought near and tied together and held by a tape. After 1-4 weeks the tip of the stock and base of the scion are cut of and detached and grown in a separate pot.

(iii) Tongue grafting:

A scion and stock having the same thickness is cut obliquely and the scion is fi into the stock and bound with a tape.

(iv) Crown grafting:

When the stock is large in size scions are cut into wedge shape and are inserted on the slits or clefts of the stock and fixed in position using graft wax.

(v) Wedge grafting:

In this method a slit is made in the stock or the bark is cut. A twig of scion is inserted and tightly bound so that the cambium of the two is joined.

c. Layering:

In this method, the stem of a parent plant is allowed to develop roots while still intact. When the root develops, the rooted part is cut and planted to grow as a new plant. Examples: Ixora and Jasminum. Mound layering and Air layering are few types of layering (Figure 1.2 b).

i. Mound layering:

The method is applied for the plants having flxible branches. The lower branch with leaves is bent to the ground and part of the stem is buried in the soil and tip of the branch is exposed above the soil. After the roots emerge from the part of the stem buried in the soil, a cut is made in parent plant so that
the buried part grow into a new plant.

ii. Air layering:

In this method the stem is girdled at nodal region and hormones are applied to this region which promotes rooting. The portion is covered with damp or moist soil using a polythene sheet. Roots emerge in these branches after 2-4 months. Such branches are removed from the parent plant and grown in a
separate pot or ground.

Advantages of conventional methods

• The plants produced are genetically uniform.
• Many plants can be produced quickly by this method.
• Some plants produce little or no seeds; in others, the seeds produced do not germinate. In such cases, plants can be produced in a short period by this method.
• Some plants can be propagated more economically by vegetative propagation. Example: Solanum tuberosum.
• Two diffrent plants with desirable characters such as disease resistance and high yield can be grafted and grown as a new plant with the same desirable characters.

Disadvantages of conventional methods

• Use of virus infected plants as parents produces viral infected new plants.
• Vegetative structures used for propagation are bulky and so they are diffilt to handle and store.

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Asexual Reproduction

The reproduction method which helps to perpetuate its own species without the involvement of gametes is referred to as asexual reproduction we know that reproduction is one of the attributes of living things and the diffrent types of reproduction have also been discussed.

Lower plants, fungi and animals show diffrent methods of asexual reproduction. Some of the methods include, formation of Conidia (Aspergillus and Penicillium); Budding (Yeast and Hydra); Fragmentation (Spirogyra); production of Gemma (Marchantia); Regeneration (Planaria) and Binary fision (Bacteria).

The individuals formed by this method is morphologically and genetically identical and are called clones.

Higher plants also reproduce asexually by diffrent methods which are given below:

Asexual reproduction is a type of reproduction that does not involve the fusion of gametes or change in the number of chromosomes. The offspring that arise by asexual reproduction from either unicellular or multicellular organisms inherit the full set of genes of their single parent.

In asexual reproduction, an individual can reproduce without involvement with another individual of that species. The division of a bacterial cell into two daughter cells is an example of asexual reproduction.

7 types of Asexual Reproduction

• Budding: A form of asexual reproduction of yeast in which a new cell grows out of the body of a parent.
• Vegetative Reproduction: Plants budding which creates a runner hich sends a clone.
• Parthenogenesis
• Binary Fission.
• Regeneration.
• Fragmentation.
• Spores.

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Techniques in Genetic Engineering

There are several techniques used in recombinant DNA technology or gene manipulation. The most frequently used methods are agarose gel electrophoresis, isolation and purification of nucleic acids, nucleic acid blotting techniques, DNA sequencing, chemical synthesis of DNA, gene transfer methods, polymerase chain reaction, construction of gene library, radiolabeling of nucleic acids etc, few of them are discussed here.

Agarose Gel Electrophoresis

Electrophoresis refers to the movement of charged molecules in an electric field. The negatively charged molecules move towards the positive electrode while the positively charged molecules migrate towards the negative electrode. Gel electrophoresis is a routinely used analytical technique for the separation and purification of specific DNA fragments.

The gel is composed of either polyacrylamide or agarose. Polyacrylamide gel electrophoresis (PAGE) is used for the separation of smaller DNA fragments while agarose electrophoresis is convenient for the separation of DNA fragments ranging in size from 100 base pairs to 20 kilobase pairs.

Gel electrophoresis can also be used for the separation of RNA molecules. A diagramatic view of the agarose gel electrophoresis unit is shown in Figure 12.30 a.

Steps

1. Gel is set with wells on one end.
2. The gel is placed in an electrophoresis apparatus and covered with buffer solution.
3. The DNA samples along with tracer dye are placed in the wells of gel.
4. Power supply is switched on and gel is run till the tracer dye reaches the end of the gel.

As the DNA is negatively charged, DNA fragments move through the gel towards the positive electrode. The rate of migration of DNA is dependent on the size and shape. In general, smaller linear fragments move faster than the larger ones.

Hence, gel electrophoresis can be conveniently used for the separation of a mixture of DNA fragments, based on their size. The bands of the DNA can be detected by soaking the gel in ethidium bromide solution (Ethidium bromide can also be added to molten agarose prior to setting the gel).

When activated by ultraviolet radiation, DNA base pairs in association with ethidium bromide, emit orange fluorescence. And in this way the DNA fragments separated in agarose electrophoresis can be identified (Figure 12.30b).

PAGE is composed of chains of acryl amide monomers crosslinked with methylene bisacryalmide units. The pore size of the gel is dependent on the total concentration of monomers and the cross links. PAGE is used for the separation of single stranded DNA molecules that differ in length by just one nucleotide.

Agarose gels cannot be used for this purpose. This is because polyacrylamide gels have smaller pore sizes than agarose gels and allow precise separation of DNA molecule from 10-1500 bp.

Polymerase Chain Reaction (PCR)

The PCR technique has already proven exceptionally valuable in many areas of molecular biology, medicine, and biotechnology. PCR technique has great practical importance and impact on biotechnology. Between 1983 and 1985 American biochemist Kary Mullis developed PCR technique that made it possible to synthesize large quantities of a DNA fragment without cloning it.

Mullis received the 1993 Nobel Prize for Chemistry for his invention. PCR is a cell free amplification technique. Figure 12.31 outlines how PCR technique works. To amplify (make large quantities) a particular DNA sequence by PCR a reaction mixture (often 100μl or less in volume) containing the following are required.

• Target DNA
• Two primers-These are synthetic oligonucleotides, usually about 20 nucleotides long. These are fragments with sequences identical to those flanking the targeted sequence.
• Thermostable DNA polymerase-Two popular enzymes employed in the PCR technique are Taq polymerase from the thermophilic bacterium.
• Thermus aquaticus and the vent polymerase from Theromococcus litoralis. These polymerases employed in PCR technique are able to function at high temperatures.
• Four deoxyribonucleoside triphosphates (dNTPs) – dCTP, dATP, dGTP, dTTP

Steps in PCR

1. Denaturation:

The target DNA containing the sequence to be amplified is heat denatured to separate its complementary strands at temperature 94 °C-95 °C.

2. Annealing:

The temperature is lowered to 37 °C-55 °C so that the primers can hydrogen bond or anneal to the DNA on both sides of the target sequence. Because the primes are present in excess the targeted DNA strands normally anneal to the primers rather than to each other.

3. Extension:

Heat resistant DNA polymerase extends the primers and synthesizes copies of the target DNA sequence using the deoxyribonucleoside triphosphate’s at 70 °C-75 °C.

The three – step cycle (Figure 12.32) is repeated to obtain copies of target DNA in large numbers. At the end of one cylcle, the targeted sequences on both strands have been copied. When the three – step cycle is repeated, the four strands from the first cycle are copied to produce eight fragments.

The third cycle yields 16 products. Theoretically, 20 cycles will Figure 12.32: Three steps PCR cycle The PCR technique has now been automated and is carried out by a specially designed machine (Figure 12.33) PCR machines are now fully automated and microprocessor controlled.

They can process up to 96 samples at a time. PCR machines can carry out 25 cycles and amplify DNA 105 times in as little as 57 minutes.

The PCR has many applications in research and in commercial arena, including generating specific DNA segments for cloning or sequencing, amplifying DNA to detect specific genetic defects, and amplifying DNA for fingerprinting in crime scene investigation.

PCR technology is improving continually. Various forms of PCR are available. RNA too can be efficiently used in PCR procedures. Cellular RNAs and RNA viruses may be studied even when the RNA is present in very small amounts (as few as 100 copies can be transcribed and amplified). Quantitative PCR is quite valuable in virology and gene impression studies.

PCR is modified as per the specific demands of the situation. Thus there are many variations in the original PCR Examples nested PCR, inverse PCR, reverse transcription PCR, time quantitative PCR, RAPD, RFLP, AFLP.

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Vectors – Types and Characteristics

Vectors are the DNA molecules, which carry a foreign DNA fragment to be cloned. They are cloning vehicles, examples of which are Plasmids, Bacteriophages, cosmids, phagemids and artificial chromosomes. The vector types differ in the molecular properties they have and in the maximum size of DNA that can be cloned into each.

Characteristics of an ideal vector.

1. Should be small in size
2. Should contain one or more restriction site
3. Should be self replicating
4. Should contain an origin of replication sequence (ori)
5. Should possess genetic markers (to detect the presence of vectors in recipient cells)

Plasmid Cloning Vectors

Bacterial plasmids are extra chromosomal elements that replicate autonomously in cells. Their DNA is circular and double stranded and carries sequences required for plasmid replication (ori sequence) and for the plasmid’s other functions. (Note: A few bacteria contain linear plasmids. Example: Streptomyces species, Borellia burgdorferi). The size of plasmids varies from 1to 500 kb. Plasmids were the first cloning vectors.

DNA fragments of about 570kb are efficiently cloned in plasmid cloning vectors. Plasmids are the easiest to work with. They are easy to isolate and purify, and they can be reintroduced into a bacteria by transformation.

Naturally occurring plasmid vectors rarely possess all the characteristics of an ideal vector. Hence plasmid cloning vectors are derivatives of natural plasmids and are “engineered” to have features useful for cloning DNA.

Examples of plasmid cloning vectors: pBR 322 (plasmid discovered by Bolivar and Rodriguez 322) and pUC 19 (plasmid from University of California). Herbert Boyer and Stanley Cohen in 1973 showed it was possible to transplant DNA segments from a frog into a strain of Escherichia coli using pSC101, a genetically modified plasmid, as the vector. The work laid the foundation for the birth of Genetech, the first company dedicated to commercialization of recombinant DNA.

Figure 12.24 a and 12.24 b shows genetic maps of plasmid cloning vectors PUC19 and PBR322 respectively. Plasmid cloning vector PUC 19 has 2,686 – bp and has following features:

1. It has a high copy number; so many copies of a cloned piece of DNA can be generated readily.
2. It has amp R (ampicillin resistant) selective marker
3. It has a number of unique restriction sites clustered in one region, called a multiple cloning site (MCS) or polylinker
4. The MCS is inserted into part of the E.coli β – galactosidase (lac Z⁺) gene. Figure 12.25 illustrates how a piece of DNA can be inserted into a plasmid cloning vector such as pUC19.
   

Bacteriophage as Cloning Vectors

They are viruses that replicate within the bacteria. A phage can be employed as vector since a foreign DNA can be spliced into phage DNA, without causing harm to phage genes. The phage will reproduce (replicate the foreign DNA) when it infects bacterial cell.

Both single and double stranded phage vectors have been employed in recombinant DNA technology. Derivatives of phage can carry fragments up to about 45 kb in length. Example PI bacteriophage and phage λ.

The main advantage of using phage vectors is that foreign DNA can be packed into the phage (invitro packaging), the latter in turn can be injected into the host cell very effectively (Note: no transformation is required). Figure 12.28 shows how a λ phage is used for cloning.

Cosmids:

Cosmids are the vectors possessing the characteristics of both plasmid and bacteriophage. The advantage with cosmids is that they carry larger fragments of foreign DNA (35-45 kb) compared to plasmids.

Phagemids:

Phagemids are the combination of plasmid and phage and can function as either plasmid or phage. Since they posses functional origins of replication of both plasmid and phage λ they can be propagated (as plasmid or phage) in appropriate E.coli.

Artificial chromosome Vectors:

Artificial chromosomes are cloning vectors that can accommodate very large pieces of DNA, producing recombinant DNA molecules resembling small chromosomes. Example: Yeast Artificial Chromosome (YAC), Bacterial Artificial Chromosomes (BACs)

Plasmid shuttle Vectors:

The plasmid vectors that are specifically designed to replicate in two or more different host organisms (say in E.coli and yeast) are referred to as shuttle vectors. The origins of replication for two hosts are combined in one plasmid.

Expression vectors:

An expression vector is a cloning vector containing the regulatory sequences (promoter sequence) necessary to allow the transcription and translation of a cloned gene or genes.

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Recombinant DNA Technology

One of the practical applications of microbial genetics and the technology arising from it is the recombinant DNA technology. The deliberate modification of an organism’s genetic information by directly changing its nucleic acid genome is called genetic engineering and is accomplished by a collection of methods known as recombinant DNA technology.

Recombinant DNA technology opens up totally new areas of research and applied biology. Thus, it is an essential part of biotechnology, which is now experiencing a stage of exceptionally rapid growth and development. In general sense, recombination is the process in which one or more nucleic acids molecules are rearranged or combined to produce a new nucleotide sequence.

Usually genetic material from two parents is combined to produce a recombinant chromosome with a new, different genotype. Recombination results in a new arrangement of genes or parts of genes and normally is accompanied by a phenotypic change.

There are many diverse and complex techniques involved in gene manipulation. However, the basic principles of recombinant DNA technology are reasonably simple, and broadly involve the following stages (Figure 12.22).

1. Isolation of DNA from the source (Donor)
2. Generation of DNA fragments and selection of the desired piece of DNA
3. Insertion of the selected DNA into a cloning vector (Example: a plasmid) to create a recombinant DNA or chimeric DNA.
4. Introduction of the recombinant vectors into host cells (Example: bacteria)
5. Multiplication and selection of clones containing the recombinant molecules
6. Expression of the gene to produce the desired product.

Cloning in the molecular biology sense (as opposed to cloning whole organisms) is the making of many copies of a segment of DNA, such as a gene. Cloning makes it possible to generate large amounts of pure DNA, such as genes, which can then be manipulated in various ways, including mapping, sequencing, mutating and transforming cells. An overview of cloning strategies in recombinant DNA technology is shown in Figure 12.23.

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Transfer of Genetic Material

Normally, genes and the characteristics they code for are passed down from parent to progeny. This is called vertical gene transfer. Bacteria and some lower eukaryotes are unique in that they can pass DNA from one cell of the same generation to another.

The exchange of genes between two cells of the same generation is referred to as horizontal gene transfer. Mechanisms like transformation, transduction and conjugation takes place naturally and may bring about genetic variation and genetic recombination.

These gene transfer mechanisms are also employed in genetic engineering to introduce desired gene into the cells. Introducing a foreign gene or recombinant DNA into the cells is one of the techniques used in genetic engineering. The success of cloning depends on the efficiency of gene transfer process.

The most commonly employed gene transfer methods are transformation, conjugation, transduction, electroporation, lipofection and direct transfer of DNA. The choice of the method depends on the type of host cell (bacteria, fungi, plant, animal). Figure 12.15 shows methods of DNA transfer.

Note: The term Transfection is used for the transfer of DNA into eukaryotic cells by various physical or chemical means.

Transformation

Transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings. Transformation occurs naturally in some species of bacteria, but it can also take place by artificial means in other cells. For transformation to happen, bacteria must be in a state of competence.

Competence refers to the state of being able to take up exogenous DNA from the environment. There are two forms of competence: natural and artificial. Transformation works best with DNA from closely-related species. The naturally-competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s).

There are some differences in the mechanisms of DNA uptake by gram positive and gram negative cells. However, they share some common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA
translocase.

Only single stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes. Figure 12.16 shows mechanism of transformation.

Artificial competence can be induced in laboratory by procedures that involve making the cell passively permeable to DNA. Typically, the cells are incubated in a solution containing divalent cations; most commonly, calcium chloride solution under cold condition, which is then exposed to a pulse of heat shock.

Electroporation is another method of promoting competence. Using this method, the cells are briefly shocked with an electric field of 10-20 kV / cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell’s membrane-repair mechanisms.

Conjugation

The initial evidence for bacterial conjugation, came from an experiment performed by Joshua Lederberg and Edward L Tatum in 1946. Later in 1950, Bernard Davis gave evidence that physical contact of the cells was necessary for conjugation. During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges (e.g. pilus) and transfer single stranded DNA (from donor to recipient).

Inside the recipient cell, the new DNA may integrate with the chromosome (rather rare) or may remain free (as is the case with plasmids). Conjugation can occur among the cells from different genera of bacteria, while transformation takes place among the cells of a bacterial genus.

A plasmid called the fertility or F factor plays a major role in conjugation. The F factor is about 100 kilobases long and bears genes responsible for cell attachment and plasmid transfer between specific bacterial strains during conjugation. F factor is made up of:-

• tra region (tra operon / transfer genes): genes coding the F pilus and DNA transfer,
• Insertion sequence: genes assisting plasmid integration into host cell chromosome.

Thus, the F factor is an episome – a genetic material that can exist outside the bacterial chromosome or be integrated into it.

During F⁺ × F^– mating or conjugation (Figure 12.17 a) the F factor replicates by the rolling circle mechanism and a copy moves to the recipient. The channel for DNA transfer could be either the hollow F pilus or a special conjugation bridge formed upon contact. The entering strand is copied to produce double – stranded DNA.

F factor can integrate into the bacterial chromosome at several different locations by recombination between homologous insertion sequences present on both the plasmid and host chromosomes. The integration of F factor into bacterial chromosome results in formation of HFR (High Frequency Recombination) cell.

When integrated, the Fplasmid’s tra operon is still functional; the plasmid can direct the synthesis of pili, carry out rolling circle replication, and transfer genetic material to an F- recipient cell.

An HFR cell is so called because it exhibits a very high efficiency of chromosomal gene transfer in comparison with F⁺ cells. In F⁺ cells the independent F factor rarely transfer chromosomal genes hence the recombination frequency is low. Figure 12.17 b shows formation of HFR cell.

When an HFR cell is mated with F^– cell the F^– recipient does not become F⁺ unless the whole chromosome is transferred as explained in Figure 12.17 c. The connection usually breaks before this process is finished. Thus, complete F factor usually is not transferred, and the recipient remains F^–.

Because the F plasmid is an episome, it can leave (deintegrate) the bacterial chromosome. Sometimes during this process, the plasmid makes an error in excision and picks up a portion of the chromosomal material to form an F′ plasmid. Figure 12.17 d shows formation of F′.

During F′XF^– conjugation (Figure 12.17 e) the recipient becomes F′ and is a partially diploid since it has two set of the genes carried by the plasmid.

The natural phenomenon of conjugation is now exploited for gene transfer and Recombinant DNA technology. In general, the plasmids lack conjugative functions and therefore, they are not as such capable of transferring DNA to the recipient cells. However, some plasmids with conjugative properties can be prepared and used.

Transduction

Transduction is the transfer of bacterial genes from one bacteria to other by viruses. Example: Bacteriophage (Bacterial viruses). To understand the role of bacteriophage in gene transfer, the lifecycle of bacteriophage is described below briefly.

After infecting the host cell, a bacteriophage (phage for short) often takes control and forces the host to make many copies of the virus. Eventually the host bacterium bursts or lyses and releases new phages. This reproductive cycle is called a lytic cycle because it ends in lysis of the host.

The lytic cycle (Figure 12.18) has four phases.

1. Attachment – Virus particle attaches to a specific receptor site on the bacterial surface.
2. Penetration – the genetic material, which is often double stranded DNA, then enters the cell.
3. Biosynthesis – After adsorption and penetration, the virus chromosome forces the bacterium to make viral componentsviral nucleic acids and proteins.
4. Assembly – Phages are assembled from the virus components. Phage nucleic acid is packed within the virus’s protein coat.
5. Release – mature viruses are released by cell lysis.
   

Bacterial viruses that reproduce using a lytic cycle often are called virulent bacteriophages (e.g. T phages) because they destroy the host cell. The genome of many DNA phages such as the lambda phage, after adsorption and penetration do not take control of its host and does not destroy the host.

Instead the viral genome remains within the host cell and is reproduced along with the bacterial chromosome. The infected bacteria may multiply for long periods while appearing perfectly normal. Each of these infected bacteria can produce phages and lyses under appropriate environmental conditions. This relationship between phage and its host is called lysogeny (Figure 12.19).

Bacteria that can produce phage particles under some conditions are said to be lysogens or lysogenic bacteria. Phages which are able to establish lysogeny are called temperate phages. The latent form of virus genome that remains within the host without destroying the host is called the prophage.

The prophage usually is integrated into the bacterial genome. Sometimes phage reproduction is triggered in a lysogenized culture by exposure to UV radiation or other factors. The lysosomes are then destroyed and new phages released – This phenomenon is called induction (Figure 12.20)

Sometimes, bacterial genes are incorporated into a phage capsid because of errors made during the virus life cycle. The virus containing these genes then infects them into another bacterium, resulting in the transfer of genes from one bacterium to the other. Transduction may be the most common mechanism for gene exchange and recombination in bacteria.

There are two very different kinds of transduction.

1. Generalized transduction
2. Specialized transduction

Generalized transduction (Figure 12.21 a) occurs during the lytic cycle of virulent and temperate phages. During the assembly stage, when the viral chromosomes are packaged into protein capsids, random fragments of the partially degraded bacterial chromosome also may be packaged by mistake. The resulting virus particles often injects the DNA into another bacterial cell but does not initiate a lytic cycle.

Thus in generalized transduction any part of the bacterial chromosome can be transferred. Once the DNA has been injected it may integrate into the recipient cell’s chromosome to preserve the transferred genes. About 70 to 90% of the transferred DNA is not integrated but is often able to survive and express itself. However, if the transferred DNA is degraded gene transfer is unsuccessful.

Specialized Transduction (Figure 12.21 b) is also called restricted transduction in which only specific portions of the bacterial genome is carried by the phage. When a prophage is induced to leave the host chromosome, exicision is sometimes carried out improperly.

The resulting phage genome contains portions of the bacterial chromosome next to the integration site. When this phage infects another bacterium, it transfers the bacterial genes from the donor bacterium along with phage DNA. Here only the bacterial genes that are close to the site of prophage are transferred. So, this transduction is called specialized.