Biology

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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.

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Formation of Mutants

The term mutant refers to an organism in which either the base sequence of DNA or the phenotype has been changed. A mutant is an organism whose genotype differs from that found in nature. The process of formation of mutant organism is called mutagenesis.

In nature and in the laboratory, mutations sometimes arise spontaneously without any help from the experimenter. This is called spontaneous mutagenesis. The two mechanisms that are most important for spontaneous mutagenesis are

1. Errors occurring during replication and
2. Spontaneous alteration of bases.

Mutations can also be induced experimentally by application of mutagens. Mutagens are agents that cause mutations.

Mutagens and their Mode of Action

Physical Mutagens

UV radiation:

UV light causes mutations because the purine and pyrimidine bases in DNA absorb light strongly in the ultraviolet range (254 to 260 nm). At this wavelength, UV light induces point mutations primarily by causing photochemical changes in the DNA.

One of the effects of UV radiation on DNA is the formation of abnormal chemical bonds between adjacent pyrimidine molecules in the same strand, or between pyrimidines on the opposite strands, of the double helix.

This bonding is induced mostly between adjacent thymines, forming what are called thymine dimers (Figure 12.10), usually designated TT. This unusual pairing produces a bulge in the DNA strand and disrupts the normal pairing of T’s (thymines) with corresponding A’s(adenines) on the opposite strand. If UV induced genetic damage is not repaired, mutations or cell death may result.

Chemical Mutagens

Chemical mutagens include both naturally occurring chemicals and synthetic substances. These mutagens can be grouped into different classes on the basis of their mechanism of action. They are

(i) Base analogs are bases that are similar to the bases normally found in DNA.
E.g. 5 – bromouracil (5-BU). TA to CG (Figure 12.11).

(ii) Base Modifying Agents are chemical that act as mutagens by modifying the chemical structure and properties of bases. The three types of mutagens that work in this way are

• A deaminating agent e.g: Nitrous acid removes amino groups (- NH₂) from the bases guanine, cytosine, and adenine.
• Hydroxylamine (NH₂ OH) is a hydroxylating mutagen that react specifically with cytosine, modifying it by adding a hydroxyl group (OH) so that it can pair solely with adenine instead of with guanine.
• Alkylating agents like methymethane sulfonate (MMS) introduces alkyl groups onto the bases at a number of location.

(iii) Intercalating agents

Acridine, proflavin, ethidium bromide are a few examples of intercalating agents. These insert (intercalate) themselves between adjacent bases in one or both strands of the DNA double helix. Intercalating agents can cause either additions or deletions.

The Ames Test: A Screen for Potential Carcinogens

Everyday we are exposed to a wide variety of chemicals in our environment, such as drugs, cosmetics, food additives, pesticides, and industrial compounds. Many of these chemicals can have mutagenic effects, including genetic diseases and cancer. Some banned chemical warfare agents (e.g. mustard gas) also are mutagens.

A number of chemicals (subclass of mutagens) induce mutations that result in tumorous or cancerous growth. These chemical agents are called chemical carcinogens. Directly testing the chemicals for their ability to cause tumors in animals is time consuming and expensive. However, the fact that most chemical carcinogens are mutagens led Bruce Ames to develop a simple, inexpensive, indirect assay for mutagens.

In general Ames test is an indicator of whether the chemical is a mutagen. The Ames test assays the ability of chemicals to revert mutant strains of the bacterium Salmonella typhimurium to wild type. The mutant strain of S.typhimurium is auxotrophic to histidine (histidine), that is it requires histidine for its growth and
cannot grow in the absence of histidine. The mutant strain is grown in a histidine deficient medium containing the chemical to be tested.

A control plate is also set up which does not contain the chemical. After incubation the control plates may have few colonies resulting from spontaneous reversion of the his – strain. Compared to the control plates if there are increased number of colonies on test plate, it indicates that the chemical has reverted the mutant strain back to wild type. This chemical is likely to be a carcinogen. Figure 12.14 shows steps in Ames test.

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Types of Mutation

The base sequence of DNA determines the amino acid sequence of a protein. The chemical and physical properties of each protein are determined by its amino acid sequence, so a single amino acid change is capable of altering the activity of, or even completely inactivating, a protein.

Genotype refers to the genetic composition of an organism. Phenotype is an observable property of organism. The functional form of a gene is called Wildtype because presumably this is the form found in nature.

Mutation is the process by which the sequence of base pairs in a DNA molecule is altered.The alteration can be a single base pair substitution, insertion or deletion. Mutations can be divided into two general categories:

1. Base – pair substitution

Base – pair substitution mutation involves a change in the DNA such that one base pair is replaced by another.

• A mutation from one purine – pyrimidine base pair to the other purine – pyrimidine base pair is a transition mutation (Figure 12.7 a). E.g. AT to GC, CG to TA.
• A mutation from a purine pyrimdine base pair to a pyrimidine – purine base pair is a transversion mutation (Figure 12.7 b). E.g. AT to TA, CG to GC.
  

2. Base pair insertion or deletions

Involves the addition or deletion of one base pair. If one or more base pairs are added to or deleted from a protein coding gene, the reading frame of an mRNA can change downstream of the mutation. An addition or deletion of one base pair, for example, shifts the mRNA’s downstream reading frame by one base, so that incorrect amino acids are added to the polypeptide chain after the mutation site.

This type of mutation, called a frame shift mutation (Figure 12.8) usually results in a nonfunctional protein.

Frame shift mutations:

• May generate new stop codons, resulting in a shortened protein.
• May result in a read through of the normal stop codon, resulting in longer than normal proteins
• Or may result in a complete alteration of the amino acid sequence of a protein.
  

Point mutations are single base changes, that do not affect the reading frame, that is, the mutation only makes a single change in a single codon, and everything else is undisturbed. Mutations can also be defined according to their effects on amino acid sequences in proteins. They are:-

1. A missense mutation (Figure 12.9 a) is a gene mutation in which a base – pair change in the DNA changes a codon in an mRNA so that a different amino acid is inserted into the polypeptide.

2. A neutral mutation (Figure 12.9 b) is a subset of missense mutations in which the new codon codes for a different amino acid that is chemically equivalent to the original and therefore does not affect the proteins function. Consequently, the phenotype does not change.

3. A silent mutation (Figure 12.9 c) is also a subset of missense mutations that occurs when a base – pair change in a gene alters a codon in the mRNA such that the same amino acid is inserted in the protein. In this case, the protein obviously has a wild type function.

4. A nonsense mutation (Figure 12.9 d) is a gene mutation in which a base – pair change in the DNA, changes a codon in an mRNA to a stop (nonsense) codon (UAG, UAA or UGA). Nonsense mutation cause premature chain termination so instead of complete polypeptides, shorter than normal polypeptide fragments (often nonfunctional) are formed.

Forward mutations change the genotype from wild type to mutant and reverse mutations (or reversions or back mutations) change the genotype from mutant to wild type or to partially wild type. An organism which has reverted is a Revertant. The effects of mutation may be diminished or abolished by a suppress or mutation.

Suppressor mutation is a mutation at a different site from that of the original mutation. A suppressor mutation masks or compensates for the effects of the initial mutation, but it does not reverse the original mutation.