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Home , Genetic recombination, Hfr cell

Chapter - III

BACTERIAL

Genetic Recombination

Gene Expression: Transcription and Translation

From to proteins

Allele: An alternate form of a . occur at loci on .

1 Genetic Recombination

Genetic material, otherwise known as DNA (DeoxyriboNucleic Acid) is composed of millions of genetic instruction sets. Each "set" is actually a specific ordered sequence of only four different amino acids (Adenine, Thymine, Guanine, and Cytosine). Each instruction set may be several hundred acid molecules long.

A "set" of instruction "code" (as it is sometimes referred to) is known as an . Connect a large number of alleles together into a long "strand" and you have half of a .

When you put two matching chromosome strands together, the "genetic code" or alleles match up on both sides, and together the two alleles are referred to as a Gene. In most body cells, chromosomes occur in pairs. This is what leads to the term "gene pair". Each "gene", therefore, is actually comprised of a pair of alleles.

A particular area or location on a chromosome, is called a "" (plural = "loci"). The combination of both alleles at a specific locus determine a particular Trait or Characteristic of the animal. Hair color and eye color are examples of traits.

Most of the cells in the body contain all of chromosomes. The chromosomes act as "instruction sets" which both tell the body how it will grow, develop and operate the traits of the individual.

Fig.1. Each "color band" pair represents a Locus. The two alleles at each location, together represent a Gene. When both alleles are the same, they are referred to as homozygous. When each allele within a pair is different, it is known as heterozygous.

Since the chromosomes occur in pairs, each "half" of the chromosome pair has the same order/arrangement of loci as the other. The exception to this, is the chromosome pair known as the Chromosomes in the specialized sex cells used for .

These chromosomes are often referred to as the X and Y chromosomes. The is shorter and contains less material than the X chromosome. The specific combination of the

2 sex chromosomes determine the gender or sex of the individual. Two X chromosomes (XX) will result in a female. The XY combination will result in a male.

The reproductive cells, known as , (which take the form of ova in females or spermatozoa in males) carry only half of the animal's genes. In all the higher (such as mammals) the offspring will receive only one half of a gene pair from each parent.

In the process of division that creates these gametes, the existing chromosomes of the parent are allowed to "cross-over". That is, some of the alleles found on one side of the chromosome pair will randomly trade places with the alleles found on the other side. After this exchange, the cell divides, and each resulting cell has only half of the genetic material of the parent. As a result, each gamete contains a unique grouping of half of the genes of the parent animal. This special process of recombining the alleles to form gametes is known as

Meiosis.

Genetic recombination is the name given to a group of reactions during which cellular machinery uses DNA to alter or "recombine" with a similar (homologous) sequence. The process involves pairing between complementary strands of DNA, and results in a physical exchange of chromosome material. Genetic information is recombined by the cell for several reasons including the repair of damaged DNA, and the production of population variability during . In some cases, recombination is known to change genes, adding new alleles to the population.

Creationists generally believe that this mechanism was designed to generate the tremendous variety that is evident within each kind, whereas evolutionists attribute such variability ultimately to random mutagenesis. However, many creationists contend that recombination processes add nothing new to the gene pool.

The position at which a gene is located on a chromosome is called a locus. In a given individual, one might find two different versions of this gene at a particular locus.

3 These alternate gene forms are called alleles. During , when the chromosomes line up along the metaphase plate, the two strands of a chromosome pair may physically cross over one another, and during these events genetic recombination is performed by the cell.

Recombination results in a new arrangement of maternal and paternal alleles on the same chromosome. Although the same genes appear in the same order, the alleles are different. This process explains why offspring from the same parents can look so different. In this way, it is theoretically possible to have any combination of parental alleles in an offspring, and the fact that two alleles appear together in one offspring does not have any influence on the statistical probability that another offspring will have the same combination. This theory of "independent assortment" of alleles is fundamental to genetic inheritance.

The frequency of recombination is actually not the same for all gene combinations. This is because recombination is greatly influenced by the proximity of one gene to another. If two genes are located close together on a chromosome, the likelihood that a recombination event will separate these two genes is less than if they were farther apart. Linkage describes the tendency of genes to be inherited together as a result of their location on the same chromosome. describes a situation in which some combinations of genes or genetic markers occur more or less frequently in a population than would be expected from their distances apart. Scientists apply this concept when searching for a gene that may cause a particular disease. They do this by comparing the occurrence of a specific DNA sequence with the appearance of a disease. When they find a high correlation between the two, they know they are getting closer to finding the appropriate gene sequence.

Evolutionary Assumptions

Chromosomes have genes arranged along their length. During meiosis, it is believed the intended function of recombination is to leave existing genes unchanged by performing reacting in the neutral regions between reading frames.

4 Recombination within genes is able to create new alleles, however, it has been assumed this is not the cell's intent, and any changes to gene sequence are believed to be resulting from mistakes during recombination or replication.

The theory of has led to the assumption that recombination originally occurred by mistake, instead of being an intelligently designed process. During sexual reproduction, gametes (egg, sperm) are produced during a cell division process called meiosis. Prior to meiotic division, homologous chromosomes unite at the axis before dividing to opposite poles. It is believed that this homologous pairing was originally performed simply to insure an equivalent division of genetic information. But, an exchange of DNA accidentally occurred during this process, which provided beneficial variability and was naturally selected to became a regular part of gamete formation. It remains generally assumed that recombination events are rather random, and therefore, the phenotypes produced by these reaction are also random.

The DNA used for meiotic recombination possess or sequences that are very similar, and also code for variations of the same characteristic. Before the chromosomal DNA is distributed into new daughter cells, the homologues pair and are spliced together at multiple locations. During these interactions, entire regions and many genes are frequently exchanged. Offspring are always genetically unique due to recombination. However, it is now clear that recombination is a powerful source of new alleles.

The knowledge of recombination comes predominantly from the E. coli, and its effect during sexual reproduction (meiosis) has been studied mostly using lower such as baker's , as well as fruit flies. Recent work with mice has provided additional information from mammals, and shown that substantial differences exist between unicellular and multicellular . The basic details and many genes involved in (HR) appear conserved among the multitude of life forms on earth. It is now widely recognized that genetic editions through HR are part of a highly coordinated process involving a cascade of specific macromolecule interactions (genes), and controlled by highly organized regulatory systems.

Non-Random Recombination

It was assumed that gene crossovers during meiosis occurred at random intervals along chromosomes. It was believed that the frequency of gene crossovers was directly related to the distance between genes, but a variety of discoveries have illustrated the existence of differential recombination rates and patterns, and forced a revision of map distances. It is now a well-known fact that recombination frequency is not constant in any one particular cell. Reactions occur more frequently in some regions of the than in others with variations of several orders of magnitude observed. These

5 hyperactive regions have been termed as "hot spots" as opposed to inert "cold spots" where little to no exchange is found.

The frequencies of recombination events are also non-random. The rates are found to be significantly higher when comparing germ-line with somatic cell types. Sex- specific differences in recombination frequency have also been elucidated. Standard linkage analysis was used to confirm that females have a higher recombination rate than males, and males recombine preferentially in the distal regions of the chromosome.

In addition to exchanges during cell division, HR is involved with many other forms of genomic DNA editing. For example, recombination is induced or shut off as a preprogrammed cell function during differentiation and development. It is also used to perform error-free DNA repair, which in this case serves to prevent unintentional variability. In fact, HR maintains the integrity of the genome through the correction of several different types of DNA damage. Homologous recombination is stimulated by double-stranded breaks during any stage of the cell cycle, and is also responsible for performing deletions, duplications, and translocations between dispersed homologous, which are frequently a response to stress. The specific details or exact required for recombination remain largely unknown, but the plethora of functions accomplished by these reactions has elevated them to the position of master mechanic responsible for virtually all forms of sequence editing and maintenance.

New Alleles

There is an interesting new class of HR only recently recognized that shares common mechanisms with meiotic crossovers, and is likely responsible for the formation of new alleles. The process known as uses template DNA to edit active sequences. During this process, pseudo genes previously referred to as junk DNA is frequently used to make these changes. Gene conversion can be easily distinguished from crossovers in most cases because only one of the homologues is altered. It has now been thoroughly documented that via gene conversion is able to create genetically altered cells, and researchers have suggested that this process can generate a gene with novel functions by rearranging various parts of the parental reading frames. DNA is also repaired through conversion when an intact copy from the sister or is used to replace the damaged region. Gene conversion is now understood to be responsible for performing many alterations that were previously attributed to mutations or other repair mechanisms.

6 Crossing-over is an exchange of sequences between two homologous regions, but during gene conversion only one of the homologues is altered. Regions elsewhere on the same chromosome are instead typically used to convert the gene, and thereby introduce new alleles into the population. This mechanism is responsible for the creation of new alleles in immuno-globulins, the MHC loci, and others.

Variable Genes

Diversification within a population occurs because the genes involved with the production of characteristics exist as a variety of alleles, and therefore traits are polymorphic or available in more than one form. Closely related species are commonly found with extremely high numbers of alleles. Evolutionists generally assume that new alleles are the result of random mutations that have accumulated gradually over millions of years. However, it was discovered that many genes in every genome are highly diverse (hypervariable) in comparison to others.

Not all genes are variable. The majority of genes in the genome is involved with housekeeping functions, and is commonly found unchanged even when comparing vastly different organisms. In contrast, variable genes change significantly from one generation to the next and show nonrandom patterns within any given gene. This diversity is systematically produced through gene conversion while under tight cellular control. For example, variable genes have hot and cold spots of activity similar to those found among gene crossovers in meiosis. A preponderance of non-synonymous substitutions over synonymous has provided even further evidence against randomness. It is becoming increasingly questionable that variability is the result of random mutations as commonly claimed by evolutionists.

Adaptation

Adaptation to a particular habitat or niche involves largely uncharacterized modifications of the genome, and much of what is learnt about genetic heredity has come from theorists who do not believe the cell was designed to perform such changes with intent. The ability of the cell to produce new alleles has probably remained misunderstood for so long because the products of these reactions are being attributed to a source that is

7 independent of cellular purpose (mutations). The mechanisms behind this type of gene conversion are not yet understood, but clearly illustrate the ability of the cell to specifically edit genes, and thereby rapidly multiply the number of alleles in a population. Further characterization should prove to be valuable evidence that cellular design governs the production of genetic variability, and adaptive evolution that occurs as a result.

The Central Dogma of genetic expression

Protein synthesis requires two steps: transcription and translation.

DNA contains codes: The simplistic diagram below illustrates the concept that three bases in DNA code for one amino acid. The DNA code is copied to produce mRNA. The order of amino acids in the polypeptide is determined by the sequence of 3-letter codes in mRNA.

Genetic Recombination: overview

The genetic information can be changed: either by or by the transfer of genes from one to another. The successful transfer of genetic information includes two elements: the introduction of genes from a donor cell into a recipient and recombination of those introduced genes into the recipient's genome. Microbial genetics are important because the genes are the basis for cell function and microorganisms are excellent tools for studying gene function.

What are Mutations?

 A mutation is any physical change in the genetic material (such as a gene or a chromosome). When a gene contains a mutation, the protein encoded by that gene will be abnormal. Some protein changes are insignificant, others are disabling.

8  More than 4,000 diseases are thought to stem from mutated genes inherited from the parents.  A mutation may or may not affect the phenotype.  A mutation is not necessarily bad. It may even be good.

Mutations are inheritable changes in the base sequence of -- the genetic material. An organism with these changes is called a mutant. Genetic recombination is the process where genes from two are combined together. A mutant will be different from its parent, its genotype or genetic makeup has been altered. The phenotype or visible properties of the mutant may or may not be altered. The genotype of a strain is indicated by use of three small italics letters followed by a capital letter and indicates the gene involved in a process (hisC indicates the gene for HisC protein). The phenotype of the strain is indicated by three letter code that ends in a +/-. For example Thr+ indicates a strain can make its own threonine while Thr- indicates that it cannot. An auxotroph is formed when a required nutritional material (amino acid for example) that the parent strain, prototroph, could make is no longer formed.

General Types of Mutations

1. Chromosomal Mutations

 Changes in chromosome structure o , duplication, inversion, or translocation.  Changes in chromosome number o Polyploidy, aneuploidy (autosomes or sex chromosomes).

2. Point Mutations

 Changes made by substituting a single base with another or by adding or deleting one or more nucleotides. o Sickle cell disease results from a single base change (Remember in RNA, the nucleotide base uracil replaces thymine).

Genetic Mutations and their Effects on Proteins

Point Mutations: Changes in single DNA nucleotides.

 A missense mutation substitutes a different amino acid for the original one.

TEMPLATE DNA code GAG (leucine - leu) -mutation-> TEMPLATE DNA code GTG (histidine - his)

 A nonsense mutation results in a stop codon being inserted someplace before the end of the gene.

9 TEMPLATE DNA code ATG (tyrosine - tyr) -mutation-> TEMPLATE DNA code ATT (STOP)

 Silent mutations are point mutations that do not change the amino acid sequence of the protein. These are most likely to have no effect. Redundancy of the Genetic Code reduces the chance that point mutations do not alter the specified amino acids.

The mRNA codons GAA and GAG code for the amino acid Glutamic Acid (Glu). The mRNA codons GCU, GCC, GCA, and GCG all code for the amino acid Alanine (Ala). The mRNA codons GGU, GGC, GGA, and GGG all code for the amino acid Glycine (Gly).

 Frameshift Mutations: Additions or deletions of one or more nucleotides. o May result in "garbage" genes, as the entire amino acid sequence in the code after the change is devastated.

Large deletions may remove a single amino acid, or an entire chunk of chromosome. The most common mutation that causes severe cystic fibrosis deletes only a single codon.

Mutations can occur spontaneously, because of mistakes during replication or due to natural radiation at a frequency of about one in 1,000,000, or may be experimentally induced using . Mutations can be chemically induced by base analogs, compounds that are structurally similar to the purines and pyrimidines in DNA. The cell incorporates them into DNA, but during subsequent replication, the analogs have a higher probability of base pairing incorrectly, thereby inserting the wrong base into the new DNA strand. Other chemical mutagens react directly with DNA to alter the bases. UV radiation is absorbed by the purines and pyrimidines in DNA, and one of its

10 effects is to form pyrimidine dimers in one strand, which prevents these thymine bases from pairing correctly during replication.

Pyrimidine dimers

DNA Lesion-Thymine Dimer

Pyrimidine dimers are molecular lesions formed from thymine or cytosine bases in DNA via photochemical reactions. light induces the formation of covalent linkages by reactions localized on the C=C double bonds. In dsRNA, uracil dimers may also accumulate as a result of UV radiation. Two common UV products are cyclobutane pyrimidine dimers (CPDs, including thymine dimers) and 6,4 photoproducts. These premutagenic lesions alter the structure of DNA and consequently inhibit polymerases and arrest replication. Dimers may be repaired by photoreactivation or nucleotide excision repair, but unrepaired dimers are mutagenic. Pyrimidine dimers are the primary cause of melanomas in human beings.

Types of dimers

Left: Spore photoproduct, Right: Cyclobutane pyrimidine dimer

A cyclobutane pyrimidine dimer (CPD) contains a four membered ring arising from the coupling of the C=C double bonds of pyrimidines. Such dimers interfere with base pairing during DNA replication, leading to mutations.

11 6,4-photoproducts, or 6,4 pyrimidine-pyrimidones, occur at one third the frequency of CPDs but are more mutagenic. Spore photoproduct lyase provides another enzymatic pathway for repair of thymine photodimers.

Ionizing radiation generates free radicals in cells, and these can react with the DNA backbone to cause breaks. Biological agents, such as transposons and the bacteriophage Mu, cause mutations by inserting DNA sequences into genes, and thereby disrupting the coding information.

Mu phage classification Group: Group I (dsDNA) Order: Caudovirales Family: Myoviridae Genus: Mu-like Species: Mu Phage

Bacteriophage Mu or phage Mu is a temperate bacteriophage, a type of virus that infects bacteria. It has an icosahedral head, a contractile tail and 6 tail fibres. It uses DNA-based transposition to integrate its genome into the genome of the host cell that it is infecting. It can then use transposition to initiate its viral DNA replication. Once the viral DNA is inserted into the bacteria, the Mu transposase protein/ in the cell recognizes the recombination sites at the ends of the viral DNA (gix-L and gix-R sites) and binds to them, allowing the process of replicating the viral DNA or embedding it into the host genome.

Fig. Schematic illustration of bacteriophage Mu based on electron microscopic observations. The letters, a, b, c, and d indicate the head, tail, baseplate, and tail fibers, respectively

Bacteriophase Mu

12 A mutation may be a change in a single base pair (point mutation) or involve large deletions or insertions of base pairs. The of a single additional base into a gene can have dramatic effects upon the amino acid sequence of the protein produced from that gene, due to the reading-frame shift this causes in the translation of the mRNA produced from the gene. It is possible to move large sections of DNA to a second location and the process is termed translocation. If the mutated gene is part of an operon the mutation may exert polar effects upon other genes in the operon.

Operon - a segment of DNA containing adjacent genes including structural genes and an operator gene and a regulatory gene. An operon is made up of 3 basic components:

 Promoter – a nucleotide sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters indicate which genes should be used for messenger RNA creation – and, by extension, control which proteins the cell produces.  Operator – a segment of DNA that a regulator binds to. It is classically defined in the lac operon as a segment between the promoter and the genes of the operon. In the case of a repressor, the repressor protein physically obstructs the RNA polymerase from transcribing the genes. A gene that activates the production of messenger RNA by adjacent structural genes.  Structural genes – the genes that are co-regulated by the operon.

Not always included within the operon, but important in its function is a regulatory gene, a constantly expressed gene which codes for repressor proteins. A gene that produces a repressor substance that inhibits an operator gene. The regulatory gene does not need to be in, adjacent to, or even near the operon.

Fig. A typical Operon

13 Fig. 1: RNA Polymerase, 2: Repressor, 3: Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7: lacY, 8: lacA. Top: The gene is essentially turned off. There is no lactose to inhibit the repressor, so the repressor binds to the operator, which obstructs the RNA polymerase from binding to the promoter and making lactase. Bottom: The gene is turned on. Lactose is inhibiting the repressor, allowing the RNA polymerase to bind with the promoter, and express the genes, which synthesize lactase. Eventually, the lactase will digest all of the lactose, until there is none to bind to the repressor. The repressor will then bind to the operator, stopping the manufacture of lactase.

In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be both co-transcribed and co-regulated to define an operon.

The effects of a specific mutation may be reversed by a second (suppressor) mutation in either the same gene, or in another gene. Note that cells do have DNA repair systems to correct damage to DNA. The SOS system is one of these, but it is error-prone and the repaired DNA may still contain mutations.

The SOS response is a state of high-activity DNA repair, and is activated by bacteria that have been exposed to heavy doses of DNA-damaging agents. Their DNA is basically chopped to shreds, and the bacteria attempts to repair its genome at any cost (including inclusion of mutations due to error-prone nature of repair mechanisms). The SOS system is a regulon; that is, it controls expression of several genes distributed throughout the genome simultaneously.

The primary control for the SOS regulon is the gene product of lexA, which serves as a repressor for recA, lexA (which means it regulates its own expression), and about 16 other proteins that make up the SOS response. During a normal cell’s life, the SOS system is turned off, because lexA represses expression of all the critical proteins. However, when DNA damage occurs, RecA binds to single-stranded DNA (single-

14 stranded when a lesion creates a gap in daughter DNA). As DNA damage accumulates, more RecA will be bound to the DNA to repair the damage.

What is interesting is that RecA, in addition to its abilities in recombination repair, stimulates the autoproteolysis of lexA’s gene product. That is, LexA will cleave itself in the presence of bound RecA, which causes cellular levels of LexA to drop, which, in turn, causes coordinate derepression (induction) of the SOS regulon genes.

As damage is repaired, RecA releases DNA; in this unbound form, it no longer causes the autoproteolysis of LexA, and so the cellular levels of LexA rise to normal again, shutting down expression of the SOS regulon genes.

One use of mutant bacterial strains has been to determine the potential mutagenicity of chemicals -- either manufactured or natural. The Ames test utilizes back mutation in a strain of bacteria that are auxotrophic for a nutrient. When auxotrophic cells (His-) are spread on a medium that lacks histidine no growth will occur. If, however, the cells are treated with a chemical that causes a reversion mutation it can then grow.

General or homologous recombination requires extensive homology and is mediated by an enzyme, RecA protein. The sequence of events are (1) nicking of a DNA molecule, (2) opening of the DNA double helix, (3) pairing between homologous single strands of two DNA molecules (requires presence of RecA), and (4) breakage and rejoining of DNA strands so that portions of the DNA molecule are exchanged. An important point is that this process leads to new genotypes only if the two molecules that are recombining differ genetically in regions outside those where breakage and rejoining occurred. In order to detect recombination or exchange of DNA, the offspring must be phenotypically different from the parent.

15 Mechanisms of genetic recombination in bacteria

In bacteria, the gene transfer that precedes recombination can occur by three mechanisms: transformation, , or conjugation. Transformation involves the uptake by a recipient of free or naked DNA released from a donor. However, cells may only be physiologically competent to take up DNA. Competence is related to changes in the cell surface that allow strong binding of DNA. In some organisms, such as E. coli, the transformation process can be enhanced by special pre-treatment of cells. The cell can undergo electroporation where small holes or pores are open in the cell. A single strand of the

16 transforming DNA is integrated into the chromosome, using general recombination mechanisms. A cell with a new genotype is generated when this strand is replicated and the resulting molecule forms the genome of a new cell, at cell division. Eukaryotic cells can also be treated to take up free DNA, although the specific treatments are different from those used in bacteria.

Transformation • Genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and it is exchanged for a piece of the recipient's DNA. • Involves 4 steps

Transformation: 4 steps

2. A fragment of DNA from the dead donor 1. A donor bacterium dies and is degraded bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium

3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the 4. Exchange is complete recipient's DNA

In transduction, the transferred DNA is carried in the capsid of a bacteriophage. The donor's DNA replaces part or all of the viral genome in the phage head. Thus, the particle is probably defective in viral replication because essential viral genes are missing. In the case of temperate phages (mild or normal) such as lambda, bacterial DNA becomes associated with the virus genome when the prophage (harmless genetic material) is excised incorrectly from the bacterial chromosome. When this occurs, the same set of bacterial genes is always incorporated into . This phenomenon is specialized transduction, because it is only effective in transducing a few special bacterial genes. In contrast, generalized transduction can transfer any bacterial gene to the recipient. This process may occur with phages that degrade their host DNA into pieces the size of viral genomes.

So, there are two types of transduction:

 generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.  specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle.

17 Generalised Transduction: 7 steps Generalised Transduction

1. A lytic bacteriophage adsorbs to a susceptible bacterium. 4. The bacteriophages are released.

2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophagecomponents and 5. The bacteriophage carrying the donor 3. Occasionally, a bacteriophage bacterium's DNA head orcapsid assembles adsorbs to a recipient around a fragment of donor bacterium bacterium'snucleoid or around a instead of a phage genome by mistake.

Generalised Transduction

6 . T he bacteriophage inserts the donor bacterium's DN A it is carrying into the recipient b ac terium

7. The donor bacterium's DNA is exchanged for some of the recipient's DNA

Some temperate phages cause phenotypic changes in the bacteria they infect even without transducing bacterial genes. In the lysogenic state, viral genes are expressed which confer new properties on the cell. Examples of this phage conversion are toxin production by such as Corynebacterium diphtheriae and surface polysaccharide structure in Salmonella anatum.

Specialised Transduction: 6 steps Specialised Transduction

3. Occasionally during spontaneous induction, a small piece of the donor 1. A temperate bacterium's DNA is bacteriophage adsorbs picked up as part of the to a susceptible phage's genome in place bacterium and injects of some of the phage its genome . DNA which remains in the bacterium's nucleoid.

4. As the bacteriophage replicates, the segment of bacterial DNA 2. The bacteriophage inserts its genome into replicates as part of the the bacterium's phage's genome. Every nucleoid to become a phage now carries that prophage. segment of bacterial DNA.

18 Specialised Transduction

5. The bacteriophage adsorbs to a recipient bacterium and injects its genome.

6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid.

Conjugation, the third means of gene transfer is mediated by special genetic elements called . Plasmids are defined as small, circular DNA molecules that reproduce autonomously. While plasmids are DNA, they control their own replication separately from that of the chromosome.

The presence of plasmids in cells can be detected by techniques that separate them from chromosomal DNA. This involves buoyant density differences due to the tight supercoiling of these rather small DNA circles; the density difference can be enhanced by adding compounds that intercalate between DNA base pairs, such as ethidium bromide. The tightly wound plasmid DNA cannot bind as much ethidium bromide as the chromosomal fragments. Adding ethidium bromide, or other treatments that affect DNA, to whole cells at the appropriate concentration may cure cells of their plasmids. If plasmid replication is more sensitive to these agents than chromosome replication, plasmids may not segregate to all progeny cells during cell division.

Some (but not all) have genes that can direct their transmission from one cell to another by conjugation. Finally, plasmids may have genes that confer novel phenotypes on cells, such as resistance to antibiotics, production of toxins, or the capacity to metabolize unusual substrates such as pesticides or industrial solvents. Antibiotic resistance is conferred by R plasmids. These plasmids have diminished the effectiveness of antibiotics in combating infectious diseases because (i) they may confer resistance to as many as five different antibiotics at once upon the cell, and (ii) by conjugation, they can be rapidly disseminated through the bacterial population. Multiple antibiotic resistance is a consequence of their construction -- they contain several transposons, each of which confers resistance to a unique antibiotic. The genes in the transposons generally specify an enzyme that inactivates the drug before it enters the cell and reaches its target. This differs from chromosomal mutations that result in antibiotic resistance. These generally are modifications of the antibiotic's target of action.

19 Transposon: A (TE) is a DNA sequence that can change its position within the genome, sometimes creating mutations and altering the cell's . Transposition often results in duplication of the TE. They are generally considered non-coding DNA. In Oxytricha, which has a unique genetic system, they play a critical role in development. They are also very useful to researchers as a means to alter DNA inside a living organism. Transposable elements (TEs) represent one of several types of . TEs are assigned to one of two classes according to their mechanism of transposition, which can be described as either copy and paste (class I TEs: retrotransposons) or cut and paste (class II TEs: DNA transposons).

Plasmids are autonomously replicating molecules. What elements are necessary to control DNA replication? There must be an origin of replication, where the frequency of replication can be regulated. The number of plasmid copies is tightly regulated at a few copies with some plasmids, whereas in others, initiation of replication is relatively uncontrolled, and twenty to thirty plasmid copies may be present in a cell. In general, the enzymes used for DNA replication are those coded by the chromosome -- it is the regulatory genes that are plasmid encoded. Conjugative plasmids initiate gene transfer by altering the cell surface to allow contact between the plasmid-containing donor cell and a plasmid-less recipient. A plasmid gene codes for the production of a sex that initiates pair formation. Subsequently, a conjugation bridge is formed through which DNA is transferred. The transfer of plasmid DNA is accompanied by its replication. That is, the donor cell does not lose its plasmid but transfers a copy to the recipient. In actual fact, replication is shared between donor and recipient. A single DNA strand is transferred as a consequence of rolling circle replication in the donor; this strand is used as a template by the recipient to generate a double stranded DNA molecule. Therefore, the consequence of conjugation is that both the donor and the recipient cells contain the plasmid. The recipient is now competent to serve as a plasmid donor in other conjugations.

So, • Bacterial Conjugation is genetic recombination in which there is a transfer of DNA from a living donor bacterium to a recipient bacterium. Often involves a sex pilus. • The 3 conjugative processes I. F+ conjugation II. Hfr conjugation III. Resistance plasmid conjugation

20 I. F+ Conjugation Process F+ Conjugation- Genetic recombination in which there is a transfer of an F+ plasmid (coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus. Other plasmids present in the cytoplasm of the bacterium, such as those coding for antibiotic resistance, may also be transferred during this process.

F+ Conjugation: 4 steps

1. The F+ male has an F+ plasmid coding 3. The sex pilus retracts and a bridge for a sex pilus and can serve as a is created between the two genetic donor bacteria. One strand of the F+ plasmid enters the recipient bacterium

4. Both bacteria make a complementary strand of the F+ plasmid and both are 2. The sex pilus adheres to an F- female now F+ males capable of producing a (recipient). One strand of the F+ sex pilus. There was no transfer of plasmid breaks donor chromosomal DNA although other plasmids the donor bacterium carries may also be transferred during F+ conjugation.

Some conjugative plasmids, such as the F factor in E. coli, can also direct transfer of chromosomal genes by conjugation. E. coli strains which have this property are Hfr strains. The F factor can integrate into the chromosome to form one DNA molecule. This occurs at regions of homology between F and the chromosome. These regions are insertion sequences located on both molecules. F factor can now transfer chromosomal genes during a conjugation, because in effect, the chromosome has become part of the F factor. It is the F factor that has the genetic information to drive gene transfer. Specifically, there is a nucleotide sequence on F that specifies the origin of transfer. The host chromosome was inserted just downstream from this region. DNA is transferred just as described above for plasmid transfer. It is important to note that chromosomal genes are transferred before any of the plasmid genes. Thus, if the cytoplasmic bridge is broken before the entire chromosome is transferred, the recipient remains.

A high-frequency recombination cell (Hfr cell) (also called an Hfr strain) is a bacterium with a conjugative plasmid (often the F-factor) integrated into its genomic DNA. The Hfr strain was first characterized by Luca Cavalli-Sforza. Unlike a normal F+ cell, hfr strains will, upon conjugation with a F− cell, attempt to transfer their entire DNA through the bridge, not to be confused with the pilus. This occurs because the F factor has integrated itself via an insertion point in the bacterial chromosome. Due to the F factor's inherent nature to transfer itself during conjugation, the rest of the is dragged along with it, thus making such cells very useful and interesting in terms of studying gene linkage and recombination. Because the genome's rate of transfer through the mating bridge is constant, molecular biologists and geneticists can use Hfr strain of bacteria (often E. coli) to study and map the chromosome. The procedure commonly used for this is called interrupted mating.

21 II. Hfr Conjugation

Genetic recombination in which fragments of chromosomal DNA from a male donor bacterium are transferred to a female recipient bacterium following insertion of an F+ plasmid into the nucleoid of the donor bacterium. Involves a sex (conjugation) pilus.

Hfr Conjugation: 5 steps 3. T he sex pilus retracts and a bridge forms between the two bacteria. One donor DNA strand begins to enter the recipient bacterium. The two cells break apart easily so the only a portion 1. An F+ plasmid inserts into the of the donor's DNA strand is donor bacterium's nucleoid to form an Hfr male. usually transferred to the recipient bacterium.

4. The donor bacterium makes a com plementary copy of the remaining DNA strand and remains an Hfr male. The recipient 2. The sex pilus adheres to an F- bacterium makes a complementary fem ale (recipient). One donor DNA strand of the transferred donor strand breaks in the middle of the DN A. inserted F+ plasmid.

5. The donor DNA fragment undergoes genetic exchange with the recipient bacterium's DNA. Since there was transfer of some donor chromosomal DNA but usually not a complete F+ plasmid, the recipient bacterium usually remains F-

The F plasmid is considered to be an episome which may become integrated into the main chromosome. When the F genes become integrated into the chromosome, the cell is said to be Hfr (high frequency of recombination). An Hfr cell may transfer F genes to an F− cell. During this transfer of genetic material, the F episome may take chromosomal DNA with it. The donor cell does not lose any genetic material as anything transferred is replicated concurrently. It is extremely rare that an Hfr cell's chromosome is transferred in its entirety. Homologous recombination occurs when the newly acquired DNA crosses over with the homologous region of its own chromosome.

22 Fig. There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance, whereas episomes, the lower example, integrate into the host chromosome.

Transposons and insertion sequences are genetic elements capable of moving within cells. Transposons differ from insertion sequences in that they contain additional genes, such as ones for antibiotic resistance. The frequency with which these elements move is rather low, but 10 to 100 fold greater than the frequency of spontaneous mutation. The ends of these elements contain repeated sequences. In addition, they code for a transposase enzyme that can insert the elements at any point into a DNA molecule. When these transposable elements insert into a DNA target sequence, that target sequence is duplicated. In addition, elements that undergo replicative transposition also are duplicated. That is, a copy remains at the original site, and the other copy is inserted at a new site. The transposase makes single strand cuts in the sequences at the ends of the transposable element, and at the target site. The element is joined to the target via the single strand ends, and the gaps are filled in by DNA replication. Finally, the cointegrate formed by recombination is resolved to generate a copy of the transposable element at the new site. In other transposons (such as Tn5), transposition is conservative, and the transposon is excised from its original location, and is reinserted at a new site. If the site of transposon insertion is within an existing bacterial gene, it is likely to be inactivated, and a mutation has occurred.

III. Resistant Plasmid Conjugation Genetic recombination in which there is a transfer of an R plasmid (a plasmid coding for multiple antibiotic resistance and often a sex pilus) from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus

23 Resistant Plasmid Conjugation: 4 steps

1. The bacterium with an R- 3. The sex pilus retracts and a plasmid is multiple antibiotic bridge is created between the resistant and can produce a sex two bacteria. One strand of pilus (serve as a genetic donor). the R-plasmid enters the recipient bacterium.

4. Both bacteria make a 2. The sex pilus adheres to an F- complementary strand of the R- female (recipient). One strand of plasmid and both are now multiple the R-plasmid breaks. antibiotic resistant and capable of producing a sex pilus.

Genetic Engineering

Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or "knocked out", using a nuclease. is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations. An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). The first GMOs were bacteria in 1973; GM mice were generated in 1974. Insulin-producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994.

Fig. Comparison of conventional with transgenic and cisgenic genetic modification

24 If genetic material from another species is added to the host, the resulting organism is called transgenic. If genetic material from the same species or a species that can naturally breed with the host is used the resulting organism is called cisgenic. Genetic engineering can also be used to remove genetic material from the target organism, creating a gene knockout organism. In Europe genetic modification is synonymous with genetic engineering while within the United States of America it can also refer to conventional breeding methods. The Canadian regulatory system is based on whether a product has novel features regardless of method of origin. In other words, a product is regulated as genetically modified if it carries some trait not previously found in the species whether it was generated using traditional breeding methods (e.g., selective breeding, cell fusion, mutation breeding) or genetic engineering. Within the scientific community, the term genetic engineering is not commonly used; more specific terms such as transgenic are preferred.

Technique

Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.

Techniques of Genetic Engineering Genetic engineering is a very young discipline, and is only possible due to the development of techniques from the 1960s onwards. Watson and Crick have made these techniques possible from our greater understanding of DNA and how it functions following the discovery of its structure in 1953. Although the final goal of genetic engineering is usually the expression of a gene in a host, in fact most of the techniques and time in genetic engineering are spent isolating a gene and then cloning it. This table lists the techniques that we shall look at in detail. Technique Purpose 1 cDNA To make a DNA copy of mRNA 2 Restriction Enzymes To cut DNA at specific points, making small fragments 3 DNA Ligase To join DNA fragments together 4 Vectors To carry DNA into cells and ensure replication 5 Plasmids Common kind of vector 6 Gene Transfer To deliver a gene to a living cells 7 Genetic Markers To identify cells that have been transformed 8 Replica Plating * To make exact copies of bacterial colonies on an agar plate 9 PCR To amplify very small samples of DNA 10 DNA probes To identify and label a piece of DNA containing a certain sequence 11 Shotgun * To find a particular gene in a whole genome 12 Antisense genes * To stop the expression of a gene in a cell 13 Gene Synthesis To make a gene from scratch 14 Electrophoresis To separate fragments of DNA * Additional information that is not directly included in AS . However it can help to consolidate other techniques.

25 1. Complementary DNA Complementary DNA (cDNA) is DNA made from mRNA. This makes use of the enzyme , which does the reverse of transcription: it synthesises DNA from an RNA template. It is produced naturally by a group of viruses called the (which include HIV), and it helps them to invade cells. In genetic engineering reverse transcriptase is used to make an artificial gene of cDNA as shown in this diagram.

Complementary DNA has helped to solve different problems in genetic engineering: It makes genes much easier to find. There are some 70,000 genes in the human genome, and finding one gene out of this many is a very difficult (though not impossible) task. However a given cell only expresses a few genes, so only makes a few different kinds of mRNA molecule. For example the b cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can be isolated from these cells and used to make cDNA of the insulin gene.

2. Restriction Enzymes

These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases because they cut the bonds in the middle of the polynucleotide chain. Some restriction enzymes cut straight across both chains, forming blunt ends, but most enzymes make a staggered cut in the two strands, forming sticky ends.

The cut ends are “sticky” because they have short stretches of single-stranded DNA with complementary sequences. These sticky ends will stick (or anneal) to another piece of DNA by complementary base pairing, but only if they have both been cut with the same restriction enzyme. Restriction enzymes are highly specific, and will only cut DNA at specific base sequences, 4-8 base pairs long, called recognition sequences.

26 Restriction enzymes are produced naturally by bacteria as a defense against viruses (they “restrict” viral growth), but they are enormously useful in genetic engineering for cutting DNA at precise places ("molecular scissors"). Short lengths of DNA cut out by restriction enzymes are called restriction fragments. There are thousands of different restriction enzymes known, with over a hundred different recognition sequences. Restriction enzymes are named after the bacteria species they came from, so EcoR1 is from E. coli strain R, and HindIII is from Haemophilis influenzae.

3. DNA Ligase

This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments. The sticky ends allow two complementary restriction fragments to anneal, but only by weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete. DNA ligase completes the DNA backbone by forming covalent bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different sources.

4. Vectors

In biology a vector is something that carries things between species. For example the mosquito is a disease vector because it carries the malaria parasite into humans. In genetic engineering a vector is a length of DNA that carries the gene we want into a host cell. A vector is needed because a length of DNA containing a gene on its own won’t actually do anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down pretty quickly. A vector gets round these problems by having these properties:  It is big enough to hold the gene we want (plus a few others), but not too big.  It is circular (or more accurately a closed loop), so that it is less likely to be broken down (particularly in prokaryotic cells where DNA is always circular).

27  It contains control sequences, such as a replication origin and a transcription promoter, so that the gene will be replicated, expressed, or incorporated into the cell’s normal genome.  It contain marker genes, so that cells containing the vector can be identified. Many different vectors have been made for different purposes in genetic engineering by modifying naturally-occurring DNA molecules, and these are now available off the shelf. For example a cloning vector contains sequences that cause the gene to be copied (perhaps many times) inside a cell, but not expressed. An expression vector contains sequences causing the gene to be expressed inside a cell, preferably in response to an external stimulus, such as a particular chemical in the medium. Different kinds of vector are also available for different lengths of DNA insert: Type of vector Max length of DNA insert Plasmid 10 kbp Virus or phage 30 kbp Bacterial Artificial Chromosome 500 kbp (BAC)

5. Plasmids

Plasmids are by far the most common kind of vector, so we shall look at how they are used in some detail. Plasmids are short circular bits of DNA found naturally in bacterial cells. A typical plasmid contains 3-5 genes and there are usually around 10 copies of a plasmid in a bacterial cell. Plasmids are copied separately from the main bacterial DNA when the cell divides, so the plasmid genes are passed on to all daughter cells. They are also used naturally for exchange of genes between bacterial cells (the nearest they get to sex), so bacterial cells will readily take up a plasmid. Because they are so small, they are easy to handle in a test tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase.

One of the most common plasmids used is the R-plasmid (or pBR322). This plasmid contains a replication origin, several recognition sequences for different restriction enzymes (with names like PstI and EcoRI), and two marker genes, which confer resistance to different antibiotics (ampicillin and tetracycline).

28 The diagram below shows how DNA fragments can be incorporated into a plasmid using restriction and ligase enzymes. The restriction enzyme used here (PstI) cuts the plasmid in the middle of one of the marker genes (we’ll see why this is useful later). The foreign DNA anneals with the plasmid and is joined covalently by DNA ligase to form a hybrid vector (in other words a mixture or hybrid of bacterial and foreign DNA). Several other products are also formed: some plasmids will simply re-anneal with themselves to re-form the original plasmid, and some DNA fragments will join together to form chains or circles. These different products cannot easily be separated, but it doesn’t matter, as the marker genes can be used later to identify the correct hybrid vector.

6. Gene Transfer

Vectors containing the genes we want must be incorporated into living cells so that they can be replicated or expressed. The cells receiving the vector are called host cells, and once they have successfully incorporated the vector they are said to be transformed. Vectors are large molecules which do not readily cross cell membranes, so the membranes must be made permeable in some way. There are different ways of doing this depending on the type of host cell.  Heat Shock. Cells are incubated with the vector in a solution containing calcium ions at 0°C. The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells to take up the vector, though no one knows why. This works well for bacterial and animal cells.  Electroporation. Cells are subjected to a high-voltage pulse, which temporarily disrupts the membrane and allows the vector to enter the cell. This is the most efficient method of delivering genes to bacterial cells.  Viruses. The vector is first incorporated into a virus, which is then used to infect cells, carrying the foreign gene along with its own genetic material. Since viruses rely on getting their DNA into host cells for their survival they have evolved many successful methods, and so are an obvious choice for gene delivery. The virus must first be genetically engineered to make it safe, so that it can’t reproduce itself or make toxins.  Three viruses are commonly used:

1. Bacteriophages (or phages) are viruses that infect bacteria. They are a very effective way of delivering large genes into bacteria cells in culture.

29 2. Adenoviruses are human viruses that causes respiratory diseases including the common cold. Their genetic material is double-stranded DNA, and they are ideal for delivering genes to living patients in gene therapy. Their DNA is not incorporated into the host’s chromosomes, so it is not replicated, but their genes are expressed.

The adenovirus is genetically altered so that its coat proteins are not synthesised, so new virus particles cannot be assembled and the host cell is not killed. 3. Retroviruses are a group of human viruses that include HIV. They are enclosed in a lipid membrane and their genetic material is double-stranded RNA. On infection this RNA is copied to DNA and the DNA is incorporated into the host’s chromosome. This means that the foreign genes are replicated into every daughter cell.

After a certain time, the dormant DNA is switched on, and the genes are expressed in all the host cells.  Plant Tumours. This method has been used successfully to transform plant cells, which are perhaps the hardest to do. The gene is first inserted into the of the soil bacterium Agrobacterium tumefaciens, and then plants are infected with the bacterium. The bacterium inserts the Ti plasmid into the plant cells' chromosomal DNA and causes a "crown gall" tumour. These tumour cells can be cultured in the laboratory and whole new plants grown from them by micropropagation. Every cell of these plants contains the foreign gene.  Gene Gun. This extraordinary technique fires microscopic gold particles coated with the foreign DNA at the cells using a compressed air gun. It is designed to overcome the problem of the strong cell wall in plant tissue, since the particles can penetrate the cell wall and the cell and nuclear membranes, and deliver the DNA to the nucleus, where it is sometimes expressed.

30  Micro-Injection. A cell is held on a pipette under a microscope and the foreign DNA is injected directly into the nucleus using an incredibly fine micro-pipette. This method is used where there are only a very few cells available, such as fertilised animal egg cells. In the rare successful cases the fertilised egg is implanted into the uterus of a surrogate mother and it will develop into a normal animal, with the DNA incorporated into the chromosomes of every cell. 

 Liposomes. Vectors can be encased in liposomes, which are small membrane vesicles (see module 1). The liposomes fuse with the cell membrane (and sometimes the nuclear membrane too), delivering the DNA into the cell. This works for many types of cell, but is particularly useful for delivering genes to cell in vivo (such as in gene therapy).

7. Genetic Markers

These are needed to identify cells that have successfully taken up a vector and so become transformed. With most of the techniques above less than 1% of the cells actually take up the vector, so a marker is needed to distinguish these cells from all the others. We’ll look at how to do this with bacterial host cells, as that’s the most common technique. A common marker, used in the R-plasmid, is a gene for resistance to an antibiotic such as tetracycline. Bacterial cells taking up this plasmid can make this gene product and so are resistant to this antibiotic. So if the cells are grown on a medium containing tetracycline all the normal untransformed cells, together with cells that have taken up DNA that’s not in a plasmid (99%) will die. Only the 1% transformed cells will survive, and these can then be grown and cloned on another plate.

31 8. Replica Plating

Replica plating is a simple technique for making an exact copy of an agar plate. A pad of sterile cloth the same size as the plate is pressed on the surface of an agar plate with bacteria growing on it. Some cells from each colony will stick to the cloth. If the cloth is then pressed onto a new agar plate, some cells will be deposited and colonies will grow in exactly the same positions on the new plate. This technique has a number of uses, but the most common use in genetic engineering is to help solve another problem in identifying transformed cells. This problem is to distinguish those cells that have taken up a hybrid plasmid vector (with a foreign gene in it) from those cells that have taken up the normal plasmid. This is where the second marker gene (for resistance to ampicillin) is used. If the foreign gene is inserted into the middle of this marker gene, the marker gene is disrupted and won't make its proper gene product. So cells with the hybrid plasmid will be killed by ampicillin, while cells with the normal plasmid will be immune to ampicillin. Since this method of identification involves killing the cells we want, we must first make a master agar plate and then make a replica plate of this to test for ampicillin resistance.

Once the colonies of cells containing the correct hybrid plasmid vector have been identified, the appropriate colonies on the master plate can be selected and grown on another plate.

32 The R-plasmid with its antibiotic-resistance genes dates from the early days of genetic engineering in the 1970s. In recent years better plasmids with different marker genes have been developed that do not kill the desired cells, and so do not need a replica plate. These new marker genes make an enzyme (actually lactase) that converts a colourless substrate in the agar medium into a blue-coloured product that can easily be seen. So cells with a normal plasmid turn blue on the correct medium, while those with the hybrid plasmid can't make the enzyme and stay white. These white colonies can easily be identified and transferred to another plate. Another marker gene, transferred from jellyfish, makes a green fluorescent protein (GFP).

9. Polymerase Chain Reaction (PCR) Genes can be cloned by cloning the bacterial cells that contain them, but this requires quite a lot of DNA in the first place. PCR can clone (or amplify) DNA samples as small as a single molecule. It is a newer technique, having been developed in 1983 by Kary Mullis, for which discovery he won the Nobel prize in 1993. The polymerase chain reaction is simply DNA replication in a test tube. If a length of DNA is mixed with the four nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, then the DNA will be replicated many times. The details are shown in this diagram:

1. Start with a sample of the DNA to be amplified, and add the four nucleotides and the enzyme DNA polymerase. 2. Normally (in vivo) the DNA double helix would be separated by the enzyme , but in PCR (in vitro) the strands are separated by heating to 95°C for two minutes. This breaks the hydrogen bonds. 3. DNA polymerisation always requires short lengths of DNA (about 20 bp long) called primers, to get it started. In vivo the primers are made during replication by DNA polymerase, but in vitro they must be synthesised separately and added at this stage.

33 This means that a short length of the sequence of the DNA must already be known, but it does have the advantage that only the part between the primer sequences is replicated. The DNA must be cooled to 40°C to allow the primers to anneal to their complementary sequences on the separated DNA strands. 4. The DNA polymerase enzyme can now extend the primers and complete the replication of the rest of the DNA. The enzyme used in PCR is derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90°C, so it is not denatured by the high temperatures in step 2. Its optimum temperature is about 72°C, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible. 5. Each original DNA molecule has now been replicated to form two molecules. The cycle is repeated from step 2 and each time the number of DNA molecules doubles. This is why it is called a chain reaction, since the number of molecules increases exponentially, like an explosive chain reaction. Typically PCR is run for 20-30 cycles.

PCR can be completely automated, so in a few hours a tiny sample of DNA can be amplified millions of times with little effort. The product can be used for further studies, such as cloning, electrophoresis, or gene probes. Because PCR can use such small samples it can be used in forensic medicine (with DNA taken from samples of blood, hair or semen), and can even be used to copy DNA from mummified human bodies, extinct woolly mammoths, or from an insect that's been encased in amber since the Jurassic period. One problem of PCR is having a pure enough sample of DNA to start with. Any contaminant DNA will also be amplified, and this can cause problems, for example in court cases.

10. DNA Probes

These are used to identify and label DNA fragments that contain a specific sequence. A probe is simply a short length of DNA (20-100 nucleotides long) with a label attached. There are two common types of label used:  A radioactively-labelled probe (synthesised using the isotope 32P) can be visualised using photographic film (an autoradiograph).  A fluorescently-labelled probe will emit visible light when illuminated with invisible ultraviolet light. Probes can be made to fluoresce with different colours. Probes are always single-stranded, and can be made of DNA or RNA. If a probe is added to a mixture of different pieces of DNA (e.g. restriction fragments) it will anneal (base pair) with any lengths of DNA containing the complementary sequence. These

34 fragments will now be labelled and will stand out from the rest of the DNA. DNA probes have many uses in genetic engineering:  To identify restriction fragments containing a particular gene out of the thousands of restriction fragments formed from a genomic library. This use is described in shotguning below.  To identify the short DNA sequences used in DNA fingerprinting.  To identify genes from one species that are similar to those of another species. Most genes are remarkably similar in sequence from one species to another, so for example a gene probe for a mouse gene will probably anneal with the same gene from a human. This has aided the identification of human genes.  To identify genetic defects. DNA probes have been prepared that match the sequences of many human genetic disease genes such as muscular dystrophy, and cystic fibrosis. Hundreds of these probes can be stuck to a glass slide in a grid pattern, forming a DNA microarray (or DNA chip). A sample of human DNA is added to the array and any sequences that match any of the various probes will stick to the array and be labelled. This allows rapid testing for a large number of genetic defects at a time.

11. Shotguning

This is used to find one particular gene in a whole genome, a bit like finding the proverbial needle in a haystack. It is called the shotgun technique because it starts by indiscriminately breaking up the genome (like firing a shotgun at a soft target) and then sorting through the debris for the particular gene we want. For this to work a gene probe for the gene is needed, which means at least a short part of the gene’s sequence must be known.

12. Antisense Genes

These are used to turn off the expression of a gene in a cell. The principle is very simple: a copy of the gene to be switch off is inserted into the host genome the “wrong” way round, so that the complementary (or antisense) strand is transcribed. The antisense mRNA produced will anneal to the normal sense mRNA forming double- stranded RNA. Ribosomes can’t bind to this, so the mRNA is not translated, and the gene is effectively “switched off”.

13. Gene Synthesis

It is possible to chemically synthesise a gene in the lab by laboriously joining nucleotides together in the correct order. Automated machines can now make this much easier, but only up to a limit of about 30bp, so very few real genes could be made this way (anyway it’s usually much easier to make cDNA). The genes for the two insulin chains (xx bp) and for the hormone somatostatin (42 bp) have been synthesisied this way. It is very useful for making gene probes.

35 14.Electrophoresis

This is a form of chromatography used to separate different pieces of DNA on the basis of their length. It might typically be used to separate restriction fragments. The DNA samples are placed into wells at one end of a thin slab of gel made of agarose or polyacrylamide, and covered in a buffer solution. An electric current is passed through the gel. Each nucleotide in a molecule of DNA contains a negatively-charged phosphate group, so DNA is attracted to the anode (the positive electrode). The molecules have to diffuse through the gel, and smaller lengths of DNA move faster than larger lengths, which are retarded by the gel. So the smaller the length of the DNA molecule, the further down the gel it will move in a given time. At the end of the run the current is turned off.

Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There are three common methods for doing this:  The gel can be stained with a chemical that specifically stains DNA, such as ethidium bromide or azure A. The DNA shows up as blue bands. This method is simple but not very sensitive.  The DNA samples at the beginning can be radiolabelled with a radioactive isotope such as 32P. Photographic film is placed on top of the finished gel in the dark, and the DNA shows up as dark bands on the film. This method is extremely sensitive.  The DNA fragments at the beginning can be labelled with a fluorescent molecule. The DNA fragments show up as coloured lights when the finished gel is illuminated with invisible ultraviolet light.

Gene Therapy

This is perhaps the most significant, and most controversial kind of genetic engineering. It is also the least well-developed. The idea of gene therapy is to genetically alter humans in order to treat a disease viz., Cystic Fibrosis (CF), Severe Combined Immunodefficiency Disease (SCID), etc. This could represent the first opportunity to cure incurable diseases. Note that this is quite different from using genetically- engineered microbes to produce a drug, or hormone to treat a disease by conventional means. Gene therapy means altering the genotype of a tissue or even a whole human.

36