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By Dr.  Noha  Saber  Shafik Lecturer of Medical Microbiology and Immunology

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Bacterial Variations  Bacterial variations include:  •Variation in morphological characters (e.g. spores and capsules formation).  •Variation in the cultural properties e.g., colonial variation between the smooth (S-form) and rough (R-form).  •Variation in the metabolic requirement and enzymatic functions.  •Variation in the biological properties, pathogenicity and virulence.

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Variations of microorganisms are divided into two groups:  A) Phenotypic variations  ( Non-hereditary  variations):  Phenotypic variation occurs in response to environmental changes. The environmental factors lead to suppression, or activation of the gene controlling process which could not take place under the previous environmental condition, the phenotypic variation is reversible, and being dependent on environmental conditions

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B) Genotypic (hereditary) variations:  Genotypic variation occurs in response to changes in the genetic structure. This variation is irreversible (i.e., heritable). A change in the bacterial genome may be caused either as a result of mutation in the cell's own DNA, or from the acquisition of additional DNA from an external source [gene transfer].

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I- Mutation  Mutation  is defined as any change in the DNA base sequence. It may produce no observable effect on structure or function of the encoded protein. In few cases, it results in effective changes (altered enzyme action, or a nonfunctional protein may be produced).

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Types of Mutations:  I. Point Mutation:  A) Substitution of one nucleotide by another:  B) Frame shift mutation II. Multi-site Mutation (Null mutation):  It includes extensive chromosomal rearrangement, multiple inversion, duplication and deletion. It results in change in the chromosomal structure  extending too many thousands of bases → resulting in complete destruction of genes' functions.

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Causes of Mutations:  1) Spontaneous Mutation:  It occurs in nature during replication of DNA, but it is immediately corrected by the repairing mechanism of the polymerase enzyme. 2) Induced Mutations:  a) Physical agents:  i ) Heat    ii) Ultraviolet light      iii) Ionizing radiation  b) Chemical agents:  i ) Nucleotide base analogs (5-bromo-uracil)  ii)  Ethedium  bromide  iii) Acridine derivatives

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II- Gene Transfer (Exchange) in Bacteria  The exchange of genetic materials between bacterial cells may occur by one of three mechanisms: 1) Transformation:  Transformation is the process of up taking and incorporating a free genetic fragment (exogenous DNA). It occurs in some bacterial species e.g. Pneumococci,  H. influenza  and certain Bacillus species.

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Mechanism of Transformation:  1) Extraction of DNA from a donor ce ll,  which may occur artificially (by treating with calcium ions at a low temperature ( 4°C ), followed by short exposure to a high temperature ( 42°C )) or released by cell lysis.  2) The foreign DNA is taken into the recipient cell.  3) The foreign DNA piece is incorporated into the host cell chromosome by a recombination process, which needs a high degree of homology.

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2) Conjugation:  Conjugation is a process in which one cell (the donor) makes contact with another cell (the recipient), and the DNA is transferred directly from the donor cell into the recipient cell.  Conjugation occur through sex pilus mediated by conjugative plasmid.

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3) Transduction:  Transduction is the transfer of bacterial DNA between bacterial cells by means of bacteriophage. The transducing phages all contain double stranded DNA. Transduction may be generalized or specialized.

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a- Generalized Transduction:  In lytic cycle of bacteriophage replication, a segment of bacterial DNA (chromosome or plasmid) is contained within the phage capsid instead of the phage genome (Error of assembly). The bacterial DNA can be transduced to another bacterial cell on infection with such phage.

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b- Specialized Transduction:  In lysogenic cycle of bacteriophage replication, the DNA of latent phage (prophage) can be inserted into the  bacterial chromosome, and replicates as a part of it. The lysogenic state is not permanent. After many generations, the phage can revert to its virulent and become excised again from the chromosome.

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Sometime, the excision is not exact (Error in excision), and the phage may pick up some of the chromosomal DNA adjacent to its insertion site.  When such phage infects another cell, it integrates into the bacterial chromosome (at a specific site).

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Transposable Genetic Elements (Transposons)  Transposons are segments of DNA able to move from one position to another in the genome, or from the chromosomal DNA to a plasmid, or the reverse. They carry the genetic information necessary for their own transfer through transposase and integrase enzymes.

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Transposons play an important role in collecting antibiotic resistant determinants in adjacent genes, and their transfer from a plasmid to a chromosomal location, leading to the development of multi-resistant bacterial strains.

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Types of Transposons:  1) Insertion sequences  are the simplest form of transposons (150-1500 base pairs). They carry only the genetic information necessary for their own transfer. They can be detected if their insertion leads to interruption or inactivation of genes, or turn on the expression of adjacent genes.

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2) Complex transposons:  These forms of transposons are carried by conjugative plasmids. In addition to genes encoding for their transposition, they carry a gene that encode for special characters (e.g., antibiotic resistance or virulence factor).  3)  Integrons :  These forms carry multiple genes (Gene cassette) that code for antibiotic resistance in bacteria, and spread in antibiotic resistant strains as a block

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4) Pathogenicity Island:  These forms are present in virulent bacteria and carry multiple genes that code for virulence factors as adhesins,  invasins  and enzymes especially in gram – ve  bacteria ( E. coli  and  H. pylori ).

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Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973.  Recombinant DNA(r DNA) :   Is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing .

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Recombinant DNA technology :  Is a technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future.

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• It is an area of medical science that is just beginning to be researched in a concerted effort .  • Recombinant DNA technology works by taking DNA from two different sources and combining that DNA into a single molecule. That alone, however, will not do much.  • Recombinant DNA technology only becomes useful when that artificially-created DNA is reproduced. This is known as  DNA cloning.

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The basic procedures of recombinant DNA technology : Recombinant DNA molecules are created in nature more often than in the laboratory; for example, every time a bacteria phage or eukaryotic virus infects its host cell and integrates its DNA into the host genome, a recombinant is created .

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Basic steps are common to most recombinant DNA experiments:  1- Isolation and purification of DNA:  2- Cleavage of DNA at particular sequences

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3- A recombinant DNA molecule is usually formed by cleaving the DNA of interest to yield inserts DNA and then ligating the insert DNA to vector DNA (recombinant DNA or chimeric DNA).

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4- Introduction of recombinant DNA into compatible host cells: In order to be propagated. 5- Replication and expression of recombinant DNA in host cells:

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Vectors- Cloning Vehicles:  Cloning vectors can be plasmids, bacteriophage, viruses, or even small artificial chromosomes. Most vectors contain sequences that allow them to be replicated autonomously within a compatible host cell, whereas a minority carries sequences that facilitate integration into the host genome.

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1- Plasmid vectors.  2- Bacteriophage vectors.  3- Virus vectors.  4- Shuttle Vectors-can replicate in either prokaryotic or eukaryotic cells.  5- Yeast Artificial Chromosomes as vectors.

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Criteria of a good cloning vector:  1- Be as small as possible.  2- Be well characterized regarding: gene location and restriction endonuclease cleavage site.  3- Be capable of autonomous replication within the host.  4- Possess non-essential regions within which the target DNA can be inserted.

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5- Carry a selectable marker (antibiotic resistance gene) so that cells transformed by the vector can be distinguished from non-transformed cells.  6- Contain single cleavage site for restriction endonuclease.  7- Display limited host range in order to reduce the biohazards associated with the recombinant molecule.

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All cloning vectors have in common at least one unique cloning site, a sequence that can be cut by a restriction endonuclease to allow site-specific insertion of foreign DNA.

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Plasmid Vectors:   Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA. These extra chromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g., yeast), exist in a parasitic or symbiotic relationship with their host cell.

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Plasmids can replicate autonomously within a host, and they frequently carry genes conferring resistance to antibiotics such as tetracycline. The expression of these marker genes can be used to distinguish between host cells that carry the vectors and those that do not

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Restriction enzymes  are endonucleases produced by bacteria that typically recognize specific 4 to 8 bp sequences, called restriction sites, and then cleave both DNA strands at this site .  Restriction enzymes are named after the bacterium from which they are isolated: For example, Eco RI is from  Escherichia coli.

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• Identification of Host Cells Containing Recombinant DNA:  • Once a cloning vector and insert DNA have been joined in vitro, the recombinant DNA molecule can be introduced into a host cell, most often a bacterial cell such as  E. coli .

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• Identification of host cells containing recombinant DNA requires genetic selection or screening or both.

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Applications of recombinant DNA technology:  1- The techniques allow extensive chromosomal mapping and gene studies.  2- Preparation of probes that are used for several diagnostic and genetic studies.

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3- The technique is also used for production of many proteins of medical importance in big amounts and with less cost such as hormones (insulin, growth hormone …), interferons, interleukins, antibiotics, monoclonal antibodies, human  antihaemophilic  factor and erythropoietin.

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4- Production of recombinant vaccines such as hepatitis B vaccine (HBsAg)  5- Gene therapy:

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DIAGNOSTIC MOLECULAR BIOLOGY METHODS  These methods apply the principle of nucleic acid hybridization and amplification in the detection, identification and characterization of microorganisms. Each bacterial species contains a specific DNA that is unique to it.

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Nucleic acid probes  Nucleic acid probes are short sequences of labeled single stranded DNA or RNA originally derived from the organism of interest.  Probes are used in hybridization experiments  to detect the presence of complementary sequences of microbial genes in the clinical specimens or in cultures.

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They are usually synthesized commercially by DNA sequencing machines and are labeled by radioactive isotopes or enzymes to facilitate their detection.

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Polymerase Chain Reaction (PCR)  Polymerase chain reaction is a method widely used in molecular biology to make many copies of a specific DNA segment. Using PCR, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.

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Principles:  PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between 0.1 and 10 kilo base pairs ( kbp ) in length.

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Applications of PCR: 1) Selective DNA isolation: 2) Amplification and quantification of DNA : 3) Medical and diagnostic applications: 4) Infectious disease applications 5) Forensic applications 6) Research applications

Bacterial Genetics part 2

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