Chapter 8. Gene Transfer.

 

Transfer of genetic material from one organism to another can have profound effects.

·         In nature, genetic material is transferred such that genetic information is introduced into recipient cells with and without the benefits of vectors (also know as vehicles).  These transfers can increase disease causing capabilities, or allow cells to become resistant to antibiotics, or increase diversity.

·         In recombinant DNA laboratories, the transfer of genetic material is extremely helpful for the investigation of mechanisms of life, or the prevention of disease or the treatment of genetic disorders.

 

Gene transfer refers to the movement of genetic information from one organism to another. 

·         In eukaryotes, one type of gene transfer occurs during sexual reproduction.  Male and female parents form gametes (haploid) which unite to form a zygote (diploid) which may develop into a new individual.

·         In bacteria, reproduction is asexual.  However, gene transfer does occur to increase diversity.  Mechanisms are transformation, transduction and conjugation.  Once the genetic information is transferred from the donor, it enters the genome of the recipient by recombination.

 

Transformation

·         Griffith experiment Streptococcus pneumoniae  - pneumococcus - Figure 8.1.

·         Griffith discovered that the pathogenic factor involved in pneumonia caused by pneumococcus was the capsule of the bacteria.

·         Avery, MacLeod and McCarty in 1944 identified the substance responsible for the transformation in Griffith’s experiment and identified it as DNA.  At this time, many scientists thought that genetic information was in the form of proteins.

·         The Hershey and Chase experiment in 1952 demonstrated that genetic information was transferred by DNA.

·         Watson and Crick determined the structure of DNA shortly after and saw the information potential.

·         Mechanism of transformation - Figure 8.2

·         DNA transferred from cell to cell by transformation is naked DNA.  The DNA is free in the extracellular space.  Cells are only competent to receive the DNA at certain periods of the life cycle.

·         A competence factor is released by the cell and facilitates the entry of the DNA.

·         The amount of DNA that enters is small - less than 5% of the cells genome.

·         The DNA is cut into small fragments during entry and made single-stranded.

·         To successfully transform cells, the DNA must be recombined into the recipient cell’s genomic material.

·         In recombinant DNA work, cells can be made “competent” to receive DNA.  Then the recipient cells can be readily transformed.

·         Not all bacteria are subject to transformation - natural or induced.

 

Transduction

·         In transduction, gene transfer is mediated by a bacteriophage (bacterial virus) vector.

·         A virulent phage infects a bacterium and eventually lyses or kills the bacterium - lytic cycle.

·         A temperate phage infects a bacterium and chooses a lytic cycle or lysogenic cycle - Figure 8.3.

·         If lysogenic cycle is chosen, the phage genome recombines into the bacterial genome and becomes a prophage.

·         Lysogenic phages are specialized transducing phages and can transduce only specific regions of the bacterial genome - Figure 8.4 - Campbell model of specialized transduction or lysogeny.

·         Generalized transducing phages undergo a lytic cycle and are capable of transducing any part of the donor’s genetic information - Figure 8.5.

·         Transduction is significant.

·         The ability of a lysogenic phage to recombine into a bacterial genome suggests a parallel evolution of phage and bacteria since there must be sequence similarities at the site of integration.

·         Transduction is a mechanism to transfer genetic material from one cell to a second.

·         The existence of a prophage suggests a paralel in human and animal cells that may function in the cause of cancer.

·         Transduction is a convenient method to map bacterial chromosomes through the use of cotransduction of phenotypic markers.

·         Certain bacteriophages specifically infect certain bacteria and cause those bacteria to produce toxins - Streptococcus pyogenes, Clostridium, and others.

 

Conjugation

·         Lederberg discovered conjugation in 1946.

·         Mechanism of conjugation

·         In one type of conjugation, the population of cells capable of conjugating contain two types of cells F+ and F-  - the former are the donor cells and the latter are the recipient cells.  The donor cells have an F plasmid.

·         Conjugation in this case is a transfer of the F plasmid from the donor to the recipient.

·         The F plasmid codes for the synthesis of pili which are instrumental in the formation of the conjugal bridge Figures 8.7, 8.8.

·         A second type of conjugation is F+ to Hfr conversion (Figure 8.9).

·         F’ plasmids are created when the Hfr plasmid recombines out of the bacterial genome imprecisely and carries with it a segment of the bacterial genome.  That segment can then be transferred to a recipient cell as in F+ conjugation Figure 8.10.

·         Significance of conjugation

·         In Hfr conjugation significant amounts of genetic material may be transferred.

·         Genetic information including determinants of pathogenicity or antimicrobic resistance may be transferred cell to cell.

·         Hfr conjugation is an excellent procedure to map genes of conjugable bacteria.

 

Other mechanisms of gene transfer

·         Plasmids

·         The F plasmid was the first to be discovered.

·         F plasmids carry genes for the synthesis of pili.

·         Resistance (R) plasmids carry resistance genes to protect bacteria from certain antibiotics and heavy metals (Figure 8.11).

·         Some plasmids carry genes to synthesize bacteriocins - bacteriocidal products.  Plasmids that code for bacteriocins are called bacteriocinogens.

·         Virulence plasmids e.g. in Salmonella.

·         Tumor inducing plasmids can cause tumors in plants Agrobacterium Ti plasmid.

 

·         Transposons. These are mobile genetic elements that can be simple or complex. They can carry in addition to the regulatory genes (promoter, operator, transposase) other genes that potentially code for toxins, antibiotic resistance, etc.

 

·         The mechanisms of gene transfer are summarized in Table 8.2

 

Genetic Engineering

 

·         Hybridoma technology

·         In mid 1970s Cesar Milstein developed a new area of science called hybridoma technology.

·         Hybridomas are hybrids of plasma cells (produce antibodies) that have a finite lifetime and a myeloma cell (immortal).  The hybrid makes a specific species of antibody which is know as a monoclonal antibody.

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