Ch. 8 Gene transfer and genetic engineering

Protoplast fusion

Is accomplished by enzymatically removing the cell walls of organisms of 2 strains and mixing the resulting protoplasts. This allows fusion of the cells and their genetic material: that is, material from one strain recombines with that from the other strain before new cell walls are produced.

Virulent phage

Is capable of causing infection and eventually the destruction and death of a bacterial cell. Because this cycle results in lysis of the infected host cell, it is called a lytic cycle.

R (resistance) plasmids

Carry genes that provide resistance to various antibiotics such as chloram-phenicol and tetracycline and to heavy metals such as arsenic and mercury

Virulence plasmids

Cause disease signs and symptoms

Protoplast Fusion (one in five fused cells recombine)

Cell wall removal Nocardia Aspergillus Ustilago maydis Yeasts

Genetic Fusion

Changes in control of genes Study the expression of genes Cloning the positive regulator of an enzyme gene

Transformation History

Fredrick Griffith (1920s) Killed versus un-killed strains "Transforming principle"

Recombinant DNA technology

Gene cloning Restriction Endonucleases DNA ligase Selection systems

Gene Amplification

High copy number plasmids -Antibiotic production -Amino acid/ protein overproduction -Overproduction of a regulator

Conjugation (Hfr)

High frequency of recombination Large sections (or all) of the chromosome transferred

In most eukaryotes, it is an essential part of the organism's life cycle and usually occurs by sexual reproduction.

Male and female parents produce gametes (sex cells), which unite to form a zygote, the first cell of a new individual. Because each parent produces many genetically different gametes, many different combinations of genetic material can be transferred to offspring.

Transformation

A change in an organism's characteristics due to uptake of naked DNA

Gene transfer for Eukaryotes

Vertical; Sexual reproduction; gamete diversity

The discovery of transformation: Griffith's experiment with pneumococcal infections in mice

When S-type pneumococci (which produce smooth-appearing colonies, due to the presence of capsules) are injected into mice, the mice die of pneumonia. The mice survive when R-type pneumococci (which produce rough-appearing colonies, due to the lack of capsules) or heat-killed S-type pneumococci are injected. But when a mixture of live R-type and heat-killed S-type pneumococci- neither of which is lethal by itself- is injected, the mice die, and live S-type organisms as well as R-types are recovered from the dead animals.

Bacteriophage (a virus that infects and replicates within a bacteria) life cycles

When a bacteriophage injects its viral DNA into a host bacterial cell, at least 2 diff. outcomes are possible. In the lytic cycle, characteristic of virulent phages, the phage DNA takes control of the cell and 1) causes it to synthesize new viral components, which are assembled into whole viral particles. The cell lyses, releasing the infective viruses, which can then enter new host cells. In the lysogenic cycle, the DNA of a temperate phage enters the host cell. 2)becomes incorporated into the bacterial chromosomes as a prophage, and replicated along with the chromosome through many cell divisions. However, a lysogenic phage can suddenly revert to the lytic life cycle. A prophage is thus a sort of "time bomb" siting inside the infected cell.

Horizontal (lateral) gene transfer

When bacteria pass genes to other microbes of their same generation.

Vertical gene transfer

When genes pass from parents to offspring

Gene amplification definition

A process by which plasmids, or in some cases bacteriophages, are induced to reproduce within cells at a rapid rate.

In conjugation, like transformation and transduction, genetic info. is transferred from one bacterial cell to another. It differs from the others though in 2 ways:

1) It requires contact between a donor and recipient cell 2) It transfers much larger quantities of DNA (occasionally whole chromosomes)

The mechanism of bacterial transformation

1. Naked DNA fragments from disintegrated cells in the area of a potential recipient cell. This cell must be of the correct genus and be in a state of competence, a proper physiologic condition, to permit entry of the DNA fragments. 2. Entry of naked DNA into competent cell 3. Recombination. Some DNA fragments replace (recombine with) original host cell DNA. the resultant recombinant cell is said to have been genetically transformed and will now express the foreign genes it has received and pass them on to all its offspring. DNA that has not recombined is broken down by enzymes.

Safety Issues

1974 biological containment: -Only using organisms with mutations when undertaking recombinant DNA studies so the organisms cannot survive outside of the laboratory. Relaxed in 1981 since: -No illnesses could be traced to recombinants -The strains of E. coli used could not infect humans or primates -Incorporation of mammalian genes into E. coli was observed in nature to no ill effect -Biosafety practices were and are in place

Genetic fusion definition

Allows transposition of genes from one location on a chromosome to another. It can also involve deletion of a DNA segment, resulting in the coupling of portions of two operons. A possible example in which the deletion of a part of a chromosome causes two different adjacent operons to be joined together. The control mechanisms of the first operon will now govern the expression of the genes that were originally part of the second operon.

A Protoplast

An organism with its cell wall removed

Conjugation

Bacteria mating via F pili 1946 Lederberg--got lucky in choice of strains

Generalized transduction

Bacteriophage infection of a host bacterium initiates the lytic cycle. The bacterial chromosome is broken into many fragments, any of which can be picked up and packaged along with phage DNA into new phage particles. When those particles are released and infect another bacterial cell, the new host acquires the genes that were brought along (transduced) from the previous bacterial host cell.

Benefits of protoplast fusion

By mixing 2 strains, each of which has a desirable characteristic, new strains that have both characteristics can be produced.

PCR cloning

Can be used if flanking DNA sequences are known Faster than traditional cloning May still involve the use of plasmids for maintaining cloned sequences

Ti (tumor inducing) plasmid

Can cause tumor formation in plants

Recombinant DNA definition

DNA that contains information from two different species of organisms

Bacteriocin plasmids

Direct the synthesis of bactericidal (bacteria-killing) proteins

F (fertility) plasmid

Direct the synthesis of proteins that self assemble into conjugation pili

1946 Lederberg

Discovered Conjugation. He used mutated strains of E. coli that were unable to synthesize certain nutrients (substances). He grew them in a nutrient-rich medium. He removed cells from each culture and washed them to remove the residue of the nutrient medium and then attempted to culture cells of each strain on agar plates that lacked the special nutrients needed by the strain. He also mixed cells from the two strains and plated them on the same medium. Whereas cells from the original cultures failed to grow, some from the mixed cultures did grow. The latter must have acquired the ability to synthesize all the substances they needed.

Fredrick Griffith (1920s)

Discovered bacterial transformation (a change in an organism's characteristics because of the transfer of genetic info), while he was studying pneumococcal infections in mice.

Gene transfer for Prokaryotes

Horizontal (lateral); Donor/recipient; recombination events

Sexual reproduction of plants and animals is what we usually think of as vertical gene transfer.

In contrast, bacteria do vertical gene transfer when they reproduce asexually by binary fission. However, bacteria can also do horizontal, or lateral, gene transfer.

Transduction (specialized)

In this process, phage DNA always inserts itself into the bacterial host chromosome at a particular site. When the phage DNA replicates, it takes bacterial genes from either side of the site and packages them with its own DNA into new phages. Only genes adjacent to the insertion site, not genes from other parts of the host chromosome, are transducer. These genes can then be introduced into the phage's next host cell, where they will confer new genetic traits.

Recombinant DNA applications

Medical/ Pharmaceutical Industrial Agricultural

Transposons

Mobile genetic elements Barbara McClintock

Temperate phage

Ordinarily doesn't cause a disruptive infection. Instead, the phage DNA is incorporated into a bacterium's DNA and is replicated with it. This phage also produces a repressor substance that prevents the destruction of bacterial DNA< and the phage's DNA doesn't direct the synthesis of phage particles. Phage DNA that is incorporated into the host bacterium's DNA is called a prophage. Persistence of a prophage without phage replication and destruction of the bacterial cell is called lysogeny, and cells containing a prophage are said to be lysogenic. Several ways to induce such cells into the lytic cycle are known, and most involve inactivation of the repressor substance.

Gene transfer

Refers to the movement of genetic information between organisms

Lederberg was lucky in choice of strains

Similar studies of other strains of E. coli failed to demonstrate conjugation. In addition to the mutations that led to synthetic deficiencies in Lederberg's organisms, he also happened to use two E. coli cell types that were capable of conjugation.

Novel catabolism plasmids

Some plasmids contain genes for catabolic enzymes. Generally, plasmids carry genes that code for functions not essential for cell growth; the chromosome carries the genes that code for essential functions.

Phages

The properties of bacteriophages. Composed of a core of nucleic acid covered by a protein coat. They infect bacterial cells (hosts) and reproduce within them. A phage capable of infecting a bacterium attaches to a receptor site on the cell wall. The phage nucleic acid enters the bacterial cell after a phage enzyme weakens the cell wall. The protein coat remains outside, attached to the cell wall. Once the nucleuc acid is in the cell further events follow one of two pathways, depending on whether the phage is virulent or temperate.

Transduction

Transfer of DNA via bacteriophage- a virus that can infect bacteria. Must understand the life cycles of bacteriophage