Plant Genetic Transformation

The study material for plant transformation techniques along with the techniques for characterization of transgenic lines can be accessed below:

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Online Interactive Lecture Series on Techniques in Plant Molecular Biology: Part V

Lecture V

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Watch Lecture V on Plant Transformation Methods on Youtube

Virtual DNA isolation experiment from the University of Utah

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Online Interactive Lecture Series on Techniques in Plant Molecular Biology: Part IV

Lecture IV

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Watch Lecture IV on Practical Aspects of Gene Cloning on Youtube

Online Interactive Lecture Series on Techniques in Plant Molecular Biology: Part III

Lecture III

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Watch Lecture III on Different Types of Cloning Vectors in Gene Cloning on Youtube

Online Interactive Lecture Series on Techniques in Plant Molecular Biology: Part II

Lecture II

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Watch Lecture II on DNA Modifying Enzymes in Gene Cloning on Youtube

Models of DNA Replication

To access and download PowerPoint presentation on 'DNA replication' click on the link below:
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Theta (θ) replication

•    The bidirectional replication of a circular chromosome in prokaryotes occurs through θ mode of replication.

•      A θ structure is an intermediate structure formed during the replication of a circular DNA molecule.

•      Two replication forks can proceed independently around the DNA ring and when viewed from above the structure resembles the Greek letter "theta" (θ).

•      Semiconservative mode of DNA replication in bacterial chromosome begins at the origin of replication called oriC.

•      DNA polymerases then move bi-directionally from origin to the termination site of DNA replication abbreviated as ter.

•    This results in the two replication forks moving in opposite directions around a circular chromosome.

•    The bidirectional replication forks move at identical speed after initiation, therefore, both replication forks meet at the termination site.

•      As a result, the two synthesized circular DNA molecules remain interlocked with each other.

•      The process ends with the topological unlinking of the two replicated DNA molecules by the action of topoisomerases.

•     Originally discovered by John Cairns, this model led to the understanding that bidirectional DNA replication.

•     The replicating cells were exposed to a pulse of tritiated thymidine, quenched rapidly and then autoradiographed.

•   Results showed that the radioactive thymidine was incorporated into both forks of the theta structure, not just one, indicating synthesis at both forks in opposite directions around the loop.

Theta model of replication

Experimental evidence supporting the theta model

Rolling circle replication

•    For some virus chromosomes, such as that of bacteriophage, a circular, double-stranded DNA replicates to produce linear DNA; the process is called rolling circle replication.

Steps:

•  The first step is the generation of a specific nick in one of the two strands at the origin of replication.

•     The end of the nicked strand is then displaced from the circular molecule to create a replication fork.

•      The free end of the nicked strand acts a primer for DNA polymerase to synthesize new DNA, using the single-stranded segment of the circular DNA as a template.

•   The displaced single strand of DNA rolls out as a free “tongue” of increasing length as replication proceeds.

•    New DNA is synthesized by DNA polymerase on the displaced DNA in the -to- direction, meaning from the circle out toward the end of the displaced DNA.

•      With further displacement, new DNA is synthesized again, beginning at the circle and moving outward along the displaced DNA strand.

•    Thus, synthesis on this strand is discontinuous because the displaced strand is the lagging-strand template.

•      As the single-stranded DNA tongue rolls out, new DNA synthesis proceeds continuously on the circular DNA template.

•   Because the parental DNA circle can continue to “roll,” a linear double-stranded DNA molecule can be produced that is longer than the circumference of the circle.

•      Example: Replication of λ phage DNA.

Model for rolling circle replication

Rolling circle replication of λ phage DNA

•   Phage genome has a linear, mostly double-stranded DNA with 12-nucleotide-long, single-stranded ends.

•      The two ends have complementary sequences—they are referred to as “sticky” ends and can base pair with one another.

•   When phage infects E. coli, the linear chromosome is injected into the cell and the complementary ends pair.

•      To produce copies of the chromosome to package in progeny phages, the now-circular phage chromosome replicates by the rolling circle mechanism.

•      The result is a multi-genome-length “tongue” of head-to-tail copies of the chromosome.

•      A DNA molecule like this, made up of repeated chromosome copies, is called a concatamer.

•      The phage chromosome has a gene called ter (terminus-generating activity), which codes for a DNA endonuclease.

•    The endonuclease binds to the cos sequence and makes a staggered cut such that linear chromosomes with the correct complementary, 12-base-long, single-stranded ends are produced.

•      The chromosomes are then packaged into the progeny phages. 

Replication of linear double-stranded DNA in eukaryotes

•      Each eukaryotic chromosome consists of one linear DNA double helix.

•   Eukaryotic chromosomes replicate efficiently and quickly because DNA replication is initiated at many origins of replication throughout the genome.

•      Eventually, each replication fork runs into an adjacent replication fork, initiated at an adjacent origin of replication.

•    The stretch of DNA from the origin of replication to the two termini of replication (where adjacent replication forks fuse) on each side of the origin is called a replicon or replication unit.

•      [The E. coli genome consists of one replicon.]

Initiation of replication:

•  In the Saccharomyces cerevisiae, replicators are approximately 100-bp sequences called autonomously replicating sequences (ARSs).

•      Replicators of more complex, multicellular organisms are less well characterized.

•      The initiator protein in eukaryotes is the multisubunit origin recognition complex (ORC).

•   The ORC binds to two different regions at one end of the replicator and recruits other replication proteins (auxiliary proteins).

•      The origin of replication is between the binding sites of ORC and other replication proteins.

Restricting replication once per cell cycle

•      DNA replication takes place during S phase of cell cycle.

•      For correct duplication of the chromosomes, each origin of replication must be used only once in the cell cycle.

•      This is accomplished by two steps.

•      The first step is replicator selection, where ORC binds to each replicator and recruits other proteins to form pre-replicative complexes (pre-RCs).

•      The pre-RCs are activated only when the cell progresses from G1 to S, the unwinding of DNA starts and then they initiate replication.

•    Limiting replication initiation to the S stage is controlled by licensing factors (MCM helicase) that are synthesized in G1 phase.

•      After one cycle of replication begins in the S phase, the licensing factors are degraded and thus, the second cycle of replication cannot initiate.

Eukaryotic replication enzymes

•      Eukaryotic cells may have 15 or more DNA polymerases.

•      Typically, replication of nuclear DNA requires three of these:

–     Pol α/primase: initiates new strands in replication by primase, making about 10 nucleotides of an RNA primer; then Pol α adds 10–20 nucleotides of DNA.

–     Pol δ:Pol δ synthesizes the lagging strand.

–     Pol ε: Pol ε appears to synthesize the leading strand

•   Other eukaryotic DNA polymerases are involved in specific DNA repair processes and yet others replicate mitochondrial and chloroplast DNA.

•   Primer removal does not involve the progressive removal of nucleotides, as is the case in prokaryotes. Rather, Pol δ continues extension of the newer Okazaki fragment; this activity displaces the RNA/DNA ahead of the enzyme, producing a flap. Nucleases remove the flap. The two Okazaki fragments are then joined by the eukaryotic DNA ligase.

Replication of 5’ ends of linear chromosomes

•      Because DNA polymerases can synthesize new DNA only by extending a primer, there are special problems in replicating the ends—the telomeres—of eukaryotic chromosomes.

•      Replication of a parental chromosome produces two new DNA molecules, each of which has an RNA primer at the end of the newly synthesized strand in the telomere region.

•   Removal of these RNA primers leaves a single-stranded stretch of parental DNA—an overhang—extending beyond the end of each new strand.

•      DNA polymerase cannot fill in the overhang.

•      If nothing were done about these overhangs, the chromosomes would get shorter and shorter with each replication cycle.

•      Most eukaryotic chromosomes have species-specific, tandemly repeated, simple sequences at their telomeres.

•      Elizabeth Blackburn and Carol W. Greider have shown that an enzyme called telomerase maintains chromosome lengths by adding telomere repeats to one strand (the one with the end), which serves as a template on previous DNA replication at each end of a linear chromosome.

•      The complementary strand to the one synthesized by the telomerase must be added by the regular replication machinery.

•       The repeated sequence in humans and all other vertebrates is 5’–TTAGGG–3’.

•      Telomerase is an enzyme made up of both protein and RNA.

•   The RNA component includes an 11-base template RNA sequence that is used for the synthesis of new telomere repeat DNA.

•    The telomerase binds specifically to the overhanging telomere repeat on the strand of the chromosome with the 3’ end .

•       The end of the RNA template sequence in the telomerase—here, 3’-CAAUC-5’— base-pairs with the 5’-GTTAG-3’ sequence at the end of the overhanging DNA strand.

•      Next, the telomerase catalyzes the addition of new nucleotides to the end of the DNA using the telomerase RNA as a template.

•    The telomerase then slides to the new end of the chromosome so that the end of the RNA template sequence—3’-CAAUC-5’—now pairs with some of the newly synthesized DNA.

•      Then, as before, telomerase synthesizes telomere DNA, extending the overhang.

•    If the telomerase leaves the DNA now, the chromosome will have been lengthened by two telomere repeats.

•      But, the process can recur to add more telomere repeats. 

•      Telomere length is regulated to an average length for every organism and cell type.

•    Mutants with a deletion in the TLC1 gene (encodes the telomerase RNA) or mutation in the EST1 or EST3 (ever shorter telomeres) genes cause telomeres to shorten continuously until the cells die.

•    This phenotype provides evidence that telomerase activity is necessary for long-term cell viability.

•    Mutations of the TEL1 and TEL2 genes cause cells to maintain their telomeres at a new, shorter-than-wild-type length, making it clear that telomere length is regulated genetically.