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Organization and Mutagenesis of Cloned Genes

Organization of cloned genes

cDNA clones have a defined organization, especially those synthesized using oligo(dT) as primer. Usually a run of A residues is present at one end of the clone which defines its 3' end, and at some variable distance upstream of this there will be an open reading frame(ORF) ending in a stop codon. As genomic clones from eukaryotes are larger, and may contain intron sequences, as well as nontranscribed sequences, they provide a great challenge to understand their organization. The genomic sequences absent in the cDNA clones are usually introns as well as sequences upstream of the transcription start site and downstream of the 3' processing site.

 

S1 nuclease mapping

S1 nuclease mapping determines the precise 5'- and 3'- ends of RNA transcripts. For 5' end mapping, an end-labeled antisense DNA molecule is hybridized to the RNA preparation (Figure-1). The hybrids are then treated with the single strand specific S1 nuclease, which will remove the single strand protrusions at each end. The remaining material is analyzed by polyacrylamide gel electrophoresis next to size markers or a sequencing ladder. The size of the nuclease- resistant band, usually revealed by autoradiography, allows the end of the RNA molecule to be deduced.

 

Figure 1: S1 nuclease mapping the 5'- end of an RNA
Figure 1: S1 nuclease mapping the 5'- end of an RNA

 

Primer extension

Primer extension is a technique whereby the 5' ends of RNA or DNA can be mapped. Primer extension can be used to determine the start site of RNA transcription for a known gene. This technique requires a radiolabelled primer (usually 20 - 50 nucleotides in length) which is complementary to a region near the 5' end of the gene (Figure-2). The primer is allowed to anneal to the RNA and reverse transcriptase is used to synthesize cDNA to RNA until it reaches the 5' end of the RNA. By running the product on a polyacrylamide gel, it is possible to determine the transcriptional start site, as the length of the sequence on the gel represents the distance from the start site to the radiolabelled primer.

 

Figure 2: Primer extension
Figure 2: Primer extension

 

Gel retardation

The technique of gel retardation shows the effect of protein binding to a labeled nucleic acid and can be used to detect transcription factors binding to regulatory sequences (Figure-3). A short labeled nucleic acid, such as the region of a genomic clone upstream of the transcription start site, is mixed with a cell or nuclear extract expected to contain the binding protein. Samples of labeled nucleic acid, with and without extract, are run on a non-denaturing gel (agarose or polyacrylamide) . If a large excess of nonlabeled nucleic acid of different sequence is also present, which will bind proteins that interact nonspecifically, then the specific binding of a factor to the labeled molecule to form one or more DNA -protein complexes is shown by the presence of slowly migrating (retarded) bands on the gel by autoradiography.

 

Figure 3: An experiment of gel retardation
Figure 3: An experiment of gel retardation

 

DNase I footprinting

Gel retardation shows that a protein is binding to a DNA molecule but it doesn't provide the sequence of the binding site which could be anywhere in the fragment used. DNase footprinting shows the actual region of sequence with which the protein interacts. An end labeled DNA fragment is required which is mixed with the protein preparation. After binding, the complex is very gently digested with DNaseI to produce on average one cleavage per molecule. In the region of protein binding, the nuclease can't easily gain access to the DNA backbone, and few cuts take place there (Figure-4). When the partially digested DNA is analyzed by PAGE, a ladder of bands is seen showing all the random nuclease cleavage positions in control DNA. In the lane where protein was added, the ladder will have a gap, or region of reduced cleavage, corresponding to the protein-binding site where the protein has protected the DNA from nuclease digestion.

 

Figure 4: DNase I footprinting
Figure 4: DNase I footprinting

 

Mutagenesis of cloned genes

Deletion mutagenesis 

Progressively deleting DNA from one end is very useful for defining the importance of particular sequences. In cDNA  clones, deletion from the ends of the coding region produces either N-terminally or C-terminally truncated proteins. The N-terminal domain of a given protein could be a DNA binding domain, the central region an ATP binding site and the C-terminal region could help the protein to interact to form dimers. In genomic clones, after the transcription start site has been identified, sequences upstream are removed progressively to discover the minimum length of upstream sequence that has promoter and regulatory function. Unidirectional deletions can be created using exonuclease III which removes one strand in a 3' to 5' direction from a recessed 3' end. A single strand-specific nuclease then creates blunt end molecules for ligation, and transformation generates the deleted clones.

 

Site-directed mutagenesis

In site-directed mutagenesis a mutation is created at a definite site of a DNA molecule. The basic procedure requires the synthesis of a short DNA primer which is complementary to the template DNA around the site where the mutation is to be introduced. The mutation may be a single base change (a point mutation), deletion or insertion, containing the desired mutation such as a base change. This synthetic primer is complementary to the template DNA around the base change so it can hybridize with the DNA containing the gene of interest. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected.

       Site-directed mutagenesis has been widely used in the study of protein functions. There are many approaches. An oligonucleotide based method was first developed by Michael Smith who was awarded a Nobel Prize for this contribution. This method involves cloning the DNA of interst into a plasmid vector. First the plasmid vector is denatured  to produce single strands. Secondly, a synthetic oligonucleotide with desired mutation is annealed to the target region. In figure, the example shows T to G point mutation. Then the mutant oligonucleotide is extended using a plsmid DNA strand as the template. Then the heteroduplex is propagated by transformation in E.coli. In theoretically, after propagation,  about 50% of the produced heteroduplexes will be mutants and the other 50% will be the "wild type" (without mutation).

       The original method using single-primer extension was inefficient due to a lower yield of mutants. The resulting mixture may contain both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of mismatch repair system which favours the methylated template DNA. Many approaches have since been developed to improve the efficiency of mutagenesis.

 

PCR site-directed mutagenesis

PCR can be used to make changes to the nucleotide sequence of DNA, this is called PCR Mutagenesis. This method can be utilized to assess the function of a promoter, and to alter amino acids to test the functions of domains in a protein. Mutations can be introduced in PCR by several methods. A pair of primers is designed that have altered sequence and which overlap by at least 20 nt. In a standard vector pGEM, SP6 and T7 are two standard primer-binding sites which can be used in combination with the mutagenic primers. Two separate PCR reactions are performed, one amplifying the 5' portion of the insert using SP6 and the reverse primer, and the other amplifying the 3' portion of the insert using the forward and T7 primers. If the two PCR products are purified, mixed and amplified using SP6 and T7 primers, then a full-length, mutated molecule is the only product that should be made. This method is nearly 100% efficient and very quick. An alternative and more convenient version involves using the forward and reverse mutagenic primers to extend all the way round the plasmid containing the target gene (Figure-5).

 

Figure 5: PCR mutagenesis
Figure 5: PCR mutagenesis

       

       At the end of the reaction, some of the product will consist of circular molecules containing the mutation in both strands and nicks at either end of the primer sequence. These molecules can be transformed directly to give clones containing mutant plasmid, without the need for restriction digests and ligation. All mutants need to be checked by sequencing before use, instead of using the whole of the PCR -generated DNA, a smaller fragment containing the mutated region could be subcloned into the equivalent sites in the original clone.

 

 

References

1. Brown, T.A. (1998).Studying gene expression. Gene cloning an introduction, 3rd Edn. Stanley Thornes (Publishers) Ltd.

2. Turner Phil, McLennan Alexander, Bates Andy and White Mike (2005).Organization of cloned genes, Mutagenesis of cloned genes. Instant notes(173-179), Molecular Biology. Taylor& Francis Group.

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