S280 Science Matters
In prokaryotes the genetic material, DNA, is not bound up with proteins to form eukaryote-like chromosomes. The DNA is essentiall 'naked'.
In E.coli the chromosome is a closed circle of naked DNA double helix. In addition to this sinlge chromosome, E. coli can contain one or more small circles of DNA called plasmids.
In general only one type of plasmid can exist inside a single bacterium.
Plasmids can serve as carriers, vectors, of foreign DNA into bacteria.
Typically, as in E. coli, transferring foreign DNA (genes) into bacteria involves a number of key steps: cutting the foreign DNA into gene-size pieces; inserting and splicing the foreign DNA fragments into opened plasmid circles to form recombitant plasmids; uptake of the recombitant plasmids by bacteria that have no plasmids.
If the protein product of the foreign gene is desired, then the foreign gene once transferred into bacteria must also be expressed.
Cutting, cleaving, the foreign DNA into gen-size fragments can be achieved using restriction enzymes. Each type of restriction enzyme has a specific target sequence in DNA that it can cleave.
Restriction enzymes do not cleave DNA from their own source (ie the same bacteria). Bases in potential target sequences in such DNA have been modified, rendering these sequences resistant to the corresponding restriction enzyme.
Restriction enzymes that have a single target sequence in plasmids can be used to open, to linearize, the plasmid circles.
Inserting foreign DNA fragments into opened plasmid circles can be achieved in several ways.
Once inserted in an opened plasmid circle, foreign DNA can be sealed in using lignase to create a recombinant plasmid.
Recombinant plasmids can be taken up by bacteria in the presence of calcium ions (Ca2+).
The overall process of gene transfer into bacteria is known variously as recombinant DNA (rDNA) technology, or gene splicing or gene cloning. Sometimes it is known simply as generic engineering but this also has a wider meaning.
To obtain the polypeptide (protein) product of a foreign gene in bacteria that gene must be expressed.
Gen expression involves two states: transcription to produce a messenger RNA (mRNA); and translation, the reading (decoding) of the mRNA to produce polypeptide. Polypeptide then folds up to yeild the ultimate protein product.
Transcription depends on having an appropriate promoter, one compatible with the RNA polymerase in the host cell. The promoter is the region in the DNA adjacent to a structural gene where RNA polymerase binds prior to transcribing the structural gene.
Most eukaryote genes are split genes. Split genes contain coding regions of DNA, exons, interspersed with non-coding regions, introns. These genes cannot be expressed in bacteria.
In eukaryotes, mechanisms exist that 'cut and splice' the primary mRNA transcripts from split genes. Thsi post-transcriptional modification gives mature functional mRNA that passes into the cytoplasm where it is translated to give polypeptide. Split genes do not occur in porkaryotes (except extremely rarely) and so neither does the machinery for such post-transcriptional modification.
The intron problem can be by-passed by tranferring cDNAs instead of gen-size pieces of foreign DNA.
A cDNA is a complementary DNA made by using a mature cytoplasmic mRNA as a template. The first step, utilizing the enzyme reverse transcriptase, produces a single-stranded DNA complementary in sequence to the mRNA template. Thsi single-stranded CNA is converted to a double-stranded cDNA using another enzyme, DNA polymerase.
A mixture of mRNAs extracted from teh cytoplasm of a suitable foreign cell can be used to produce a mixture of cDNAs. This mixture is then used as the 'source of genes', individual cDNAs being spliced into plasmids to create recombinant plasmids. The recombinant plasmids are then taken up into E. coli cells which are then grown to produce individual clones.
To get expression of cloned cDNAs we must provide a compatible promoter adjacent to the cDNA. This can be done using an expression vector. An expression vector can be a plasmid that contains a promoter compatible with the RNA polymerase of the host species.
Techniques whereby gene-size pieces of foreign DNA (generated by restriction enzymes) or mixtures of cDNAs are cloned are known as shotgun techniques. They result in the foramtion of gene or cDNA libraries. The problem is then to find the right 'book', the clone with the desired foreign gene of cDNA. This is done by screening individual clones. We can calculate how many clones will probably need to be screened.
Individual clones can be grown by separating individual cells by spreading them onto the surface of nutrient jelly; a technique called plating. Each cell grows and divides where it sits to produce a clone of its descendants visible as a mound or colony.
If the transferred foreign gene is expressed, yeilding a protein product, the colonies containing this gene can be located by screening for the product, the protein.
If the protein prouct is an enzyme it may be conventiant to detect it by using a suitable specific enzyme assay. Where no such suitable assay exists, or the protein is not an enzyme, it may be detected by using a specific antibody prepared against that protein.
Alternatively, the gene itself can be detected directly by means of a gene probe. This is particularly useful where the gene is not expressed. A gene probe is a polynucleotide complementary in base sequence to (one or other of the two strands of) the gene sought. The gene probe binds specifically to DNA extracted from the clone containing the desired gene.
Clones can be probed en masse by making a replica of the oclonies on nitrocellulose and exposing the DNA in situ giving single-stranded DNA prints.
Several techniques exist for producing probes. For example, mRNA (or its cDNA equivalent), short 'synthetic mRNAs' (ie orgonucleotides) or related genes can all be used as gene probes.
In some cases the shotgun approach can be avoided by chemically producing a synthetic gene and transferring this into bacteria. To chemically synthesize a gene we must know its base sequence, something possible if the gene has already been isolated and its base sequence analysed. Or if the amino acid sequence of its polypeptide product is known we can deduce the sequence of the gene or an equivalent one.
Somatostatin was an early example of a foreign peptide produced in bacteria by transfer of a synthetic gene. The initial polypeptide produced was a hybrid, somatostatin being released by treatment of this with cyanogen bromide.
E. coli is not the only host for foreign genes, nor are plasmids the only vectors.
Bacteriophage λ has been much used as a vector for E.coli. . It can tolerate larger prices of spliced-in foreign DNA. It can be used to clone foreign genes in the phage itself - i.e. constructing gene or cDNA libraries in complete, packaged, phage.
Other microbial hosts may be needed because: they are better understood for industrial use; they present no safety hazards such as might the cell wall toxin of E. coli; they can secrete certain proteins; eukaryote microbes, such as yeast, may carry out some post-translational modification of polypeptides, such as attaching sugars to amino acid R groups.
Animal and plant cells can be grown as single cells in tissue culture. However the technique is tricky and relatively expensive as compared to growing bacteria or yeast.
Tissue culture cells can provide a source of animal and plant host cells. These may carry out post-translational modification needed to get some eukaryote protein products in biologically active form.
A variety of vectors have been developed for host cells other than E. coli, both for other projaryote cells and eukaryote ones too. Some vectors are based on plasmids, others on viruses. Some virus-derived vectors also behave like plasmids, co-existing with but replicating independently of the host cell genome. Some cells can take up DNA without vectors.
In many host cells the introduced foreign DNA ends up spliced permanently into the host cell's chromosomal DNA where it can replicate and be passed on generation after generation of cell.
Suitable promoters can be found to get expression of foreign gnes in a variety of host cells.
Whatever host cells vectors are to be used for, it is often convenient to carry out the manipulation of such vectors in E. coli. Some vectors, called shuttle vectors, can replicate in both E. coli and another host cell. These allow manipulation and production of the vector (plus foreign genes) to be done in E. coli, followed by transfer of the genes, via the vector, to the other host cell.
Producing whole genetically engineered multicellular animals and plants has some technical problems different from engineering single cells: all the cells in the organism must receive the foreign gene, it must be passed on from cell to cell at mitosis; the gene must be passed on to other generations of organisms at sexual reproduction; the gene must be expressed in some tissues and not others.
Genetic engineering of plants depends often on the ability to regenerate whole plants from single cells or tissue emplants.
Plant regeneration often proceeds via an undifferentiated mass of cells called a callus.
Plantlets can be produced from a callus by altering hormone levels appropriately.
Agrobacterium tumefaciens, the cause of crown gall disease, is the source of important vectors.
The tumour (gall)-inducing capacity of agrobacterium tumefaciens is by virtue of a plasmid called Ti plasmid. A region of the plasmid DNA, the T-DNA or T-region is inserted into the plant cell chromosomal DNA during natural infection.
Vectors that have lost their tumour-inducing capacity can be derived from the Ti plasmid. Foreign genes inserted in the T-DNA of such vectors can be carried into plant cells and then integrate into the host cell DNA.
Foreign genes can be expressed in plant cells by providing suitable attached promoters. Tissue-specific expression can be achieved by also including tissue-specific regulatory (DNA) sequences adjacent to the structural gene.
A combination of regeneration techniques and A. tumefaciens-mediated gene transfer can work well in dicots (eg tobacco, potato, etc.) but not generally in monocots (eg cereals).
Protoplasts, plant cells stripped of their cell walls, can take up DNA, and this is true for monocots as well as dicots. But some monocots, cereals in particular, are hard to regenerate from protoplasts; some can be regenerated, through (eg rice).
Biolistics, the shooting of DNA carried on tiny particles, is another way of getting foreign DNA into plants.
Whole animals cannot be regenerated from tissue explants nor from single cells.
Injecting DNA into a pronucleus of a newly fertilized egg is a way of introducing foreign genes into animals. The animal derived from such an egg contains the foreign gene in its chromosomal DNA. Given a suitable promoter and regulatory DNA sequences, the foreign genes can be expressed in a tissue-specific manner. They can also be passed on to offspring by sexual reproduction.
Such techniques were pioneered in experiments transferring genes into mice, notably genes for rat or human growth hormone. The expression of such genes was manifest dramatically by greater growth of animals derived from treated eggs.
Also, chimeras and cloning from the supplement.
Genetic engineering is likely to make a big impact on the pharmaceutical industry.
Drug production is laready heavily laboratory-based, so the adoption of genetic engineering techniques does not represent the major hurdle that itmight to other industries.
Diagnostics can exploit molecular markers of disease. Genetic engineering can call upon the high specificity of biological recognition to detect such markers.
Protein markers can be recognized by antibiotics. Monoclona antibodies, produced by single clones of spleen cells, are particularly useful for this.
Base sequences characteristic of desease can be recognized by gene probes. These sequences can be from pathogens, or can be mutant human genes.
Prenatal diagnosis may be possible for some diseases.
Cheap, accurate diagnostics raise the possibility of widespread screening, not only for deseases, but also for susceptibility to diseases. This may raise ethical problems.
The Human Genome Project is under way to identify the complete base sequence of the human genome (see supplement). This may increase our understanding of predisposition to various diseases.
Vaccination can prevent individuals from falling prey to specific diseases. Genetic engineering techniques can improve the effectiveness of vaccination by allowing the production of new and better vaccines.
Genetic engineering has revolutionized drug production. The first drug to be made by rDNA techniques was human insulin.
Genetic engineering is particularly suited to obtaining natural substances, normally produced in very small amounts, which can help the body to fight disease using its own, natural, chemicals. The prime example of this is interferon..
Mini-antibodies (single-domain antibodies) can be used for therapy as well as for diagnosis. They can be humanized so that they are more effective in the patient's body.
Genetic engineering promises completely new approaches to therapy, correcting the effects of faulty genes by isnerting normal, undamaged genes into the patient's cells.
A whole range of new approaches to disease control is being applied to cystic fibrosis. This ranges from diagnosis using gene probes to proposed gene therapy using the CFTR gene.
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