Following excision, a conjugative transposon can follow different courses:. This ability to transfer themselves and other genes not only to closely related but also to genetically remote strains makes conjugative transposons important elements of communication, exchange and adaptation in bacteria.
Their transfer also occurs in nature Salyers et al. A rather intriguing observation is that in some bacteria e. Bacteroides spp certain antibiotics appear to act as stimulants for the conjugative transfer of transposons carrying genes of resistance to antibiotics. No longer is there a risk of not finding a matching homology zone. The choice is not biased towards minor genetic change, important innovations are also possible. This kind of transformation based on the acceptance of small replicons by the mechanisms of competence is called transfection.
The name was formed by combining the prefix from transformation with the suffix from infection, since for some time the small replicons have wrongly been considered to be infectious parasites of bacteria. Even today many biologists still consider the temperate phages merely as viruses, although they are typical self-transmissible small replicons. By contrast there are no natural horizontal gene transfers between eukaryotes other than by sexuality which does not usually cross the species barrier.
Learning from the observations made with bacteria, biologists have been able to realize artificial transformation in eukaryotes, even to perform controlled, selected gene changes with the resulting production of recombinant substances of great commercial and medical value.
These developments in biotechnology and genetic engineering have been inspired, greatly helped and accelerated by the knowledge of basic gene exchange processes in prokaryotes. They always bring along, for the possible benefit of the host cell, a few potentially useful accessory genes as hereditary supplements converting genes and they are the main participants in the horizontal exchange of genes which may benefit prokaryotic cells.
The importance of these essential elements of the prokaryotic world is underestimated by biologists including the majority of microbiologists Wellington and Van Elsar, An important difference between eukaryotes and prokaryotes resides in the latter having access to and benefiting from a very active free market of all their genes, a market mostly maintained by small replicons.
Hence, in contrast with eukaryotes in which the basic self-replicating sub-units are the cells, the prokaryotes also possess a supplement of much smaller autonomous basic entities, the small replicons. These have a profound influence on the dissemination and the timely use of all prokaryotic hereditary properties.
Prokaryotic cells provide them with the facilities allowing the visiting small replicons to function as if they were their own genes. As far as we know, the number of small replicons varies from one to seventeen Fox, in different strains. When a small replicon is no longer useful to its host, being disposable, it is eliminated curing and usually replaced by another temporarily more helpful one. The same strain may thus be successively endowed with several different biochemical abilities depending on the type of combination of its small replicons.
Until recently, many of these differently modified strains were considered to be different species Le Minor, Small replicons and prokaryotic strains are surprisingly polyvalent in their capacities to associate in countless opportunistic combinations, as observed in nature.
This contributes to diversity and to adaptable variations, and constitutes a rich reserve of a large and readily available variety of genes in different habitats: soil, oceans, etc. There is a limit to the number of small replicons that a prokaryotic cell can harbor and the cell may become increasingly vulnerable as their number increases Karska-Wysocki and Sonea, ; Sonea et al.
The small replicons may be eliminated from their host cells by some chemical substances, for example the acridines Damsker and Sonea, ; Dobardzic and Sonea, Small replicons that are very closely related and whose combined presence would not confer any additionnal advantage to the host cell will not generally be kept together in the host cell; one of them will be lost during subsequent cell replications.
It never remains static because not only do small replicons have their own evolution, but even the same type may present itself with many variations by obtaining converting genes from previous hosts. In the absence of such transfer genes they are non self-transmissible NST.
They are formed by two kinds of genes: replicator genes and converting genes Fig. The replicator genes allow them to replicate autonomously in different types of prokaryotic cells. Added to them are a few converting genes, all of them accessory but possibly useful to some of the visited strains.
These genes were very probably picked-up by their NST plasmids on the occasion of visits to former host cells. This could have happened recently or centuries earlier. The NST plasmids do not have active mechanisms to send some of their own copies from one strain to another; they are nonetheless involved in numerous horizontal gene transfers by transfection, when their DNA is liberated by a dying cell close to one receptive and competent prokaryotic cell.
Moreover, NST plasmids are often carried along by ST small replicons when these leave their common host cell for another one. The mechanisms involved are called mobilization when performed by ST plasmids and transduction when temperate bacteriophages the transfer form of the prophages act as carriers.
The frequency of these horizontal exchanges for the genes of NST plasmids is remarkable. This collaboration between the two categories of small replicons added to transformation has raised the importance of gene exchanges in prokaryotes to the level of an efficient free market for genes a global genome. Proportion of the different types of genes according to their general activities in the small replicons.
Symbols representing surface receptors for horizontal gene transfer by self-transmissible ST small replicons between different types of prokaryotic strains. They accept the tip of the tubules pili or tails of the phages necessary for the active transfer of ST small replicons which together perform most of the horizontal transfer of genes between prokaryotic strains. Every strain possesses surface receptors for temperate phages TPh which carry prophages and sometimes other genes from the donor cell.
Only about half of the strains have surface receptors for the tips of pili which participate in the active transfer of self-transmissible plasmids STP and, occasionnally, other genes from the donor cell. The repressor also prevents the multiplication of any other similar small replicon that would subsequently come into the same cell.
This is called immunity for the prophages and exclusion of entry for the ST plasmids. When, for different reasons, the repressor is not rapidly synthesized and the transfer operon is consequently still derepressed when arriving in a new cell, it encodes the synthesis of copies of its ST small replicon independently of the usual division concomitant with that of the large replicon of the host cell.
It also encodes very small proteinic tubes only visible with EM which will help the copies of the ST small replicons reach a receptive cell and penetrate its cell wall at a receptor site specific for the tip of that tube. Such receptors exist at the surface of all prokaryotic cells for receiving different types of ST small replicons Fig. This contact is directly realized by the tubular formation pilus while the transfer operon directs a single newly synthesized strand copied from a ST plasmid into the recipient cell.
The new copy is guided by the pilus which originates in the donor cell and is attracted to one of the many surface receptors for pili presented by the receiving cell Fig. The donor cell, now equipped with one or two pili, functions as a syringe to inject the genes of the ST plasmid and those taken along from the donor cell. The latter process is called conjugation for genes from its large replicon, and mobilization when small replicons are also involved. As already, noted they may be eliminated later curing if they become useless.
The genes originating from the large replicon of the donor cell carried along on such an occasion will, in general, be accepted in the receiving cell, particularly if they find a homology zone on the large replicon of the new host conjugation , as is also the case in transformation. The first small replicons that have been widely accepted by biologiste as carriers of useful genes between different types of prokaryotic cells have been the ST plasmids.
Their involvement in the spreading of genes of resistance to antibiotics used against pathogenic strains which cause serious, contagious diseases was first shown decades ago and it has been confirmed in hundreds of unchallenged scientific publications afterwards. Soil bacteriologiste and ecologists later discovered similar types of ST plasmids which transferred resistance genes against different toxic substances from one bacterial strain to another.
ST plasmids possess a few limitations: they are active mostly in Gram negative strains and they need a very close contact between donor and recipient cells; therefore they are much more successful in very concentrated populations of prokaryotic strains.
These are found in animal digestive tracts, in soil, in rich aquatic sediments, at the surface of still water expanses, etc. As a consequence, their decisive role in gene exchanges among prokaryotes is ignored or underestimated.
The ability of the prophages to carry beneficial hereditary changes is only briefly, if at all, mentioned although it probably plays the most important role in prokaryotic gene transfer. They both need specific receptors at the surface of the bacteria they will visit Fig. Most significant is the fact that there are receptors for prophages at the surface of all known strains. Like ST plasmids, prophages also possess complex transfer operons which are usually repressed, but capable of derepression, often when surrounding conditions are not favorable enough for their host cells.
In this case, copies of these types of small replicons are synthesized independently from the rythmic division of the large replicon chromosome of the host cell. The prophages possess the most complex mechanisms for gene transfer among ST small replicons. Both types of ST small replicons form, however, a family of specialized biological entities with the same general type of results in gene transfer. These will assemble and become small biological syringes, the temperate phages Fig.
Soon afterwards they are liberated by the encoded enzymes, e. Thus, these newly formed prophages with their proteic capsid and tail have become transfer robots, called temperate phages. They are able to reach even far away receptive cells which present appropriate surface receptors, and to subsequently lysogenize them. This means that they may be accepted as stable small replicons with their transfer operon repressed.
In most cases, when the transfer operon is repressed lysogeny Lwoff, and only the converting genes of the prophages are expressed, the activity of the host cell is modified Cleckner et Sonea, In other cases, cells injected with a prophage will be lysed by the activity of its derepressed transfer operon.
For the same reason, some lysogenic cells, following derepression of their transfer operon, produce many temperate phages. The majority of the cells in the population of lysogenic strains have their transfer operons repressed, and only in a few of them do they become derepressed in a well-controlled way. However, when one looks at the prophages as the most complex and efficient of the small replicons, spontaneous induction appears logical and of great biological significance.
This shifting from the lysogenic to the lytic phase happens, on every division, at least in one or a few cells in every thousand. This maintains in a lysogenic strain a constant production of temperate phages by spontaneous induction, accompanied by the liberation of all the other DNA of their former host.
It is a repetitive and abundant offer of genes for other strains. The temperate phages built in this way are carrying the prophages genes and, often, along with them, other types of genes: other small replicons or stable genes from the large replicon of the donor cell transduction.
This constant liberation of genes freed in nature and available for exchanges is one of the most generalized stable mechanisms of gene spreading in astronomical numbers, all ready to enter a different cell. Specialists in marine microbiology have shown several years ago that ocean waters contain surprisingly high concentrations of tailed bacteriophages more than half of which are carrying prophages Bergh et al. Transfer of converting genes by lysogenisation is evidently more frequent than it was believed up to now.
Moreover, transduction, the transfer by a temperate phage of genes other than its own, present in the donor cell can happen in lakes, oceans, rivers, soil and sewage treatment facilities. Work by R. Miller and his group has shown that very high concentrations of bacteriophages can be found in some fresh and marine water samples.
Again, since temperate bacteriophages can transduce DNA to several different strains their role in broadcasting bacterial genes on a high scale and to faraway places is certainly much greater than formerly estimated.
Transduction is also known to occur in soil, plant surfaces, shellfish, etc. Miller, There is no reason to exclude the possibility that it often takes place in almost any bacterial community. When one of these phages reaches a prokaryotic strain containing the useful gene for the lysogenic strain in need and bearing an adequate receptor, it will inject its prophage into one cell which, generally, will start a lytic cycle with the result that numerous copies of its temperate phage are produced and liberated.
Such copies will in turn continue to inject progressively millions of other receptive cells and to produce appropriate quantities of transducing phages carrying the needed gene for the original lysogenic strain.
There is a reasonable possibility that a few such temperate phages, containing their prophages enriched with the needed gene, will return to the original strain and help it to solve its problem.
Experimentally, such a process can easily be performed and proved possible. In the case of ST plasmids, tubules pili remain attached to the donor cell DC and can get in contact, by their other end, with receptors R on the surface of the receiving cells RC. When such a liberated phage approaches the pilus of another cell bearing a compatible receptor it sticks to it and injects its DNA into the receiving cell RC.
Later, when a phage finds cells with appropriate surface receptors, the end of the phage tube tail clings to one of them and its DNA content, usually the prophage, is injected into the receiving cell. When they lyse cells during their synthesis, they also favor to the utmost transformation and transfection in their community.
No System or mechanism equivalent to the prophages exists in the eukaryotic world. It is interesting to recall that another type of DNA phage, the filamentous one, may carry converting genes and establish a kind of lysogenic-like situation fig. For example, a whole genetic region on the large replicon of Vibrio cholerae, the bacterium responsible for the serious contagious disease cholera, codes for different substances including the enterotoxin and the factor directing the phage parts assembly.
This genetic region also called a genetic cassette apparently comes to V. The filamentous phages have a linear DNA molecule containing replicator genes and other genes, for transfer. Their DNA is also a transposon and it has been shown to contain converting genes.
This is a different type of gene exchange. It is based on elements other than those in prophages and plasmids but with a somewhat similar gene transfer activity and a few other characteristics reminescent of ST plasmids and the prophage-temperate phage complex. Together they possess and spread a complete collection of all the genetic information carried by prokaryotic genes, a memory of all its biochemical diversity and of the numerous possibilities for combining all the tested ways to cope with different situations.
A global communication network has emerged from this arrangement, supported by the gigantic genetic information bank on which superior prokaryotic functions are based. All these genetic blueprints represent the entire genetic patrimony of prokaryotes, kept open for use by any strain Sonea, Once we realize this, it becomes less surprising that it took only a few decades for infectious bacteria to acquire resistance genes to dozens of antibiotics, one after another, all around the world.
The latter, as well as the mixed prokaryotic communities may temporarily change their composition when needed. These rearrangements are made within the global prokaryotic entity which acts as the reservoir of all these genes and strains, and thus remains the basic stable element of the prokaryotes from which diversity and change originate and spread.
In this giant pool are distributed numerous genes of each kind randomly in different sites where they are available for use and exchange. Their survival is ensured. In the same way, prokaryotic cells may be accepted in different communities of mixed strains if this can participate in division of labor and contribute to the survival of these communities. It follows that there are astronomical numbers of prokaryotic cells and also of separated genes in our biosphere making up a permanently renewed pool of candidates for advantageously replacing the less successful basic elements in the different regions of the prokaryotic world.
Therefore, one can talk of a giant common genome of prokaryotes, representing the totality of their genetic patrimony, available to cells or communities of mixed strains which might use it profitably. This allows prokaryotes to perform complex functions similar to those of computers Sonea, a, As a result, the blue-prints of hereditary information of the entire prokaryotic world are constantly offered and replenished, supported by the giant reservoir of genes available through the agency of exchange mechanisms.
Even if these mechanisms work more frequently between closely related strains, the astronomical numbers of cells and small replicons present in different places and situations in the biosphere will, in time, provide solutions where they are needed. The selection of the best information is performed at the reception site: the mixed community doing it for cells and the individual cell for the genes.
If a new addition or replacement helps the receiver and improves its functioning, the latter with its new hereditary stock, will multiply abundantly. The best immediate solutions are, in a sense, constantly selected everywhere and, at the global level, this results in exceptional capacities for the prokaryotic superorganism, a powerful half of the living world. Open competition chooses the best solution. They maintain its stability and its life-sustaining qualities. As mentioned previously, the larger concentrations of prokaryotic cells exist as mixed strains in fertile soils, ocean beds, marshes, alimentary tracts of all animals, etc.
These communities are not uniform combinations, they are different from one place to another and even vary in time, and with the season. Their cells are often entangled with eukaryotes which live immersed in the prokaryotic world which fills our biosphere. This crowding by prokaryotic cells favors the collaborating strains and discourage the development of less sociable cells.
Moreover, such high concentrations make the stabilizing action of the prokaryotes on the biosphere continuous and more efficient. This favours successful settlement and survival in favorable niches and results in major contributions to the cycles of organic and inorganic substances, the production of recuperable waste and the maintenance of homeostasis. The stability of our atmosphere, its chemical composition, results in large part from prokaryotic activities and it has been so for at least 1.
Small size is one such feature. It allows the production of enormous numbers of different cells, often billions, in the same neighborhood, increasing the likelihood of exchanges of cells and genes, and cross-feeding.
Also, the speed of chemical reactions and the pace of growth and cellular division all contribute to the development of stable communities. The larger the number of types and cells, the greater the probability of useful exchanges and collaboration. They are highly specialized in their biochemical roles, yet also able to function in associations where division of labor is practiced.
Their cells are equally small, surrounded by a resistant wall, and harbor the two types of replicons which function in the same way as those of eubacteria. We believe that the two categories, archaea and eubacteria, taken together, are part of the global prokaryotic superorganism with a lifestyle entirely different from that of eukaryotes.
Inside, these cells throw together all they need to survive. They let all their cell parts hang out together. Their DNA — the instruction manuals that tell these cells how to build everything they need — just floats around in the cells.
Prokaryotes are masterful survivors. Bacteria and archaea have learned to make meals of everything from sugars and sulfur, to gasoline and iron. They can get energy from sunlight or the chemicals spewed from deep-sea vents.
Archaea in particular love extreme environments. They can be found in high-salt springs, rock crystals in caves or the acidic stomachs of other organisms. That means that prokaryotes are found on and in most places on Earth — including within our own bodies. Eukaryotes are the third domain of life. Animals, plants and fungi all fall under this umbrella, along with many other single-celled organisms, such as yeast.
Prokaryotes might be able to eat almost anything, but these eukaryotes have other advantages. These cells keep themselves tidy and organized. Eukaryotes tightly fold and pack their DNA into a nucleus — a pouch inside each cell. The cells have other pouches, too, called organelles. These neatly manage other cell functions.
For example, one organelle is in charge of protein-making. Another disposes of trash. In most cases, bacteria must be made artificially competent in the laboratory by increasing the permeability of the cell membrane. This can be achieved through chemical treatments that neutralize charges on the cell membrane or by exposing the bacteria to an electric field that creates microscopic pores in the cell membrane. These methods yield chemically competent or electrocompetent bacteria, respectively.
Following the transformation protocol, bacterial cells are plated onto an antibiotic-containing medium to inhibit the growth of the many host cells that were not transformed by the plasmid conferring antibiotic resistance. A technique called blue-white screening is then used for lacZ -encoding plasmid vectors such as pUC Blue colonies have a functional beta-galactosidase enzyme because the lacZ gene is uninterrupted, with no foreign DNA inserted into the polylinker site.
These colonies typically result from the digested, linearized plasmid religating to itself. However, white colonies lack a functional beta-galactosidase enzyme, indicating the insertion of foreign DNA within the polylinker site of the plasmid vector, thus disrupting the lacZ gene.
Thus, white colonies resulting from this blue-white screening contain plasmids with an insert and can be further screened to characterize the foreign DNA. The bacterial process of conjugation see How Asexual Prokaryotes Achieve Genetic Diversity can also be manipulated for molecular cloning. F plasmids , or fertility plasmids, are transferred between bacterial cells through the process of conjugation.
Recombinant DNA can be transferred by conjugation when bacterial cells containing a recombinant F plasmid are mixed with compatible bacterial cells lacking the plasmid. F plasmids encode a surface structure called an F pilus that facilitates contact between a cell containing an F plasmid and one without an F plasmid.
On contact, a cytoplasmic bridge forms between the two cells and the F-plasmid-containing cell replicates its plasmid, transferring a copy of the recombinant F plasmid to the recipient cell. Once it has received the recombinant F plasmid, the recipient cell can produce its own F pilus and facilitate transfer of the recombinant F plasmid to an additional cell.
The use of conjugation to transfer recombinant F plasmids to recipient cells is another effective way to introduce recombinant DNA molecules into host cells. Alternatively, bacteriophages can be used to introduce recombinant DNA into host bacterial cells through a manipulation of the transduction process see How Asexual Prokaryotes Achieve Genetic Diversity.
In the laboratory, DNA fragments of interest can be engineered into phagemids , which are plasmids that have phage sequences that allow them to be packaged into bacteriophages. Bacterial cells can then be infected with these bacteriophages so that the recombinant phagemids can be introduced into the bacterial cells. Molecular cloning may also be used to generate a genomic library. Having such a library allows a researcher to create large quantities of each fragment by growing the bacterial host for that fragment.
These fragments can be used to determine the sequence of the DNA and the function of any genes present. One method for generating a genomic library is to ligate individual restriction enzyme-digested genomic fragments into plasmid vectors cut with the same restriction enzyme Figure 5. After transformation into a bacterial host, each transformed bacterial cell takes up a single recombinant plasmid and grows into a colony of cells.
All of the cells in this colony are identical clones and carry the same recombinant plasmid. Figure 5. The generation of a genomic library facilitates the discovery of the genomic DNA fragment that contains a gene of interest.
To construct a genomic library using larger fragments of genomic DNA, an E. Then, these recombinant phage DNA molecules can be packaged into phage particles and used to infect E. During infection within each cell, each recombinant phage will make many copies of itself and lyse the E. Thus, each plaque from a phage library represents a unique recombinant phage containing a distinct genomic DNA fragment.
Plaques can then be screened further to look for genes of interest. One advantage to producing a library using phages instead of plasmids is that a phage particle holds a much larger insert of foreign DNA compared with a plasmid vector, thus requiring a much smaller number of cultures to fully represent the entire genome of the original organism.
Figure 6. These recombinant phage DNA molecules are packaged into phage particles and allowed to infect a bacterial lawn. Each plaque represents a unique recombinant DNA molecule that can be further screened for genes of interest. Whereas all cells in a single organism will have the same genomic DNA, different tissues express different genes, producing different complements of mRNA.
This means that the introns, control sequences such as promoters, and DNA not destined to be translated into proteins are not represented in the library. The focus on translated sequences means that the library cannot be used to study the sequence and structure of the genome in its entirety.
The construction of a cDNA genomic library is shown in Figure 7. Figure 7. The use of bacterial hosts for genetic engineering laid the foundation for recombinant DNA technology; however, researchers have also had great interest in genetically engineering eukaryotic cells, particularly those of plants and animals. The introduction of recombinant DNA molecules into eukaryotic hosts is called transfection.
Genetically engineered plants, called transgenic plants , are of significant interest for agricultural and pharmaceutical purposes. The first transgenic plant sold commercially was the Flavr Savr delayed-ripening tomato, which came to market in Genetically engineered livestock have also been successfully produced, resulting, for example, in pigs with increased nutritional value [1] and goats that secrete pharmaceutical products in their milk.
Compared to bacterial cells, eukaryotic cells tend to be less amenable as hosts for recombinant DNA molecules. Because eukaryotes are typically neither competent to take up foreign DNA nor able to maintain plasmids, transfection of eukaryotic hosts is far more challenging and requires more intrusive techniques for success.
One method used for transfecting cells in cell culture is called electroporation. A brief electric pulse induces the formation of transient pores in the phospholipid bilayers of cells through which the gene can be introduced. Figure 8. Electroporation is one laboratory technique used to introduce DNA into eukaryotic cells. Figure 9. Microinjection is another technique for introducing DNA into eukaryotic cells.
A microinjection needle containing recombinant DNA is able to penetrate both the cell membrane and nuclear envelope. An alternative method of transfection is called microinjection.
Because eukaryotic cells are typically larger than those of prokaryotes, DNA fragments can sometimes be directly injected into the cytoplasm using a glass micropipette, as shown in Figure 9. Transfecting plant cells can be even more difficult than animal cells because of their thick cell walls.
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