Generalized Recombination and RecA
In homologous recombination, a type of genetic recombination, nucleotide sequences are exchanged between two similar molecules of DNA.
Explain the process of homologous recombination in bacteria
- Homologous recombination can vary among different organisms and cell types, but most forms involve the same basic steps.
- Homologous recombination is a major DNA repair process in bacteria. It is also important for producing genetic diversity in bacterial populations.
- Homologous recombination has been most studied and is best understood for Escherichia coli.
- recombination: The formation of genetic combinations in offspring that are not present in the parents
- genetic: Relating to genetics or genes.
- homologous: Showing a degree of correspondence or similarity.
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.
Homologous recombination can vary among different organisms and cell types, but most forms involve the same basic steps. After a double-strand break occurs, sections of DNA around the 5′ ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3′ end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. After strand invasion, one or two cross-shaped structures called Holliday junctions connect the two DNA molecules. Depending on how the two junctions are cut by enzymes, the type of homologous recombination that occurs in meiosis results in either chromosomal crossover or non-crossover. Homologous recombination that occurs during DNA repair tends to result in non-crossover products, in effect restoring the damaged DNA molecule as it existed before the double-strand break.
Homologous recombination is conserved across all three domains of life as well as viruses. Homologous recombination is also used in gene targeting, a technique for introducing genetic changes into target organisms.
Homologous recombination is a major DNA repair process in bacteria. It is also important for producing genetic diversity in bacterial populations. Homologous recombination has been most studied and is best understood for Escherichia coli. Double-strand DNA breaks in bacteria are repaired by the RecBCD pathway of homologous recombination. Breaks that occur on one of the two DNA strands, known as single-strand gaps, are thought to be repaired by the RecF pathway. Both the RecBCD and RecF pathways include a series of reactions known as branch migration, in which single DNA strands are exchanged between two intercrossed molecules of duplex DNA, and resolution, in which those two intercrossed molecules of DNA are cut apart and restored to their normal double-stranded state.
The RecBCD pathway is the main recombination pathway used in bacteria to repair double-strand breaks in DNA. These double-strand breaks can be caused by UV light and other radiation, as well as chemical mutagens. Double-strand breaks may also arise by DNA replication through a single-strand nick or gap. Such a situation causes what is known as a collapsed replication fork and is fixed by several pathways of homologous recombination including the RecBCD pathway.
In this pathway, a three-subunit enzyme complex called RecBCD initiates recombination by binding to a blunt or nearly blunt end of a break in double-strand DNA. After RecBCD binds the DNA end, the RecB and RecD subunits begin unzipping the DNA duplex through helicase activity. The RecB subunit also has a nuclease domain, which cuts the single strand of DNA that emerges from the unzipping process. This unzipping continues until RecBCD encounters a specific nucleotide sequence (5′-GCTGGTGG-3′) known as a Chi site.
Upon encountering a Chi site, the activity of the RecBCD enzyme changes drastically. DNA unwinding pauses for a few seconds and then resumes at roughly half the initial speed. This is likely because the slower RecB helicase unwinds the DNA after Chi, rather than the faster RecD helicase, which unwinds the DNA before Chi. Recognition of the Chi site also changes the RecBCD enzyme so that it cuts the DNA strand with Chi and begins loading multiple RecA proteins onto the single-stranded DNA with the newly generated 3′ end. The resulting RecA-coated nucleoprotein filament then searches out similar sequences of DNA on a homologous chromosome. The search process induces stretching of the DNA duplex, which enhances homology recognition (a mechanism termed conformational proofreading). Upon finding such a sequence, the single-stranded nucleoprotein filament moves into the homologous recipient DNA duplex in a process called strand invasion. The invading 3′ overhang causes one of the strands of the recipient DNA duplex to be displaced, to form a D-loop. If the D-loop is cut, another swapping of strands forms a cross-shaped structure called a Holliday junction.Resolution of the Holliday junction by some combination of RuvABC or RecG can produce two recombinant DNA molecules with reciprocal genetic types, if the two interacting DNA molecules differ genetically. Alternatively, the invading 3′ end near Chi can prime DNA synthesis and form a replication fork. This type of resolution produces only one type of recombinant (non-reciprocal).
Transformation is the direct uptake, incorporation and expression of exogenous genetic material from its surroundings.
Differentiate between natural and artificial transformation
- Transformation results in the genetic alteration of the recipient cell.
- Exogenous DNA is taken up into the recipient cell from its surroundings through the cell membrane (s).
- Transformation occurs naturally in some species of bacteria, but it can also be affected by artificial means in other cells.
- eukaryotic: Having complex cells in which the genetic material is organized into membrane-bound nuclei.
- transformation: In molecular biology transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
- expression: Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product.
- exogenous: Produced or originating outside of an organism.
- translocase: An enzyme that assists in moving another molecule, usually across a membrane.
In molecular biology, transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells. For transformation to happen, bacteria must be in a state of competence, which might occur as a time-limited response to environmental conditions such as starvation and cell density. Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell; the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact), and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium).
“Transformation” may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because “transformation” has a special meaning in relation to animal cells, indicating progression to a cancerous state, the term should be avoided for animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA into eukaryotic cells is often called “transfection“.
Bacterial transformation may be referred to as a stable genetic change, brought about by the uptake of naked DNA (DNA without associated cells or proteins ). Competence refers to the state of being able to take up exogenous DNA from the environment. There are two forms of competence: natural and artificial.
About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; more may be able to take it up in their natural environments. DNA material can be transferred between different strains of bacteria in a process that is called horizontal gene transfer.
Some species, upon cell death, release their DNA to be taken up by other cells; however, transformation works best with DNA from closely-related species. These naturally-competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogeneous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.
GRAM-POSITIVE AND GRAM-NEGATIVE DIFFERENCES
Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells. However, most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase. Only single-stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process.
In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence however, its role is uncertain. The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.
Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA, by exposing it to conditions that do not normally occur in nature. Typically, the cells are incubated in a solution containing divalent cations; most commonly, calcium chloride solution under cold condition, which is then exposed to a pulse of heat shock. However, the mechanism of the uptake of DNA via chemically-induced competence in this calcium chloride transformation method is unclear.
The surface of bacteria such as E. coli is negatively-charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively-charged. One function of the divalent cation therefore, would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface. It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.
Electroporation is another method of promoting competence. Using this method, the cells are briefly shocked with an electric field of 10-20 kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell’s membrane-repair mechanisms.
O. T. Avery, et al. were first to demonstrate that “rough” colonies of S. pneumoniae could be transformed to “smooth” (capsule producing) colonies by addition of DNA extracts of the former to the latter, thus “transforming” them. (See Lederberg below)
- Lederberg, Joshua (1994). The Transformation of Genetics by DNA: An Anniversary Celebration of AVERY, MACLEOD and MCCARTY(1944) in Anecdotal, Historical and Critical Commentaries on Genetics. The Rockfeller University, New York, New York 10021-6399. PMID 8150273.
Transduction is the process by which DNA is transferred from one bacterium to another by a virus.
Differentiate between generalized and specialized transduction
- Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation), and it is DNAase resistant.
- Transduction happens through either the lytic cycle or the lysogenic cycle.
- Transduction is especially important because it explains one mechanism by which antibiotic drugs become ineffective due to the transfer of antibiotic-resistance genes between bacteria.
- lytic cycle: The normal process of viral reproduction involving penetration of the cell membrane, nucleic acid synthesis, and lysis of the host cell.
- lysogenic cycle: A form of viral reproduction involving the fusion of the nucleic acid of a bacteriophage with that of a host, followed by proliferation of the resulting prophage.
- transduction: Transduction is the process by which DNA is transferred from one bacterium to another by a virus.
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation), and it is DNAase resistant (transformation is susceptible to DNAase). Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell’s genome.
When bacteriophages (viruses that infect bacteria) infect a bacterial cell, their normal mode of reproduction is to harness the replicational, transcriptional, and translation machinery of the host bacterial cell to make numerous virions, or complete viral particles, including the viral DNA or RNA and the protein coat.
Transduction is especially important because it explains one mechanism by which antibiotic drugs become ineffective due to the transfer of antibiotic-resistance genes between bacteria. In addition, hopes to create medical methods of genetic modification of diseases such as Duchenne/Becker Muscular Dystrophy are based on these methodologies.
The Lytic Cycle and the Lysogenic Cycle
Transduction happens through either the lytic cycle or the lysogenic cycle. If the lysogenic cycle is adopted, the phage chromosome is integrated (by covalent bonds) into the bacterial chromosome, where it can remain dormant for thousands of generations. If the lysogen is induced (by UV light for example), the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles. The lytic cycle leads to the production of new phage particles which are released by lysis of the host.
Transduction is a method for transferring genetic material. The packaging of bacteriophage DNA has low fidelity and small pieces of bacterial DNA, together with the bacteriophage genome, may become packaged into the bacteriophage genome. At the same time, some phage genes are left behind in the bacterial chromosome.
There are generally three types of recombination events that can lead to this incorporation of bacterial DNA into the viral DNA, leading to two modes of recombination.
Generalized transduction is the process by which any bacterial gene may be transferred to another bacterium via a bacteriophage, and typically carries only bacterial DNA and no viral DNA. In essence, this is the packaging of bacterial DNA into a viral envelope. This may occur in two main ways, recombination and headful packaging.
If bacteriophages undertake the lytic cycle of infection upon entering a bacterium, the virus will take control of the cell’s machinery for use in replicating its own viral DNA. If by chance bacterial chromosomal DNA is inserted into the viral capsid which is usually used to encapsulate the viral DNA, the mistake will lead to generalized transduction.
If the virus replicates using “headful packaging,” it attempts to fill the nucleocapsid with genetic material. If the viral genome results in spare capacity, viral packaging mechanisms may incorporate bacterial genetic material into the new virion.
The new virus capsule, now loaded with part bacterial DNA, continues to infect another bacterial cell. This bacterial material may become recombined into another bacterium upon infection.
Fates of DNA Inserted into the Recipient Cell
When the new DNA is inserted into this recipient cell it can fall to one of three fates: the DNA will be absorbed by the cell and be recycled for spare parts; if the DNA was originally a plasmid, it will recirculate inside the new cell and become a plasmid again; if the new DNA matches with a homologous region of the recipient cell’s chromosome, it will exchange DNA material similar to the actions in conjugation. This type of recombination is random and the amount recombined depends on the size of the virus being used.
Specialized transduction is the process by which a restricted set of bacterial genes are transferred to another bacterium. The genes that get transferred (donor genes) depend on where the phage genome is located on the chromosome. Specialized transduction occurs when the prophage excises imprecisely from the chromosome so that bacterial genes lying adjacent to the prophage are included in the excised DNA. The excised DNA is then packaged into a new virus particle, which can then deliver the DNA to a new bacterium, where the donor genes can be inserted into the recipient chromosome or remain in the cytoplasm, depending on the nature of the bacteriophage.
When the partially encapsulated phage material infects another cell and becomes a “prophage” (is covalently bonded into the infected cell’s chromosome), the partially coded prophage DNA is called a “heterogenote. ” Example of specialized transduction is λ phages in Escherichia coli, which was discovered by Esther Lederberg.
Prokaryotes reproduce asexually by binary fission; they can also exchange genetic material by transformation, transduction, and conjugation.
Distinguish among the types of reproduction in prokaryotes
- Binary fission is a type of reproduction in which the chromosome is replicated and the resultant prokaryote is an exact copy of the parental prokaryate, thus leaving no opportunity for genetic diversity.
- Transformation is a type of prokaryotic reproduction in which a prokaryote can take up DNA found within the environment that has originated from other prokaryotes.
- Transduction is a type of prokaryotic reproduction in which a prokaryote is infected by a virus which injects short pieces of chromosomal DNA from one bacterium to another.
- Conjugation is a type of prokaryotic reproduction in which DNA is transferred between prokaryotes by means of a pilus.
- transformation: the alteration of a bacterial cell caused by the transfer of DNA from another, especially if pathogenic
- transduction: horizontal gene transfer mechanism in prokaryotes where genes are transferred using a virus
- binary fission: the process whereby a cell divides asexually to produce two daughter cells
- conjugation: the temporary fusion of organisms, especially as part of sexual reproduction
- pilus: a hairlike appendage found on the cell surface of many bacteria
Reproduction in prokaryotes is asexual and usually takes place by binary fission. The DNA of a prokaryote exists as as a single, circular chromosome. Prokaryotes do not undergo mitosis; rather the chromosome is replicated and the two resulting copies separate from one another, due to the growth of the cell. The prokaryote, now enlarged, is pinched inward at its equator and the two resulting cells, which are clones, separate. Binary fission does not provide an opportunity for genetic recombination or genetic diversity, but prokaryotes can share genes by three other mechanisms.
In transformation, the prokaryote takes in DNA found in its environment that is shed by other prokaryotes. If a nonpathogenic bacterium takes up DNA for a toxin gene from a pathogen and incorporates the new DNA into its own chromosome, it, too, may become pathogenic. In transduction, bacteriophages, the viruses that infect bacteria, sometimes also move short pieces of chromosomal DNA from one bacterium to another. Transduction results in a recombinant organism. Archaea are not affected by bacteriophages, but instead have their own viruses that translocate genetic material from one individual to another. In conjugation, DNA is transferred from one prokaryote to another by means of a pilus, which brings the organisms into contact with one another. The DNA transferred can be in the form of a plasmid or as a hybrid, containing both plasmid and chromosomal DNA.
Reproduction can be very rapid: a few minutes for some species. This short generation time, coupled with mechanisms of genetic recombination and high rates of mutation, result in the rapid evolution of prokaryotes, allowing them to respond to environmental changes (such as the introduction of an antibiotic) very rapidly.
Complementation refers to a relationship between two different strains of an organism which both have homozygous recessive mutations.
Explain the mechanism of genetic complementation
- A complementation test answers the question: “Does a wild-type copy of gene X rescue the function of the mutant allele that is believed to define gene X? “.
- Complementation arises because loss of function in genes responsible for different steps in the same metabolic pathway can give rise to the same phenotype.
- When strains are bred together, offspring inherit wildtype versions of each gene from either parent.
- Complementation: In genetics, complementation refers to a relationship between two different strains of an organism which both have homozygous recessive mutations that produce the same phenotype (for example, a change in wing structure in flies) but which do not reside on the same (homologous) gene.
- mutation: Any heritable change of the base-pair sequence of genetic material.
- homozygous: of an organism in which both copies of a given gene have the same allele
In genetics, complementation refers to a relationship between two different strains of an organism which both have homozygous recessive mutations that produce the same phenotype (for example, a change in wing structure in flies) but which do not reside on the same (homologous) gene.
These strains are true breeding for their mutation. If, when these strains are crossed with each other, some offspring show recovery of the wild-type phenotype, they are said to show “genetic complementation”. When this occurs, each strain’s haploid supplies a wild-type allele to “complement” the mutated allele of the other strain’s haploid, causing the offspring to have heterozygous mutations in all related genes. Since the mutations are recessive, the offspring will display the wild-type phenotype.
A complementation test (sometimes called a “cis-trans” test) refers to this experiment, developed by American geneticist Edward B. Lewis. It answers the question: “Does a wild-type copy of gene X rescue the function of the mutant allele that is believed to define gene X?”. If there is an allele with an observable phenotype whose function can be provided by a wild type genotype (i.e., the allele is recessive), one can ask whether the function that was lost because of the recessive allele can be provided by another mutant genotype. If not, the two alleles must be defective in the same gene. The beauty of this test is that the trait can serve as a read-out of gene function even without knowledge of what the gene is doing at a molecular level.
Complementation arises because loss of function in genes responsible for different steps in the same metabolic pathway can give rise to the same phenotype. When strains are bred together, offspring inherit wildtype versions of each gene from either parent. Because the mutations are recessive, there is a recovery of function in that pathway, so offspring recover the wild-type phenotype. Thus, the test is used to decide if two independently derived recessive mutant phenotypes are caused by mutations in the same gene or in two different genes. If both parent strains have mutations in the same gene, no normal versions of the gene are inherited by the offspring; they express the same mutant phenotype and complementation has failed to occur.
In other words, if the combination of two haploid genomes containing different recessive mutations yields a mutant phenotype, then there are three possibilities: Mutations occur in the same gene; One mutation affects the expression of the other; One mutation may result in an inhibitory product. If the combination of two haploid genomes containing different recessive mutations yields the wild type phenotype, then the mutations must be in different genes.
Gene Transfer in Archaea
Archaea are distinct from bacteria and eukaryotes, but genetic material can be transferred between them and between Archaea themselves.
Describe the mechanisms of gene transfer in Archaea
- Archaea while being very different from eukaryotes and bacteria, there are many commonalities at the the genetic level between them.
- Horizontal gene transfer can explain the similarities between the genes found in the three domains of life and indeed there is evidence that horizontal gene transfer occurs with Archaea species.
- Archaea can be infected by double-stranded DNA viruses, which can account for gene transfers, as well like bacteria, Archaea may conjugate.
- archaea: a taxonomic domain of single-celled organisms lacking nuclei that are fundamentally from bacteria.
- translation: Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text.
- transcription: Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes.
Archaea are genetically distinct from bacteria and eukaryotes, but are poorly understood: many of the genes that Archaea encode are of unknown function. Transcription and translation in archaea resemble the same processes more closely in eukaryotes than in bacteria, with the archaean RNA polymerase and ribosomes being very close to their equivalents in eukaryotes.
Although Archaea only have one type of RNA polymerase, its structure and function in transcription is similar to that of the eukaryotic RNA polymerase II, with similar protein assemblies (the general transcription factors) directing the binding of the RNA polymerase to a gene’s promoter. However, other archaean transcription factors are closer to those found in bacteria. Post-transcriptional modification is simpler than in eukaryotes, since most archaean genes lack introns, although there are many introns in their transfer RNA and ribosomal RNA genes, and introns may occur in a few protein-encoding genes. This is all to say there are many similarities in the genes shared between Archaea and the other domains of life, suggesting there was a transfer of genetic material between the domains of life. This phenomenon is described as horizontal gene transfer.
Horizontal gene transfer (HGT) refers to the transfer of genes between organisms in a manner other than traditional reproduction. Also termed lateral gene transfer, it contrasts with vertical transfer, the transmission of genes from the parental generation to offspring via sexual or asexual reproduction. HGT has been shown to be an important factor in the evolution of many organisms, including bacteria, plants and humans.
Archaea show high levels of horizontal gene transfer between lineages. Some researchers suggest that individuals can be grouped into species-like populations given highly similar genomes and infrequent gene transfer to/from cells with less-related genomes, as in the Archaea genus Ferroplasma. On the other hand, studies in Halorubrum found significant genetic transfer to/from less-related populations. These gene transfers are identified by sequencing the DNA of various Archaea species; through the similarities and differences of the DNA of the different types of Archaea it is determined if the gene was perfectly transferred or from a common ancestor. The elucidation of this can be controversial.
How genetic material can move from one Archaea to another is poorly understood. In bacteria the natural ways in which this occurs is through either bacterial conjugation or viral transfer, also known as transduction. Conjugation is where two (sometimes distantly related) bacteria transfer genetic material by direct contact. Transduction occurs when a virus “picks up” some DNA from its host and when infecting a new host, moves that genetic material to the new host. It is thought that conjugation can occur in Archaea, though unlike bacteria the mechanism is not well understood. As well Archaea can be infected by viruses. In fact Archaea can be infected by double-stranded DNA viruses that are unrelated to any other form of virus and have a variety of unusual shapes, including bottles, hooked rods, or teardrops. Taken together it is clear that gene transfer happens in Archaea, and probably is similar to horizontal gene transfer seen in the other domains of life.