Term
| When can errors in DNA replication be heritable? |
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Definition
| These errors can be heritable if the error is made in germ cells. |
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Term
| If genetic mutations are generally bad for an individual, why do they occur? |
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Definition
| Because environmental conditions on earth are constantly changing. If a population of organisms is genetically diverse, it is more likely that an individual will exist that can thrive under a new set of environmental conditions. Therefore, genetic diversity provides fuel for natural selection and is of clear benefit to the population. |
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Term
| Are babies born without any mutations? |
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Definition
| It is very unlikely. Because of the error rate of DNA replication (1 in 10^6-10^9) and the large size of the human genome (~3X10^9), it is likely to have many new mutations in its genome. |
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Term
| How do new mutations occur? |
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Definition
| Most frequently they arise due to mistakes made during DNA replication and DNA repair. These tend to be small lesions such as point mutations and small deletions or insertions. Less frequently, larger lesions are created by unequal crossovers during recombination and the insertion of large pieces of DNA in the form of mobile DNA elements (transposons) and viral genomes. |
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Term
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Definition
| A form of mating in bacteria where the transfer of genetic material between bacterial cells is by direct cell-to-cell contact or by a bridge-like connection between two cells. |
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Term
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Definition
| A hairlike appendage found on the surface of many bacteria. Pili connect a bacterium to another of its species, or to another bacterium of a different species, and build a bridge between the interior of the cells. This enables the transfer of plasmids between the bacteria. An exchanged plasmid can code for new functions, e.g., antibiotic resistance. The pilus is made up of the protein pilin. |
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Term
| What initiates the pilus production? |
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Definition
| Genes located on the F plasmid. |
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Term
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Definition
| A conjugative plasmid found in F+ (male) bacterial cells that leads with high frequency to its transfer, and much less often to transfer of the bacterial chromosome, to an F− (female) cell lacking such a plasmid. |
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Term
| T or F: Any E. coli cell can mate with any other E. coli cell. |
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Definition
| Bacteria that carry a particular F plasmid can only mate with other E. coli cells that do not carry that F plasmid. |
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Term
| Rolling circle replication |
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Definition
| A process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids. Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin, or DSO. The initiator protein remains bound to the 5' phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as a primer for DNA synthesis by DNA polymerase III. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Displacement of the nicked strand is carried out by a host-encoded helicase called PcrA (the abbreviation standing for plasmid copy reduced) in the presence of the plasmid replication initiation protein. |
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Term
| Rolling circle replication (figure) |
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Definition
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Term
| What genes are carried on the F plasmid? |
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Definition
| Genes that allow for mating and pilus production, but also sequences that allow it to be replicated and transferred to the new host. |
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Term
| Which end of the F plasmid is transferred to the recipient? |
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Definition
| The 5' end is transferred to the recipient cell, causing DNA synthesis to occur in both the donor and recipient cells, resulting in a copying of the plasmid. |
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Term
| How could a rifampicin resistant bacterium transfer its rifampicin resistance gene to a new host? |
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Definition
| The bacterium integrates its F plasmid into the rifampicin-resistant chromosome. Mating of that bacterium with a rifampicin-sensitive bacterium could result in transfer of the resistant gene to the new host. |
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Term
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Definition
| The genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surrounding and taken up through the cell membrane(s). |
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Term
| Bacterial gene transfer by F plasmid (figure) |
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Definition
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Term
| How could a bacterium obtain antibody resistance via transformation? |
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Definition
| Exogenous DNA can originate from a dead and lysed neighbor and may contain antibiotic resistance genes. Therefore, this represents another mechanism by which medically important gene transfer can occur in bacteria. |
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Term
| Bacterial homologous recombination |
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Definition
| A frequent method of reciprocal genetic exchange between bacterium. These are catalyzed by the rec enzymes. The process initiates with the alignment in register of two homologous, double stranded DNA sequences. A nick is then made in one strand allowing it to invade and anneal to the complementary strand of the homologue (called strand exchange). Then the displaced strand is nicked and it anneals to the other homologue at which point the ends are ligated to complete formation of the cross-strand exchange a.k.a. the Holliday junction. At this point, if the overlapping strands were cut and re-ligated then no exchange would occur. To achieve an actual crossover exchange, the DNA strands must go through the "gymnastics (see lecture slides)". The point of these gymnastics is to get the outside strands to cross each other. This is achieved by the end of step B, shown above. At this point, the crossing strands are cut and ligated to each other to complete the exchange. This is done with impressive fidelity and mistakes are not often made at the recombination junction. |
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Term
| What catalyzes recombination in bacteria? |
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Definition
| A set of enzymes referred to as the rec enzymes (rec A,B,C, and D). |
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Term
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Definition
| A mobile junction between four strands of DNA. The splitting up of the junction can result in crossing over. |
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Term
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Definition
| The process by which DNA is transferred from one bacterium to another by a virus. Bacteriophages can transfer genes, such as antibiotic resistance genes, between bacteria by picking up flanking host DNA during a sloppy excision event. This host DNA can then become packaged with the new viral particles that will infect new bacterial hosts thus converting them to antibiotic resistance. |
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Term
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Definition
| A small, parasitic infectious agent that can replicate only inside the living cells of organisms. |
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Term
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Definition
| The protein shell of a virus that serves to protect and transmit the viral DNA to new hosts.. |
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Term
| What is generally included in viral DNA? |
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Definition
| Genes for the capsid proteins, enzymes required to integrate the viral genome into the host genome and in some cases proteins needed to help direct the host machinery to replicate and/or express the viral genome. In addition, bacterial viruses may carry genes picked up from previous hosts. |
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Term
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Definition
| Viruses that infect bacteria. |
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Term
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Definition
| A virus with linear, dsDNA that infects E. coli. |
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Term
| What are the two paths that can be taken by viral DNA once injected into a host and circularizing? |
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Definition
The two possible fates are:
1) The Lytic pathway: Immediately be transcribed to make the proteins required to assemble new viral particles, the viral genome gets replicated, new virus particles are assembled and the cell is lysed to release the new viral particles into the environment.
2) The prophage pathway: A virally encoded integrase catalyzes a site-specific recombination event leading to integration of the viral DNA into the bacterial chromosome. Note this is different than homologous recombination, the integrase mediates recombination between specific but unrelated sequence elements in the bacterial chromosome and the viral DNA sequence. Once integrated, the virus can remain in a latent state (called the prophage) until an induction event, such as an environmental insult, leads to excision of the virus. Excision occurs by the reverse reaction of integration. Once excised, the viral DNA can then enter the lytic cycle as described above. |
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Term
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Definition
| The process by which circular viral DNA is immediately transcribed to make the proteins required to assemble new viral particles, the viral genome gets replicated, new virus particles are assembled and the cell is lysed to release the new viral particles into the environment. |
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Term
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Definition
| A virally encoded integrase catalyzes a site-specific recombination event leading to integration of the viral DNA into the bacterial chromosome. Note this is different than homologous recombination, the integrase mediates recombination between specific but unrelated sequence elements in the bacterial chromosome and the viral DNA sequence. Once integrated, the virus can remain in a latent state (called the prophage) until an induction event, such as an environmental insult, leads to excision of the virus. Excision occurs by the reverse reaction of integration. Once excised, the viral DNA can then enter the lytic cycle as described above. |
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Term
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Definition
| A phage (viral) genome inserted and integrated into the circular bacterial DNA chromosome. |
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Term
| Where can homologous recombination occur in bacteria? |
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Definition
| Anywhere in the region of homology. |
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Term
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Definition
| Relatively short sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's genome size. Transposons code for at least a transposase that catalyzes transposition and may also carry antibiotic resistance genes that can be transferred to other cells by hopping into plasmids or bacteriophages. |
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Term
| What are three of the most common transposons of E. coli and what do they encode for? |
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Definition
IS3, Tn3 and Tn10. All encode for an enzyme that
catalyzes transposition, called the
transposase, and are flanked by short
sequence elements recognized by that
transposase. |
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Term
| How do transposons contribute to genetic diversity? |
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Definition
1) May also carry antibiotic resistance genes:
– Multiple transposition events can lead to amplification of the antibiotic resistance gene.
– Transposons can become infective by hopping into a plasmid or into a bacteriophage.
2) Insertion can disrupt a gene.
3) Since the transposon carries promoters, it can effect the expression of neighboring genes.
4) Repeat sequences can confuse the homologous recombination apparatus leading to rearrangement of the bacterial chromosome. |
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Term
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Definition
| An enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. |
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Term
| Replicative transposition |
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Definition
| "Copy and paste" the donor DNA into the target DNA. The result of this is the amplification of the transposon. |
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Term
| Non-replicative transposition |
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Definition
| The transposon is cut from one donor DNA molecule and pasted into a new target DNA molecule. In these instances, there is no amplification of the transposon. |
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Term
| Which method allows for amplification of the transposon? |
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Definition
| Replicative transposition. |
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Term
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Definition
| Alternative forms of a genetic locus; a single allele for each locus is inherited separately from each parent (e.g., at a locus for eye color the allele might result in blue or brown eyes). Note that “wild type” is an allele. |
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Term
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Definition
| The specific allelic composition of a cell, either of the entire cell or more commonly for a certain gene or a set of genes. The various alleles of genes that an organism possesses. |
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Term
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Definition
| The detectable outward manifestations of a specific genotype. The observable attributes of an organism. |
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Term
| According to Mendel's 2nd law, how many possible gametes exist for each parent? |
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Definition
| There are 2^23 or 8.4X10^6 possible gametes for each parent. |
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Term
| Describe the general process of eukaryotic homologous recombination. |
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Definition
In eukaryotes, homologous recombination is induced by a double strand DNA break (step 1). The Rad50 complex then resects the 5’ ends via a 5’ to 3’ exonuclease activity (step 2). The exposed 3’ overhangs can then find their homologous sequences and via help from the RecA homologue Rad51 carry out strand invasion (step 3). The
gaps from the resection are then filled in by DNA synthesis (step 4). This creates the Holliday junction (a.k.a. cross-strand exchange) which is very similar to that described for bacterial recombination. Finally, a similar set of gymnastics as described for bacteria, leads to the other strands crossing each other at which point they are cut and ligated to each other to complete the exchange (steps 5 and 6). |
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Term
| Homologous recombination in human cells (figure) |
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Definition
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Term
| When does recombination occur in the human? |
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Definition
| During meiosis after DNA replication. The replicated homologues pair in register with the help of proteins called cohesins, then recombination occurs between paired arms of the homologous chromosomes. |
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Term
| What separates in meiosis I? |
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Definition
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Term
| What separates in meiosis II? |
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Definition
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Term
| How much crossing over happens per meiosis, on average? |
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Definition
| On average there are three crossovers per chromosome. |
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Term
| What are the three major transposons in humans? |
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Definition
1) The LINE-1 elements are ~6-8 kb in length, are found in a great number of copies (850,000) and on
average can collectively account for 21% of
the human genome sequence. They encode for their own reverse transcriptase and are still capable of transposition.
2) The SINEs are smaller than the LINEs (100-300 bp), are found in more copies (1.5 million), constitute ~13% of the genome and use the reverse
transcriptase from LINEs to move.
3) The Alu sequences are also small (~300 nucleotides), constitute 5% of the genome (500,000 copies) but most are no longer competent to transpose. |
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Term
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Definition
| ~6-8 kb in length, are found in a great number of copies (850,000) and on average can collectively account for 21% of the human genome sequence. They encode for their own reverse transcriptase and are still capable of transposition. |
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Term
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Definition
| Transpositions that are smaller than the LINEs (100-300 bp), are found in more copies (1.5 million), constitute ~13% of the genome and use the reverse transcriptase from LINEs to move. |
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Term
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Definition
| Small (~300 nucleotides), constitute 5% of the genome (500,000 copies) but most are no longer competent to transpose. |
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Term
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Definition
| Move via an RNA intermediate. First the element is transcribed into an RNA, then reverse transcriptase, the gene for which lies in the LINE-1 elements, synthesizes a double stranded DNA copy which then inserts itself into a new site in the genome. These elements are induced to transpose only in the germ line: 1/100-1/10 sperm bear a new site of retro-transposition. |
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Term
| How do retrotransposons contribute to human genetic diversity? |
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Definition
1) They can disrupt gene function by inserting within the exon of a gene; an L1 insertion into the Factor VIII gene causes one form of hemophilia.
2) They can affect the expression of genes. For example it was recently shown that Line-1 elements lower gene expression by decreasing rates of transcription elongation.
3) Perhaps most importantly they create sites for illegitimate homologous recombination known as unequal cross-overs. |
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Term
| How can transposon insertions lead to a misalignment of homologues during recombination? |
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Definition
[image]
Transposon insertions that flank a gene can lead to mis-alignment of homologues during recombination. This is cartooned above. One product from this unequal cross-over will have a duplication of the globin gene, the other a deletion. Repeated events such as these have created a globin gene family in humans. As you can see, the globin genes have transposable elements between them that are believed to have served as the sites for unequal cross-overs. Through genetic drift, these different globin genes have become specialized to be expressed at distinct stages of human development.
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Term
| How do human transposons differ from bacterial transposons? |
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Definition
| Human transposons are what are known as retrotransposons, meaning they move via an RNA intermediate. |
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Term
| What is the mechanism by which retrotransposons act during meiosis? |
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Definition
[image]
1) RNA polymerase makes an RNA copy of the transposon.
2) The RNA copy is reverse transcribed to a DNA copy by an enzyme encoded by the transposon.
3) That DNA copy is integrated back into a chromosome. |
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