* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Transposons ※ Transposons are DNA elements that can hop, or
Human genome wikipedia , lookup
Epigenetics wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Minimal genome wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
DNA profiling wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
DNA polymerase wikipedia , lookup
SNP genotyping wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Genetic engineering wikipedia , lookup
Designer baby wikipedia , lookup
Genomic library wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Genealogical DNA test wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Primary transcript wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Transposable element wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Point mutation wikipedia , lookup
Microevolution wikipedia , lookup
Genome editing wikipedia , lookup
Epigenomics wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Molecular cloning wikipedia , lookup
Non-coding DNA wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
DNA vaccination wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
DNA supercoil wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Transposons ※ Transposons are DNA elements that can hop, or transpose, from one place in DNA to another. They are also called “jumping genes”. They carry the enzyme, transposase responsible for transposition, the movement by a transposon. ※ They are discovered by Barbara McClintock in the early 1950s. ※ The transposons now exist in all organisms on the earth, including human. ※ Transposons may offer a way of introducing genes from one bacterium into the chromosome of another bacterium to which it has little DNA sequence homology, so they obviously play an important role in evolution. ※ Transposition must be tightly regulated and occur only rarely; otherwise, the cellular DNA would become riddled with the transposons, which would have many deleterious effects. In fact, the frequency of transposition varies from once in every 103 to once in every 108 cell division, depending on the type of transposon. It is not higher than the chance of a gene inactivated by other mutation. Almost half of human genome may be the transposons. ※ Genome – The complete DNA sequence of an organism. 人類基因體計畫 1. 人類的染色體為 23 對,其一半即為構成人類的基因體,約含有 3 X 109 鹼基對 (bp) ,其大小約為大腸桿茵(4.2 X 106 bp)的 1,000 倍,這是否表示人類的 基因體所含的基因數目為大腸桿茵的 1,000 倍(大腸桿茵有 2,000 個基因)。 答案是否定的,人類基因體計畫的完成顯示人類的基大約有 35,000 個。所以 人類基因體含有大量的〞廢物 DNA(junk DNA)〞 ,約佔人類總 DNA 的 97%, 這些 DNA 包括基因中的隱子,基因間的重覆序列及所謂的跳躍基因。 2. The largest of component of the human genome consists of transposons. Other repetitive sequences include large duplications and simple repeats. • Overview of transposition Types of bacterial tansposons 1. Insertion sequence (IS) elements (1) These transposons are usually only about 750 ~ 2000 bp long and encode little more than the transposae that promote they transposition. (2) Repeats at ends, usually inverted repeats. (3) IS3 consists of two open reading frames (ORFA and ORFB). (4) ORFB is shifted -1 relative to ORFA, but a programmed -1 (2) Fig.9.2 is the structure of the IS3 element, which contains 1 frameshift causes the synthesis of a fusion protein, ORFAB, which is the active transposase. (5) Smaller protein made from ORFA when the frameshift does not occur regulates transcription of transposase. (6) The target site sequence that is duplicated on the insertion of IS3 is 3 bp long (ex., ATT). The length of such direct repeats is characteristic of each type of transposon. Structure of the insertion sequence element IS3 and its related family member 1. The inverted repeats are shown as arrows, and the 3-bp target sequence that is duplicated after transposition is boxed. 2. OFRA and OFRB encode the N terminus and C terminus of the transposase, which are translated in different reading frames and are not active by themselves. 3. A programmed -1 frameshift put both ORFA and ORFB in the same frame and makes the active transposase. The C terminus of the IS3 transposase contains the DDE motif characteristic of this type of transposase. 2. Composite transposons (1) A larger transposon: Two IS elements of the same type bracket other genes, usually the antibiotic resistant gene(s). (2) Transposition * Each IS element of the same transposon can transpose independently as long as the transposase acts on both of its ends. * Two IS elements are often not completely autonomous, because the active transposase of one IS element can act on the outside ends to promote transposition of the composite transposon when the transposase of the other element is inactive due to a mutation. i. Outside-end transposition When a transposase acts on the inverted repeats at the farthest ends of a composite transposon, the two IS elements transpose as a unit along with the genes between them. ii. Inside-end transposition A transposase encoded by one IS element can also act on the inside ends of both IS elements. Structures of some composite transposons 1. The active transposase gene is in one of the two IS elements. 2. The IS elements can be in either the same or opposite orientation (arrows). 2 • Two IS elements can transpose any DNA between them • Either the outside or inside ends of the IS elements in a composite transposon can be used for transposition (a) Transposition with the outside ends of IS10 element would move Tn10, with the tetracycline resistance gene (Tetr) to another DNA. • Either the outside or inside ends of the IS elements in a composite transposon can be used for transposition (a) Transposition with the outside ends of IS10 element would move Tn10, with the tetracycline resistance gene (Tetr) to another DNA. • Rearrangements of DNA caused by composite transposons through inside-ends transposition - neighboring sequences between the original site of insertion of the transposon and the site into which it is trying to transpose will be either deleted or inverted. (B, C, D) 4. Methods have been developed to select tet-sensitive derivatives of E. coli haerboring the Tn10 transposon. Most of these tet-sensitive derivatives have deletions or inversions of DNA next to the site of insertion of Tn10 element. 5. Inside-end transposition is presumably responsible for most of the often-observed instability of DNA (rearrangement) caused by composite transposon. 6. Some composite transposons have mechanisms to avoid inside-end transposition. Ex., adenines of inside-ends of Tn5 are methylated so that they are recognized less well by the transposase. 7. Assembly of plasmids by IS elements - Many of the resistance gene on plasmids are bracketed by the same IS element. Apparently, the plasmid was assembled in nature by resistance genes hopping onto the plasmid from some other DNA via the bracketing IS elements. 3 III. Mechanisms of transposition IIIa. A molecular model for transposition of Tn3 (A replicative transposition) 1. Breaks are made in the target DNA and at the ends of the transposon, respectively (1 and 2). 2. The 3’ OH ends of the transposon (dots) are ligated to 5’ PO4 ends of the target DNA (3). 3. The free 3’ ends of the target DNA prime replication in both directions over the transposon to form the cointegrate (4). 4. The cointegrate is resolved by recombination promoted by the resolvase TnpR at the res sites (5) 5. Resolution of the cointegrate give rise to two copies of the transposons, one at the former (or donor) site and a new one at the target site. (The A and B in the target DNA illustrate how the target DNA is reversed in the step 3 for ease of drawing.) 6. The transposase cuts the target and donor DNAs and promotes ligation of the ends. 7. The resolvase specifically promotes recombination between the res elements in cointegrate. 8. Mu phage replicate itself and insert itself around the chromosome of its bacterial host by a mechanism similar to Tn3. (1) It does not resolve the cointegrate and soon the chromosome becomes riddled with Mu genome. (2) These genomes are packaged directly from chromosomal DNA into the phage head, discarding the bacterial chromosomal DNA between the inserted Mu genomes. IIIb. Transposition by Tn10 and Tn5 • Transposition by a cut-and-paste mechanism (also known as conservative mechanism or nonreplicative transposition) • The transposon is moved from one place and inserted into another place. • Transposon produces a short duplication of target DNA at the ends of the transposon. • Donor DNA probably leaves break and is consequently degraded. • There is no cointegrate formation as it does in the replicative mechanism. Details of the mechanism of transposition by Tn5 1. Single copies of the transposae (TnP) bind to each end of the transposon, 4 and then bind to each other, bring the two ends of transposon together (synapsis). 2. Transposase bound at one end cuts the DNA at the other end and vice versa to leave 3’ OH ends at each end of transposon. 3. These activated 3’ OH ends attack the phosphodiester bond on the other strand, forming 3’-5’ phosphodiester hairpins. This cuts the transposon out of the donor DNA. 4. When the transposase binds to the target DNA, it cuts the two hairpin ends again and the 3’ OH ends attack phosphodiester bonds 9 bp apart in the target DNA, cutting them, and the 5’ phosphate ends in the target DNA are joined to the 3’ OH ends in the transposon, inserting the transposon into the target DNA. 5 .The 9-bp single-stranded gaps on each side of transposon are filled in by DNA polypomerase to make the 9-bp repeats in the target DNA. Details of the mechanism of transposition by Tn7 • The cut and paste transposon Tn7 can be converted into a replicative transposon by a single amino acid change in one subunit of transposase. • Different subunits of transposase make the cuts in the opposite strands of DNA at the ends of transposon. - TnsA cuts at the 5’ and, and TnsB cuts at 3’ end, - They cut the donor DNA only in the presence of the target DNA. • If the TnsA subunit that makes the cut that leaves the 5’ hydroxyl end is altered by a mutation, transposase will cut only the other strand, leaving a free 3’ OH like a replicative transposase. * Apparently, the transposases need only make the appropriate cuts and joinings, and the replication apparatus of the cell does the rest. * The linear Tn7 transposon does not cut itself out of the donor DNA unless the target DNA is already bound to the transposase, so the 5’ ends are not left exposed for long. The major difference between replicative and nonreplicative transposition 1. Replicative transposase cuts only one strand at the junction. 2. Nonreplicative transposase makes cuts in both strands in the junction. The similarity between replicative and nonreplicative transposition 5 1. The cut 5’ ends of the target DNA are joined to the free 3’ ends of the transposon. 2. The free 3’ ends of target DNA are used as primers for replication that proceeds until a free 5’ end in the donor DNA is reached (The only different is whether the replication has to proceed over the entire transposon (replicative ) or only over the short region that is duplicated (cut and paste). IV. General properties of transposons • Target specificity 1 . No transposable element inserts completely randomly into target DNA: Target specificity of some transposons are relaxed and some are stringent. 2. Tn7 transposes with a high frequency into only one site in E. coli DNA, called attTn7, just downstream of the glmS gene. (1) The transposition machinery consists of five proteins: i. TnsA and TnsB – make up the transposase that cuts and joins the DNA strands. ii. Other proteins play ancillary roles: (i) TnsD – may direct the Tn7 to the target sequence, attTn7. It may induce changes representative of triple-stranded structures in the attTn7 site. (ii) TnsC – (i) event directs TnsC to stimulate transposition into the site. (iii) TnsE – In the absence of TnsD, TnsE stimulates transposition into other site on chromosome. This transposition is inefficient but random. (2) The glmS gene is highly conserved. i. The product of glmS performs an important step in cell wall biosynthesis. ii. The insertion site of Tn7 is downstream the gene, and has no effect on cell only its transcription termination site. • Effects on genes adjacent to the insertion site – could be negative or positive (Tn5 and Tn10 contain promoter near their termini) • Regulation of transposition – transposition of most transposons occurs rarely because they self- regulate their transposition. The regulatory mechanisms differ greatly: 1. Tn3 – The TnpR protein represses the transcription of the 6 transposase gene (Tnp). 2. Tn10 – transposition occurs primarily just after a replication fork has passed through the element. (1) Newly replicated E. coli DNA is hemimethylated at GATC sites, and it not only activates the transposase promoter but also increases the activity of the transposon ends. (2) The translation of transposase is also repressed by an antisense RNA. 3. Tn5 - Using a truncated transposase version to inhibit the active one. (1) Two similar IS50 elements flank the antibiotic resistance genes. (2) An N terminally truncated Tnp (transposase) inhibit the active one. (3) Dam methylation of the inside ends (IEs) of the IS50 prevents the transposase from cutting IEs and transposing the individual IS50 elements. • Target immunity – Some transposons prefer not to hop close in the DNA to another transposon of the same type. Immunity can extend over 100,000 bp of DNA. (1) If two transposons were to insert close to each other, would cause large deletions and often lead to the death of the cell. Also, the presence of two transposons close to each other can cause instability in chromosome. (2) Only Mu, Tn3 and Tn7 families of transposons are known to exhibit target immunity. i. MuB protein seems to be indirectly resposible for the immunity. ii. The binding of MuB to a DNA make it a target for the MuA transposase, which then promotes transposon into DNA. iii. The binding of MuA then cause MuB to dissociate from DNA. iv. Once a transposon has inserted, a copy of MuA may remain bound to the end of the inserted transposon, and prevent the binding of other MUB to the same target DNA and other transposition into that DNA. v. A similar mechanism may explain target site immunity by Tn7, and the resposible proteins are TnsB and TnsC. V.Transposon mutagenesis • Transposons are useful for mutagenesis should have the following properties: 7 1. It should transpose at a fairly high frequency. 2. It should not be very selective in its target sequence. 3. It should carry an easily selectable gene, such as one for resistance to an antibiotic. 4. It should have a broad host range for transposition if it is to be used in several different kinds of bacteria. • Transposon Tn5, for example in many types of G – bacteria. • There are no equally universal methods for G + bacteris. (A) A standard protocol for transposon mutagenesis of G- bacteria: 1. A suicide ColE1-derived plasmid contains a mob site and transposon Tn5 . 2. The relaxase of this suicide plasmid recognizes the coupling protein of promiscuous plasmid RP4. 3. This suicide plasmid is mobilized into the bacterium by the products of the RP4 transfer genes, which are inserted in the chromosome. 4. Tn5 hops into the chromosome of the recipient cell, and the ColE1 plasmid is lost because it can not replicate. (B) Random transposon mutagenesis of a plasmid 1. Transposon Tn5 is introduced into cells on a suicide vector. 2a. The culture is incubated, allowing the Tn5 time to hop, either into the chromosome (large circle) or into a plasmid (small circle). 2b. Plating on kanamycin-containing medium results in the selection of cells in which a transposition has occurred. 3. Plasmid is prepared from Kanr cells and used to transform Kans cells. 4. Selection for Kanr allows the identification of cells that has acquired a Tn5-carrying plasmid. • Cloning genes mutated with a transposn insertion 1. A transposon used for mutagenesis of a chromosome contains a plasmid origin of replication (ori). 2. The chromosome is cut with a restriction enzyme, ex., EcoRI which no cut in transposon, and religated. 3. Transform E. coli with ligated mix, the resulting plasmid in the Ampr transformants will contain the chromosomal sequences that flanked the transposon . 8 Transformation • DNA can be exchanged among bacteria in three ways: 1. Conjugation – a plasmid or other self-transmissible DNA element transfers it self and sometimes other DNA into other bacterial cell. 2. Transduction – a phage carries DNA from one bacterium to another. 3. Transformation – cells take up free DNA directly from their envirnment. • Naturally transformable bacterium (Most types of cells cannot take up DNA efficiently unless they have been exposed to special chemical or electrical treatments to make them more permeable.) 1. Naturally transformable bacterium (or naturally competent bacterium) – They can take up DNA from the environment without requiring special treatment. 2. About 40 species have been found to be naturally competent or transformable. 3. Bacillus subtilis, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria gonorrhoeae, Helicobacter pylori, Acinetobacter baylyi, and some species of marine cyanobacteria. • Artificially induced competence Bacteria can be sometimes be made competent by certain chemical treatments or DNA can be forced into bacteria by a strong electric field in a process called electroporation. 1. Treatment with calcium ions. (1) Chemically induced transformation is usually inefficient, and only a small percentage of the cells are ever trnasformed. (2) Accordingly, the cells must be plated under conditions selective for the transformed cells. (3) Therefore, the DNA used for the transformation should contain a selectable gene such as one encoding resistance to an antibiotic. 2. Electroporation (1) The bacteria are mixed with DNA and briefly exposed to a strong electric field. (2) The bacteria must first be washed extensively in buffer with very low ionic strength such as distilled water. The buffer usually also contains a nonionic solute such as glycerol to prevent osmotic shock. 9 (3) The brief electric field across the cellular membranes might create artificial pore of H2O lined by phospholipid head groups. pass through these temporary hydrophilic pores. (4) Electroporation requires specialized equipment. DNA can Discovery of transformation 1. In 1928, Fred Griffith found that one form of the pathogenic pneumococci (now called Streptococcus pneumoniae) could be mysteriously “transformed” into another form. 2. Griffith made a conclusion that the dead pathogenic bacteria gave off a “transforming principle” that changed the live nonpathogenic rough-colony-forming bacteria into the pathogenic smooth-colony form. 3. Later, other researchers did an experiment in which they trnasformed rough-colony-forming bacteria into the pathogenic smooth-colony form by mixing the rough forms with extracts of the smooth forms in a test tube. 4. About 16 years later after Griffith did his experiment with mice, Oswald Avery and his collaborators purified the “transforming principle” from extracts of smooth-colony formers and showed that it is DNA. Avery and his colleagues were first to demonstrate that DNA, and not protein or other factors in the cell, is the hereditary material. Competence • The ability of some bacteria to take up naked DNA from their environment. • It is genetically programmed. Generally, more than a dozen genes are involved, encoding both regulatory and structural components. • The general steps that occur in natural transformation differ somewhat in Gram-negative and positive bacteria. • The followings are two examples for G – and G + , respectively. • The steps for DNA uptake 1. Binding of double-stranded DNA to the outer cell surface of bacterium. 2. Movement of DNA across the cell wall and outer membrane (no outer membrane in G + bacterium). 3. Degradation of one of the DNA strands. 4. Translocation of the remaining single strand of DNA into cytoplasm of the cell across inner membrane. 5. Once in the cell, the single-stranded transforming DNA might synthesize the complementary strand and reestablish itself as a plasmid, stably integrate into the chromosome, or degraded. • While the DNA uptake system of G + and G – bacteria have 10 features in common, they do seem to differ in certain important respects. 1. There are many proteins involved in transformation in bacteria. 2. They are discovered on the basis of isolation of mutants that are completely lacking in the ability to take up DNA. 3. The genes affected in the mutants were named com (for competence defective). (1) The com genes are organized into several operons. (2) The products of these, including the comA and comK operons, are involved in regulation of competence. (3) Others, including the products of genes in the comE, comF, and comG operons, become part of the competence machinery in the membrane that takes DNA up into the bacteria. (4) The genes in these operons are given two letters, the first for the operon and the second for the position of gene in the operon, ex., comFA is the first gene of the comF operon. • Steps in natural transformation 1. ComEA encoded by the first gene of the comE operon, binds directly extracellular double-stranded DNA. 2. The comF genes encode proteins that translocate the DNA into the cell. ComFA is an ATPase that may provide the energy for translocation of DNA through the membrane (not shown). 3. ComEA, ComEC, and ComFA form a sort of ATP-binding cassette (ABC) transporter, which transports DNA into the cell. 4. The genes in the comG operon encode proteins that might form a “pseudopilus” which helps move DNA through the ComEC channel. They might bind to extracellular DNA, perhaps acting through the ComEA DNA-binding protein, and then retract, drawing the DNA into the cell. 5. The comE, comF and comG operons are all under the transcrptional control of ComK, a transcription factor that is itself regulated by ComA. 6. Some of genes involved in the transformation process are not designated as com, because such genes were first discovered on the basis of their involvement in other processes. (1) The nucA gene product makes double-strand breaks in extracellular DNA. The free DNA ends become the substrates for the 11 competence proteins. (2) Other examples are single-stranded-DNA binding protein (SSB), and RecA functions in the recombination of transforming DNA with chromosome DNA. • The lengths of single-stranded DNA incorporated into the recipient chromosome are about 8.5 to 12 kb based on cotransformation of genetic markers, and the incorporation takes only few minutes to be completed. • The proteins in shaded boxes are analogous in G + and G – bacteria. ComEC of B. subtilis is an ortholog of ComA protein of Neisseria. • The DNA is shown running through the cell wall alongside the pseudopilus (ComG in B. subtilis; PilE in G – systems that are related to type II protein secretion systems.) • Natural transformation of Gram-positive bacteria • The comF genes encode proteins that translocate the DNA into the cell. • ComEA, ComEC, and ComFA form a sort of ATP-binding cassette (ABC) transporter. • The genes in the comG operon encode proteins that might form a “pseudopilus” which helps move DNA through the ComEC channel, and the ComECs retract,drawing the DNA into the cell. Natural transformation of Gram-negative bacteria 1. ComA protein of Neisseria is an ortholog of ComEC of B. subtilis. 2. The DNA is shown running through the cell wall alongside the pseudopilus (ComG in B. subtilis; PilE in G – systems). 3. In most G – bacteria specific sequences are required for the binding of DNA, so that these species usually take up DNA only of the same species. Regulation of competence in B. subtilis It is achieved through a two-component regulatory system: a sensor protein (ComP) and a response regulator (ComA) protein. 1. When the cell runs out of nutrients and the population reach a high density registered by ComP. 2. ComP autophosphorylates itself. 3. ComP ~P transfer ~P to ComA. 4. ComA~P is an active transcriptional activator for several genes, including some required for competence. 5. Eventually, another transcriptional activator, ComK is made. 12 It is directly responsible for activating the transcription of other com genes, including those that form the transformation machinery. How does the cell know that other B. subtilis cells are nearby and that it should induce competence? 1. High cell density is signaled through small peptides, competence pheromones that are excreted by the bacteria as they multiply. 2. Cells become competent only in the presence of high concentrations of these peptides. 3. This is a phenomenon called quorum sensing. The small molecules are known as including homoserine lactones that signal cell density in some G – bacteria. 4. In B. subtilis, the major competence pheromone peptide is called ComX and is cut out of a longer polypeptide, the product of the comX gene. 5. The product of gene comQ which is immediately upstream of comX is a protease that cut the longer polypeptide. 6. Once the peptide has been cut out of the longer molecule, it binds to the ComP protein in the membrane and trigger its autophosphorylation. 7. At best, only about 10% of B. subtilis cells ever become competent, no matter how favorable the conditions or how high the cell density. This has been called a bistable state and seems to be determined somehow by autoregulation of the ComK activator protein. Regulation of competence development in B. subtilis by quorum sensing A 1. ComP in the membrane senses a high concentration of the ComX peptide, and phosphorylates itself by transferring a phosphate from ATP. 2. The phosphate is then transferred to ComA. 3. ComA activates the transcription of many genes including comK. 4. ComK is an activator of the com genes. B 1. In another pathway, a peptide sometimes called CSF (competence-stimulating factor) processed from the signal sequence of another protein (PhrC) is imported into the cell by the SpoOK oligopeptide permease. 2. CFS indirectly activates ComA~P by inactivating RapC. 13 Relationship between competence, sporulation, and other cellular states 1. About the same time as B. subtilis reaches the stationary phase, some cells acquire competence and some cells sporulate, forming the endospore. 2. Sporulation allows a bacterium to enter a dormant state and survive adverse conditions, such as starvation, irradiation and heat. 3. To coordinate sporulation and competence, B. subtilis cells may produce other competence peptide. (1) There are at least two such peptides that regulate ComA indirectly by inhibiting proteins, Rap proteins, which bind to the C-terminal DNA-binding domain of ComA~P and prevent it from binding to DNA and activating transcription. (2) These peptides (CSF) are processed from the signal sequences of longer polypeptides, the products of the phr genes, and are transported into cell by the oligopeptide permease, SpoOK. (3) The spoOK gene is an example of a regulatory gene that is required for sporulation and also for the development of competence. Three questions for natural transformation A. How efficient is DNA uptake? - Donor DNA is radioactively labeled by growing the cells in medium containing 32P. - The radioactive DNA is then extracted and mixed with competent cells. - The mixture is treated with DNase at various times. - Any DNA that is not degraded and survives intact must have been taken up by the cells, where it is protected from the DNase. - Collect cells on filter and count the radioactivity. Degraded DNA will pass through filter. - The radioactivity on the filter is compared with the total radioactivity of the DNA that was added to the cell. - This kind of experiment shows that some competent bacteria take up DNA very efficiently. B. Can only DNA of the same species enter a given cell? - The same experiment demonstrates that some types of bacteria take up DNA from only their own species (ex., Neisseria gonorrhoeae and Haemophilus influenzae) whereas others (B. subtilis) can take up DNA from any source. - Bacteria that preferentially take up the DNA of their own species do so because their DNA contains specific uptake 14 sequences. Transformation in Streptococcus pneumoniae 1. Competence-stimulating peptide accumulates as the cells reach a high density. 2. Double-stranded DNA binds to the cell, and one strand is degraded. Transformation in Haemophilus influenzae 3. The basic transformation scheme may differ among different types of naturally competent bacteria. 4. In H. influenzae, the double-stranded DNA may first take up in subcellular compartments called transformsomes. C. Are both of the DNA strands taken up and incorporated into the cellular DNA? - Experiments have shown that only double-stranded DNA can bind to specific receptors on the cell surface, i.e., single-stranded DNA can not transform cells and yield recombinant types. - However, the transforming DNA enters a “eclipse” period for a short time after it is added to competent cells, as expected if it enters the cell in a single-stranded state. - The following is the design for experiment: Whether both of the DNA strands taken up and incorporated into the cellular DNA? As shown in Fig. 6.8, transforms were observed depending on the time the DNA was extracted from the cells. 1. Time 1, the DNA is still outside the cells and accessible to the DNase. No Arg+ transformants are observed because the Arg+ donor DNA is all destryed by DNase. 2. Time 2, some of DNA is now inside the cells, where it can not be degraded by the DNase, but this DNA is single-stranded. It has not yet recombined with bacterial chromosomal DNA, and so no Arg+ transformants observed in step 4. 3. Time 3, when some of the DNA has recombined with bacterial chromosomal DNA, and so is again double-stranded, do transformants appear in step 4. ■ Thus, the transformingf DNA enters the eclipse period for a short time after it is added to competent cells, as expected if it enters the cells in a single-stranded state. Neither plasmids nor phage DNAs can be efficiently introduced into naturally competent cells for two reasons: 15 1. They must double stranded to replicate. Natural transformation requires breakage of double-stranded DNA and degradation of one of the two strands so that a linear single strand can enter the cells. 2. They must recyclize. However, pieces of plasmid or phage DNA can not recyclize if there are no repeated or complementary sequences at their ends. - To overcome the problem, they are usually dimerized and multimerized into long concatemers. - If a dimerized plasmid or phage DNA is cut only once, it still has complementary sequences at its ends that can recombine to recyclize the plasmid. - Evidences to support: Most preparations of plasmid or phage DNAs contain some dimers. Role of natural transformation 1. Nutrition – Organisms may take up DNA for use as a carbon and nitrogen source. 2. Repair – Cells may take up DNA from other cells to repair damage to their own DNA. 16