Genética Microbiana y de Levaduras II Dra. Mónica Vásquez Figures from Brock Biology of Microorganisms

Maps of the transposable elements IS2 and Tn5 Maps of the transposable elements IS2 and Tn5. The red arrows underneath each map indicate the inverted repeats. The arrows above the maps show the direction of transcription of any genes on the elements. Tnp is the gene encoding the transposase. The transposase genes of these two elements are not closely related. (a) IS2 is an insertion sequence of 1327 base pairs with inverted repeats of 41 base pairs at its ends. (b) Tn5 is a composite transposon of 5.7 kilobase pairs with the insertion sequences IS50L and IS50R at its left and right ends, respectively. IS50L is not capable of independent transposition because there is a nonsense mutation (see Section 10.2) marked by a blue cross in its transposase gene. Figure: 10-28a-b Caption: Maps of the transposable elements IS2 and Tn5. The red arrows underneath each map indicate the inverted repeats. The arrows above the maps show the direction of transcription of any genes on the elements. Tnp is the gene encoding the transposase. The transposase genes of these two elements are not closely related. (a) IS2 is an insertion sequence of 1327 base pairs with inverted repeats of 41 base pairs at its ends. (b) Tn5 is a composite transposon of 5.7 kilobase pairs with the insertion sequences IS50L and IS50R at its left and right ends, respectively. IS50L is not capable of independent transposition because there is a nonsense mutation (see Section 10.2) marked by a blue cross in its transposase gene. Otherwise, the two IS50 elements are very nearly identical. Note that these two IS50 elements are inverted with respect to each other. The genes kan, str, and bleo, confer resistance to the antibiotics kanamycin (and neomycin), streptomycin, and bleomycin. Interestingly, streptomycin resistance is not expressed in Escherichia coli. Otherwise, the two IS50 elements are very nearly identical. Note that these two IS50 elements are inverted with respect to each other. The genes kan, str, and bleo, confer resistance to the antibiotics kanamycin (and neomycin), streptomycin, and bleomycin. Interestingly, streptomycin resistance is not expressed in Escherichia coli.

Figure: 10-29 Caption: Transposition. Insertion of a transposable element generates a duplication of the target sequence. Note the presence of inverted repeats (IRs) at the ends of the transposable element. Figure 10.30 shows more detailed models of the mechanism of transposition. Transposition. Insertion of a transposable element generates a duplication of the target sequence. Note the presence of inverted repeats (IRs) at the ends of the transposable element. Figure 10.30 shows more detailed models of the mechanism of transposition.

Mechanisms of transposition Mechanisms of transposition. (a) In both conservative and replicative transposition the transpose makes cuts (marked with arrows) in the DNA strands at the end of the transposable element (orange) and at the target site (red). The number and location of the cuts may vary depending on the mechanism. (b) The target site becomes ligated to the transposable element. The black dots indicate free ends of DNA strands at which replication can occur (Section 7.5). (c) In conservative tranposition further cuts are made before DNA replication/repair occurs, and the transposable element is lost from the donor DNA. (d) Repair leads to duplication of the target site and completion of transposition to the new site. (e) In replicative transposition, replication occurs without the cutting of the transposable element from the donor site leading to two copies of the transposable element as part of a cointegrate. Note, however, this has led to the joining of the donor (light green) and target (dark green) DNA molecules together. (f) These molecules are separated (resolved) in a further reaction. Resolution of cointegrates is shown in more detail in Figure 10.31. Figure: 10-30a-f Caption: Mechanisms of transposition. (a) In both conservative and replicative transposition the transpose makes cuts (marked with arrows) in the DNA strands at the end of the transposable element (orange) and at the target site (red). The number and location of the cuts may vary depending on the mechanism. (b) The target site becomes ligated to the transposable element. The black dots indicate free ends of DNA strands at which replication can occur (Section 7.5). (c) In conservative tranposition further cuts are made before DNA replication/repair occurs, and the transposable element is lost from the donor DNA. (d) Repair leads to duplication of the target site and completion of transposition to the new site. (e) In replicative transposition, replication occurs without the cutting of the transposable element from the donor site leading to two copies of the transposable element as part of a cointegrate. Note, however, this has led to the joining of the donor (light green) and target (dark green) DNA molecules together. (f) These molecules are separated (resolved) in a further reaction. Resolution of cointegrates is shown in more detail in Figure 10.31.

Figure: 10-31 Caption: Replicative transposition. After the formation of single-strand cuts, a cointegrate structure arises by association of the two molecules (see Figure 10.30). After recombination, resolution of the cointegrate structure leads to the release of the original transposon and duplication of the transposon in the target molecule. Replicative transposition. After the formation of single-strand cuts, a cointegrate structure arises by association of the two molecules (see Figure 10.30). After recombination, resolution of the cointegrate structure leads to the release of the original transposon and duplication of the transposon in the target molecule.

Transposon mutagenesis. The transposon moves into the middle of gene 2 Transposon mutagenesis. The transposon moves into the middle of gene 2. Gene 2 is now disrupted by the transposon and is inactivated. Gene A of the transposon will be expressed in both locations. Figure: 10-32 Caption: Transposon mutagenesis. The transposon moves into the middle of gene 2. Gene 2 is now disrupted by the transposon and is inactivated. Gene A of the transposon will be expressed in both locations.

Structure of two naturally occurring integrons from Pseudomonas Structure of two naturally occurring integrons from Pseudomonas. The integron In0 has the basic set of genes: intI1, encodes integrase; attI, the site where site-specific integration can occur; P, a promoter; and sulI, a gene conferring sulfonamide resistance that contains its own promoter. The integron In7 contains all of these genes, but in addition, a gene cassette has been integrated. All cassettes contain a site (blue square) for site-specific recombination. This cassette contains aadB, which confers resistance to certain aminoglycoside antibiotics. Figure: 10-33 Caption: Structure of two naturally occurring integrons from Pseudomonas. The integron In0 has the basic set of genes: intI1, encodes integrase; attI, the site where site-specific integration can occur; P, a promoter; and sulI, a gene conferring sulfonamide resistance that contains its own promoter. The integron In7 contains all of these genes, but in addition, a gene cassette has been integrated. All cassettes contain a site (blue square) for site-specific recombination. This cassette contains aadB, which confers resistance to certain aminoglycoside antibiotics.

Figure: 14-01 Caption: Schematic, cut-away view of a eukaryotic cell. Although all eukaryotic cells contain a nucleus, not all organelles or other structures shown are present in all eukaryotic cells. Schematic, cut-away view of a eukaryotic cell. Although all eukaryotic cells contain a nucleus, not all organelles or other structures shown are present in all eukaryotic cells.

Photomicrograph of a field of cells of the baker’s yeast Saccharomyces cerevisiae. (a) Bright-field.

Life cycle of a typical yeast, Saccharomyces cerevisiae Life cycle of a typical yeast, Saccharomyces cerevisiae. A haploid cell of S. cerevisiae contains 16 chromosomes. Figure: 14-08 Caption: Life cycle of a typical yeast, Saccharomyces cerevisiae. A haploid cell of S. cerevisiae contains 16 chromosomes.

Life cycle of yeasts Budding Yeast Fission Yeast

Mating Haploid cells produce specific mating factors, which bind to specific cell-surface receptors Changes gene expression Induces cell fusion, which produces a diploid cell

Yeast sexual cycle Caught in the act: cell attachment, cell fusion and nuclear fusion in an electron micrograph Haploid cells produce mating peptide pheromones, i.e. a-factor and alpha-factor, to which the mating partner responds to prepare for mating

Mating-type (MAT) locus Two alleles that determine mating type MAT MATa How do these alleles determine mating type? How do these alleles switch?

Figure: 14-09 Caption: The cassette mechanism involved in the switch in yeast from mating type a to a and back again. Whichever “cassette” is inserted at the active locus (reading head) determines the mating type. The process shown is reversible, so type a can also revert to type a . The cassette mechanism involved in the switch in yeast from mating type a to a and back again. Whichever “cassette” is inserted at the active locus (reading head) determines the mating type. The process shown is reversible, so type a can also revert to type a .

Mating-type switching Cells switch mating type (almost) every generation Only mother cells (not buds) switch

Figure: 14-10a Caption: Electron micrographs of the mating process in a yeast, Hansenula wingei. (a) Two cells have fused at the point of contact and have sent out protuberances toward each other. Electron micrographs of the mating process in a yeast, Hansenula wingei. (a) Two cells have fused at the point of contact and have sent out protuberances toward each other.

Figure: 14-10b Caption: Electron micrographs of the mating process in a yeast, Hansenula wingei. (b) Late stage of mating. The nuclei of the two cells have fused, and the diploid bud has formed at a right angle to the conjugation tube. This bud eventually separates and becomes the forerunner of a diploid cell line. A single cell of Hansenula is about 10 µm in diameter. Electron micrographs of the mating process in a yeast, Hansenula wingei. (b) Late stage of mating. The nuclei of the two cells have fused, and the diploid bud has formed at a right angle to the conjugation tube. This bud eventually separates and becomes the forerunner of a diploid cell line. A single cell of Hansenula is about 10 µm in diameter.

Growth by budding division in Saccharomyces cerevisiae Growth by budding division in Saccharomyces cerevisiae. Note the pronounced nucleus. Phase-contrast micrograph. A single cell of S. cerevisiae is about 8 µm in diameter. Figure: 14-22 Caption: Growth by budding division in Saccharomyces cerevisiae. Note the pronounced nucleus. Phase-contrast micrograph. A single cell of S. cerevisiae is about 8 µm in diameter.

HO, which codes for an endonuclease Genetic analysis (i.e. the use of mutants) shows that the following genes are required for mating-type switching to occur: MAT HO, which codes for an endonuclease HMLalpha (for MATa -> MATalpha switch) HMRa (for MATalpha -> MATa switch) All except HO are on yeast chromosome 3. The "cassette" model was proposed to explain mating-type switching, which occurs at too high a frequency to be due to normal mutation events. The model proposes that the HMLalpha and HMRa loci contain "silent copies" of alpha and a mating type genes. Replicas of either can be copied into MAT, the active locus, where they are expressed.

The gene products shown are regulatory proteins, that control the ability of the yeast to mate (and other aspects of its phenotype). alpha1 is a positive regulator, that switches on genes required for the alpha phenotype, including alpha factor, a secreted pheromone alpha2 is a negative regulator that turns off a-specific genes in diploid cells, a1 and alpha2 combine to inhibit alpha1 (and hence all the genes it regulates) and repress HO, and turn on the meiosis pathway if the diploid cells are starved