Chapte r 17 Rearrangement of DNA

17.1 Introduction 17.2 The mating pathway is triggered by pheromone-receptor interactions 17.3 The mating response activates a G protein 17.4 Yeast can switch silent and active loci for mating type 17.5 The MAT locus codes for regulator proteins 17.6 Silent cassettes at HML and HMR are repressed 17.7 Unidirectional transposition is initiated by the recipient MAT locus 17.8 Regulation of HO expression 17.9 Trypanosomes switch the VSG frequently during infection New VSG sequences are generated by gene switching VSG genes have an unusual structure The bacterial Ti plasmid causes crown gall disease in plants T-DNA carries genes required for infection Transfer of T-DNA resembles bacterial conjugation Selection of amplified genomic sequences Transfection introduces exogenous DNA into cells Genes can be injected into animal eggs ES cells can be incorporated into embryonic mice Gene targeting allows genes to be replaced or knocked out

Amplification refers to the production of additional copies of a chromosomal sequence, found as intrachromosomal or extrachromosomal DNA. Transgenic animals are created by introducing new DNA sequences into the germline via addition to the egg Introduction

Amplification refers to the production of additional copies of a chromosomal sequence, found as intrachromosomal or extrachromosomal DNA. Transgenic animals are created by introducing new DNA sequences into the germline via addition to the egg Introduction

Figure 17.1 Mating type controls several activities The mating pathway is triggered by signal transduction

Figure 17.2 The yeast life cycle proceeds through mating of MATa and MATa haploids to give heterozygous diploids that sporulate to generate haploid spores The mating pathway is triggered by signal transduction

Figure 17.3 Either a or a factor/receptor interaction triggers the activation of a G protein, whose bg subunits transduce the signal to the next stage in the pathway The mating pathway is triggered by signal transduction

Figure 17.4 The same mating type response is triggered by interaction of either pheromone with its receptor. The signal is transmitted through a series of kinases to a transcription factor; there may be branches to some of the final functions The mating pathway is triggered by signal transduction

Figure Homologous proteins are found in signal transduction cascades in a wide variety of organisms The mating pathway is triggered by signal transduction

Figure 17.5 Changes of mating type occur when silent cassettes replace active cassettes of opposite genotype; when transpositions occur between cassettes of the same type, the mating type remains unaltered Yeast can switch silent and active loci for mating type

Figure 17.6 Silent cassettes have the same sequences as the corresponding active cassettes, except for the absence of the extreme flanking sequences in HMRa. Only the Y region changes between a and a types Yeast can switch silent and active loci for mating type

Figure 17.7 In diploids the a1 and a2 proteins cooperate to repress haploid-specific functions. In a haploids, mating functions are constitutive. In a haploids, the a2 protein represses a mating functions, while a1 induces a mating functions Yeast can switch silent and active loci for mating type

Figure 17.8 Combinations of PRTF, a1, a1 and a2 activate or repress specific groups of genes to correspond with the mating type of the cell Yeast can switch silent and active loci for mating type

Figure 9.10 RNA polymerase initially contacts the region from -55 to +20. When sigma dissociates,the core enzyme contracts to -30; when the enzyme moves a few base pairs, it becomes more compactly organized into the general elongation complex. 9.4 Sigma factor controls binding to DNA

Figure 17.6 Silent cassettes have the same sequences as the corresponding active cassettes, except for the absence of the extreme flanking sequences in HMRa. Only the Y region changes between a and a types Silent cassettes at HML and HMR are repressed

Figure 17.9 HO endonuclease cleaves MAT just to the right of the Y region, generating sticky ends with a base overhang Unidirectional transposition is initiated by the recipient MAT locus

Figure Cassette substitution is initiated by a double-strand break in the recipient (MAT) locus, and may involve pairing on either side of the Y region with the donor (HMR or HML) locus Unidirectional transposition is initiated by the recipient MAT locus

Figure 14.5 Recombination is initiated by a double- strand break, followed by formation of single- stranded 3 ends, one of which migrates to a homologous duplex. 9.4 Sigma factor controls binding to DNA

Figure Switching occurs only in mother cells; both daughter cells have the new mating type. A daughter cell must pass through an entire cycle before it becomes a mother cell that is able to switch again Regulation of HO expression

Figure Three regulator systems act on transcription of the HO gene. Transcription occurs only when all repression is lifted Regulation of HO expression

Figure A trypanosome passes through several morphological forms when its life cycle alternates between a tsetse fly and mammalian host Trypanosomes rearrange DNA to express new surface antigens

Figure The C- terminus of VSG is cleaved and covalently linked to the membrane through a glycolipid Trypanosomes rearrange DNA to express new surface antigens

Figure VSG genes may be created by duplicative transfer from an internal or telomeric basic copy into an expression site, or by activating a telomeric copy that is already present at a potential expression site Trypanosomes rearrange DNA to express new surface antigens

Figure Internal basic copies can be activated only by generating a duplication of the gene at an expression-linked site 17.7 Trypanosomes rearrange DNA to express new surface antigens

Figure Telomeric basic copies can be activated in situ; the size of the restriction fragment may change (slightly) when the telomere is extended Trypanosomes rearrange DNA to express new surface antigens

Figure The expression-linked copy of a VSG gene contains barren regions on either side of the transposed region, which extends from ~1000 bp upstream of the VSG coding region to a site near the 3 terminus of the mRNA Trypanosomes rearrange DNA to express new surface antigens

Figure An Agrobacterium carrying a Ti plasmid of the nopaline type induces a teratoma, in which differentiated structures develop. Photograph kindly provided by Jeff Schell Interaction of Ti plasmid DNA with the plant genome

Figure Ti plasmids carry genes involved in both plant and bacterial functions Interaction of Ti plasmid DNA with the plant genome

Figure T-DNA is transferred from Agrobacterium carrying a Ti plasmid into a plant cell, where it becomes integrated into the nuclear genome and expresses functions that transform the host cell Interaction of Ti plasmid DNA with the plant genome

Figure Nopaline and octopine Ti plasmids carry a variety of genes, including T- regions that have overlapping functions 17.8 Interaction of Ti plasmid DNA with the plant genome

Figure The vir region of the Ti plasmid has six loci that are responsible for transferring T-DNA to an infected plant Interaction of Ti plasmid DNA with the plant genome

Figure Acetosyringone (4-acetyl-2,6- dimethoxyphenol) is produced by N. tabacum upon wounding, and induces transfer of T-DNA from Agrobacterium Interaction of Ti plasmid DNA with the plant genome

Figure The two- component system of VirA-VirG responds to phenolic signals by activating transcription of target genes Interaction of Ti plasmid DNA with the plant genome

Figure T-DNA has almost identical repeats of 25 bp at each end in the Ti plasmid. The right repeat is necessary for transfer and integration to a plant genome. T-DNA that is integrated in a plant genome has a precise junction that retains 1-2 bp of the right repeat, but the left junction varies and may be up to 100 bp short of the left repeat Interaction of Ti plasmid DNA with the plant genome

Figure T-DNA is generated by displacement when DNA synthesis starts at a nick made at the right repeat. The reaction is terminated by a nick at the left repeat Interaction of Ti plasmid DNA with the plant genome

Figure T-DNA is generated by displacement when DNA synthesis starts at a nick made at the right repeat. The reaction is terminated by a nick at the left repeat Interaction of Ti plasmid DNA with the plant genome

Amplification refers to the production of additional copies of a chromosomal sequence, found as intrachromosomal or extrachromosomal DNA Selection of amplified genomic sequences

Figure The dhfr gene can be amplified to give unstable copies that are extrachromosomal (double minutes) or stable (chromosomal). Extrachromosomal copies arise at early times Selection of amplified genomic sequences

Figure Amplified copies of the dhfr gene produce a homogeneously staining region (HSR) in the chromosome. Photograph kindly provided by Robert Schimke Selection of amplified genomic sequences

Figure Amplified extrachromosomal dhfr genes take the form of double-minute chromosomes, as seen in the form of the small white dots. Photograph kindly provided by Robert Schimke Selection of amplified genomic sequences

Figure Amplified extrachromosomal dhfr genes take the form of double-minute chromosomes, as seen in the form of the small white dots. Photograph kindly provided by Robert Schimke Selection of amplified genomic sequences

Transfection of eukaryotic cells is the acquisition of new genetic markers by incorporation of added DNA. Transgenic animals are created by introducing new DNA sequences into the germline via addition to the egg Exogenous sequences can be introduced into cells and animals by transfection

Figure Transfection can introduce DNA directly into the germ line of animals Exogenous sequences can be introduced into cells and animals by transfection

Figure A transgenic mouse with an active rat growth hormone gene (left) is twice the size of a normal mouse (right). Photograph kindly provided by Ralph Brinster Exogenous sequences can be introduced into cells and animals by transfection

Figure Hypogonadism of the hpg mouse can be cured by introducing a transgene that has the wild- type sequence Exogenous sequences can be introduced into cells and animals by transfection

Figure ES cells can be used to generate mouse chimeras, which breed true for the transfected DNA when the ES cell contributes to the germ line Exogenous sequences can be introduced into cells and animals by transfection

Figure A transgene containing neo within an exon and TK downstream can be selected by resistance to G418 and loss of TK activity Exogenous sequences can be introduced into cells and animals by transfection

Figure Transgenic flies that have a single, normally expressed copy of a gene can be obtained by injecting D. melanogaster embryos with an active P element plus foreign DNA flanked by P element ends Exogenous sequences can be introduced into cells and animals by transfection

17.11 Summary Yeast mating type is determined by whether the MAT locus carries the a or sequence. Additional, silent copies of the mating- type sequences are carried at the loci HML and HMRa. Trypanosomes carry >1000 sequences coding for varieties of the surface antigen.

Agrobacteria induce tumor formation in wounded plant cells. The wounded cells secrete phenolic compounds that activate vir genes carried by the Ti plasmid of the bacterium. Endogenous sequences may become amplified in cultured cells. Exposure to methotrexate leads to the accumulation of cells that have additional copies of the dhfr gene. New sequences of DNA may be introduced into a cultured cell by transfection or into an animal egg by microinjection Summary