We describe a general method for analyzing the genetic fine structure of plasmid-borne genes in yeast. Previously we had reported that a linearized plasmid is efficiently rescued by recombination with a homologous restriction fragment when these are co-introduced by DNA-mediated transformation of yeast. Here, we show that a mutation can be localized to a small DNA interval when members of a deletion series of wild-type restriction fragments are used in the rescue of a linearized mutant plasmid. The resolution of this method is to at least 30 base pairs and is limited by the loss of a wild-type marker with proximity to a free DNA end. As a means for establishing the nonidentity of two mutations, we determined the resolution of two-point crosses with a mutant linearized plasmid and a mutant homologous restriction fragment. Recombination between mutations separated by as little as 100 base pairs was detected. Moreover, the results indicate that exchange within a marked interval results primarily from one of two single crossovers that repair the linearized plasmid. These approaches to mapping the genetic fine structure of plasmids should join existing methods in a robust approach to the mutational analysis of gene structure in yeast.
We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.
The molecular products of DNA double strand break repair were investigated after transformation of yeast (Saccharomyces cerevisiae) with linearized plasmid DNA. DNA of an autonomous yeast plasmid cleaved to generate free ends lacking homology with the yeast genome, when used in transformation along with sonicated non-homologous carrier DNA, gave rise to transformants with high frequency. Most of these transformants were found to harbor a head-to-head (inverted) dimer of the linearized plasmid. This outcome of transformation contrasts with that observed when the carrier DNA is not present. Transformants occur at a much reduced frequency and harbor either the parent plasmid or a plasmid with deletion at the site of the cleavage. When the linearized plasmid is introduced along with sonicated carrier DNA and a homologous DNA restriction fragment that spans the site of plasmid cleavage, homologous recombination restores the plasmid to its original circular form. Inverted dimer plasmids are not detected. This relationship between homologous recombination and a novel DNA transaction that yields rearrangement could be important to the cell, as the latter could lead to a loss of gene function and lethality.
The formation of an inverted dimer plasmid on transformation with linear molecules is formally analogous to the fusion of the daughters of a broken chromosome at their broken ends. In the latter case, this leads to the formation of a dicentric chromosome, which could break at anaphase. Hence the process is cyclic. Similarly, when our linear molecules are modified by the addition of a cloned yeast centromere, dicentric inverted dimers are not obtained. Instead, we obtain monocentric plasmids with partial duplication and deletion that apparently derive from a process of fusion, bridge-breakage, and fusion. This is not surprising, since it is known that dicentric plasmids undergo breakage in yeast (Mann and Davis 1983). However, any apparent similarity of this process to that which occurs with a broken chromosome in maize must be tempered by the special nature of the transformation process. Most significantly, inverted dimers are rare when sonicated carrier DNA is not present during the transformation. This requirement is not understood, but it is a condition that may not be met in a yeast cell harboring a broken chromosome. It is possible that carrier DNA induces a repair process that results in fusion. On the other hand, a property of the transformation process that results in an inhibition of fusion may be overcome by the presence of carrier DNA. Most inverted dimers are apparently formed from an interaction between two input linear molecules. We cannot rule out the possibility that a minor fraction derive from a single molecule. Thus, the fusion of two input molecules is a much more efficient process than a replicative process that could occur with single linear molecule. For a similar fusion process to occur with a broken yeast chromosome, replication would be required. We do not know if a broken yeast chromosome can replicate. Evidence consistent with the presence of a breakage-fusion-bridge process in yeast has been obtained through the formation of dicentric chromosomes via meiotic recombination (Haber et al. 1984). Spores from these meioses sometimes give rise to a clone that is mixed for markers of the chromosome that could have been dicentric. A process of fusion-bridge-breakage could account for the formation of some of these mixed clones. However, the dicentric chromosomes apparently often survive meiotic disjunction and break in the spore's first mitotic anaphase or possibly in a later generation. Thus, the interpretation of the origin of these mixed clones is uncertain. Some aspects of the fusion process are especially intriguing.(ABSTRACT TRUNCATED AT 400 WORDS)
Chi recombinational hotspots are sites around which the rate of Rec-promoted recombination in bacteriophage lambda is elevated. Examination of a derivative of lambda into which the plasmid pBR322 was inserted reveals that pBR322 lacks Chi sites. Using this lambda-pBR322 hybrid, we obtained mutations creating Chi sites at three widely separated loci within pBR322. Nucleotide sequence analysis reveals that the mutations are single base-pair changes creating the octamer 5' GCTGGTGG 3'. This sequence is present at three previously analyzed Chi sites in lambda, and all analyzed mutations creating or inactivating these Chi sites occur within this octamer. We conclude that Chi is 5' GCTGGTGG 3', or its complement, or both.
The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as approximately 45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved.
Drosophila olfactory aversive conditioning has served as a powerful model system with which to elucidate the molecular and neuronal mechanisms underlying memory formation. In the typical protocol, flies are exposed to a constant odor stream while receiving a pulsed electric shock in the conditioning tube of a manual apparatus. We have devised a simple, low-cost semi-automated conditioning apparatus that computationally controls the delivery of odor and shock. A semiconductor-based odor sensor is employed to monitor the change of odor concentration in the training tube. The system thus allows electric shocks to be precisely matched with odor concentration in the training tube. We found that short-term memory performance was improved with a pulsed odor flow protocol, in which odor is presented in short pulses, each paired with electric shock, rather than as a constant flow. The effect of pulsed odor flow might be ascribed to the phenomenon of 'conditioned approach', where approach toward an odor is induced when the electric shock is presented before odor pulse ends. Our data shows that the system is applicable to the study of olfactory memory formation and to the examination of conditioning parameters at a level of detail not practical with a manual apparatus.
The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. Here we show that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. We conclude that endocytosis is required for the activation of the Toll signaling pathway.
The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor's phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time.