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Chromosome stability models are usually qualitative models derived from molecular-genetic mechanisms for DNA repair, DNA synthesis, and cell division. While qualitative models are informative, they are also challenging to reformulate as precise quantitative models. In this report we explore how (A) laboratory experiments, (B) quantitative simulation, and (C) seriation algorithms can inform models of chromosome stability. Laboratory experiments were used to identify 19 genes that when over-expressed cause chromosome instability in the yeast Saccharomyces cerevisiae To better understand the molecular mechanisms by which these genes act, we explored their genetic interactions with 18 deletion mutations known to cause chromosome instability. Quantitative simulations based on a mathematical model of the cell cycle were used to predict the consequences of several genetic interactions. These simulations lead us to suspect that the chromosome instability genes cause cell-cycle perturbations. Cell-cycle involvement was confirmed using a seriation algorithm, which was used to analyze the genetic interaction matrix to reveal an underlying cyclical pattern. The seriation algorithm searched over 10(14) possible arrangements of rows and columns to find one optimal arrangement, which correctly reflects events during cell cycle phases. To conclude, we illustrate how the molecular mechanisms behind these cell cycle events are consistent with established molecular interaction maps.

A healthy cell must maintain chromosome integrity during each phase of the cell cycle. Yet during the cell cycle, defects in DNA repair, DNA synthesis, and chromosome transmission can cause chromosome instability. Here, we build a cyclical model for chromosome maintenance by assaying the genetic interactions of pairs of genes that each normally functions to maintain chromosomes. First, we confirm that 19 genes affect chromosome maintenance by overexpressing the respective genes using an assay for chromosome instability. We then introduce plasmids containing these 19 genes into 18 deletion mutant strains each of which is known to cause chromosome maintenance defect. By conditionally overexpressing each of the 19 genes in each of the 18 deletion strains, we observe 34 cases of synthetic dosage lethality, 58 of synthetic dosage sickness, and 26 of suppression (out of 349 combinations tested). Ordinarily, given a large matrix of genetic interactions, it is computationally intractable (and NP hard) to compute all permutations of the arrangements of the rows and columns, which is necessary for finding an optimum arragement of entries for a given pattern of functional significance. Here, using our small yet dense matrix we rearrange the matrix into an optimal cycle. By optimally reordering the matrix representation of these genetic interactions using integer linear programming, we find that the optimal order of the functions of the genes approximates the temporal order of processes during the yeast cell cycle. To further validate this theoretical approach, we computed in silico genetic interactions derived from a predetermined cyclical model and confirmed that the predicted cycle was consistent. These results suggest that matrices from other genetic experiments could be computationally rearranged using this method to reveal an underlying cycle that is biological meaningful.

A temperature-sensitive lethal mutant ofStaphylococcus aureuswas found to harbor a mutation in the uncharacterized two-component histidine kinase (HK)-response regulator (RR) pair encoded byyycFG; orthologues ofyycFGcould be identified in the genomes ofBacillus subtilisand other gram-positive bacteria. Sequence analysis of the mutant revealed a point mutation resulting in a nonconservative change (Glu to Lys) in the regulator domain of the RR at position 63. To confirm that this signal transduction system was essential, a disrupted copy of either the RR (yycF) or the HK (yycG) was constructed with a set of suicide vectors and used to generate tandem duplications in the chromosome. Resolution of the duplications, leaving an insertion in either theyycFor theyycGcoding region, was achieved only in the presence of an additional wild-type copy of the two open reading frames. Phenotypic characterization of the conditional lethal mutant showed that at permissive growth conditions, the mutant was hypersusceptible to macrolide and lincosamide antibiotics, even in the presence of theermBresistance determinant. Other mutant phenotypes, including hypersensitivity to unsaturated long-chain fatty acids and suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations, suggest that the role of the two-component system includes the proper regulation of bacterial cell wall or membrane composition. The effects of this point mutation are strongly bactericidal at the nonpermissive temperature, indicating that this pathway provides an excellent target for the identification of novel antibiotics.

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governingStaphylococcus aureuspathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300S. aureusinsertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important forS. aureusin vivo survival. Our findings extend the understanding of pathogenic mechanisms employed byS. aureusto ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

A genetic strategy identified Salmonella typhimurium strains carrying large (> 40 kb) Tn 10-catalyzed inversions; the inverted segments were characterized by XbaI digestion and pulsed field gel electrophoresis. Two size classes of large inversions were found. More than half of the inversions extended 40-80 kb either clockwise or counterclockwise of the original Tn10 site. The remaining inversions extended up to 1620 kb (33% of the genome), but the distal endpoints of these inversions were not randomly scattered throughout the chromosome. Rather, each Tn10 repeatedly yielded similar (though not identical) inversions. The biased endpoint selection may reflect the limited search for target DNA sequences by the Tn10 transposase, and the spatial proximity of the donor and target regions in the folded S. typhimurium nucleoid. Using this interpretation, the data suggest that DNA sequences 40-80 kb clockwise and counterclockwise of the insertion site are in spatial proximity with the insertion, perhaps reflecting the organization of DNA into approximately 120-kb nucleoid domains. In addition, the data predict the spatial proximity of several distant DNA regions, including DNA sequences equidistant from the origin of DNA replication.

The murB gene of Salmonella typhimurium was cloned and found to be 75% and 82% identical to the DNA and protein sequences, respectively, of the same gene in Escherichia coli. These identities are among the lowest recorded between the two bacteria. Nevertheless, wild-type S. typhimurium murB complemented the known temperature-sensitive E. coli mutant, and wild-type E. coli murB complemented three temperature-sensitive mutants of S. typhimurium. The 5S rRNA gene, rrfB, and the region between murB and rrfB were also cloned and sequenced. The rrfB gene of S. typhimurium differs from rrfB of E. coli in only 2 of 120 nt, but the region between murB and rrfB has diverged greatly and includes a sequence that elosely resembles a repetitive extragenic palindrome of the type normally associated with E. coli. Previous comparisons of gene divergence have suggested that the chromosomal mutation rate is lower in the vicinity of the origin of replication. However, the S. typhimurium murB gene, located 6 map minutes from the origin of replication, is highly substituted at synonymous sites and the sequence between murB and rrfB is significantly modified as well. Thus, murB is an exception to the general observation that genes near the origin of replication show less divergence than do genes elsewhere in the bacterial chromosome.

Two internal promoters in the his operon of Salmonella typhimurium have been precisely mapped genetically. The internal promoters are found in, or very close to, gene border regions in the his operon. The his operon was examined for the presence of additional internal promoters whose transcripts were sensitive to rho-mediated transcription termination and therefore had escaped detection. No new internal promoters were found. It is argued that the internal promoters described here are not likely to be fortuitous message start sites, but may play a physiologically important role in operon expression.

Generalized transducing fragments that have redundant sequences in direct order can circularize during transduction events. The length of the required redundant sequences can be at least as short as IS10 (1.4 kb) (KLECRNER 1977).The circular transduced fragment is able to recombine with homologous sequences in the chromosome. Circularization and insertion of transduced fragments allow addition of segments to the bacterial chromosome rather than replacement of recipient segments as in a normal transductional cross. It also provides a method for translocation of bacterial genes to a variety of specific sites on the chromosome in either orientation. The significance of these events to bacterial eiolution is discussed.

Promoters located within the Tn10 insertion element cause transcription of "host" sequences adjacent to both ends of the inserted Tn10 element. These promoters are usually not observed in genetic experiments because their transcripts are efficiently terminated at nearby rho-dependent termination sites. The observations presented here provide an explanation for several confusing aspects of transposon behavior and suggest the possibility that many transposons possess promoters that have escaped detection for similar reasons.

A number of genetic methods for the isolation, characterization and manipulation of large chromosomal inversions in Salmonella typhimurium are described. One inversion-carrying mutant is characterized in detail and used to demonstrate a number of unique genetic properties of bacterial inversions.—Contrary to expectation, it was found that large inversion mutations can be repaired by generalized transduction. The repair results from the simultaneous introduction of two wild-type transduced fragments into a single recipient cell. Homologous recombination between the two transduced fragments and the two inversion breakpoints causes the inverted segment to be reinverted. This results in regeneration of the wild-type orientation of this chromosome segment. Similar recombination events allow a large inversion mutation to be introduced into a wild-type strain; two transduced fragments from an inversion strain cause recombination events resulting in inversion of a large chromosome segment.—Genetic methods for mapping the extent of a large inversion mutation by generalized transduction are described and tested. The methods are operationally simple and allow good resolution of the two inversion breakpoints.

In bacteria there appear to be two distinct levels of DNA condensation: the organization of independently supercoiled domains of DNA (averaging 100 kbp in size) and the organization of shorter segments of DNA (60–120 bp) with abundant DNA-binding proteins.

We have isolated 440 mutants of Salmonella typhimurium that show temperature-sensitive growth on complex medium at 44 degrees. Approximately 16% of the mutations in these strains have been mapped to 17 chromosomal locations; two of these chromosomal locations seem to include several essential genes. Genetic analysis of the mutations suggests that the collection saturates the genes readily mutable to a ts lethal phenotype in S. typhimurium. Physiological characteristics of the ts lethal mutants were tested: 6% of the mutants can grow at high temperature under anaerobic conditions, 17% can grow when the medium includes 0.5 M KCl, and 9% of the mutants die after a 2-hr incubation at the nonpermissive temperature. Most ts lethal mutations in this collection probably affect genes required for growth at all temperatures (not merely during high temperature growth) since Tn10 insertions that cause a temperature-sensitive lethal phenotype are rare.