Genomic mismatch scanning: a new approach to genetic linkage mapping.

Genomic mismatch scanning (GMS) is a new method of genetic linkage analysis that does not require conventional polymorphic markers or gel electrophoresis. GMS is ideally suited to affected-relative-pair mapping. DNA fragments from all regions of identity-by-descent between two relatives are isolated based on their ability to form extensive mismatch-free hybrid molecules. The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step. Here we demonstrate the practicality of GMS in a model organism, Saccharomyces cerevisiae. GMS is likely to be applicable to other organisms, including humans, and may be of particular value in mapping complex genetic traits.

Ion-pair reorganization regulates reactivity in photoredox catalysts.

Cyclometalated and polypyridyl complexes of d6 metals are promising photoredox catalysts, using light to drive reactions with high kinetic or thermodynamic barriers via the generation of reactive radical intermediates. However, while tuning of their redox potentials, absorption energy, excited-state lifetime and quantum yield are well-known criteria for modifying activity, other factors could be important. Here we show that dynamic ion-pair reorganization controls the reactivity of a photoredox catalyst, [Ir[dF(CF3)ppy]2(dtbpy)]X. Time-resolved dielectric-loss experiments show how counter-ion identity influences excited-state charge distribution, evincing large differences in both the ground- and excited-state dipole moment depending on whether X is a small associating anion (PF6-) that forms a contact-ion pair versus a large one that either dissociates or forms a solvent-separated pair (BArF4-). These differences correlate with the reactivity of the photocatalyst toward both reductive and oxidative electron transfer, amounting to a 4-fold change in selectivity toward oxidation versus reduction. These results suggest that ion pairing could be an underappreciated factor that modulates reactivity in ionic photoredox catalysts.

Genome-wide association analysis of clinical vs. nonclinical origin provides insights into Saccharomyces cerevisiae pathogenesis.

Because domesticated Saccharomyces cerevisiae strains have been used to produce fermented food and beverages for centuries without apparent health implications, S. cerevisiae has always been considered a Generally Recognized As Safe (GRAS) microorganism. However, the number of reported mucosal and systemic S. cerevisiae infections in the human population has increased and fatal infections have occurred even in relatively healthy individuals. In order to gain insight into the pathogenesis of S. cerevisiae and improve our understanding of the emergence of fungal pathogens, we performed a population-based genome-wide environmental association analysis of clinical vs. nonclinical origin in S. cerevisiae. Using tiling array-based, high-density genotypes of 44 clinical and 44 nonclinical S. cerevisiae strains from diverse geographical origins and source substrates, we identified several genetic loci associated with clinical background in S. cerevisiae. Associated polymorphisms within the coding sequences of VRP1, KIC1, SBE22 and PDR5, and the 5' upstream region of YGR146C indicate the importance of pseudohyphal formation, robust cell wall maintenance and cellular detoxification for S. cerevisiae pathogenesis, and constitute good candidates for follow-up verification of virulence and virulence-related factors underlying the pathogenicity of S. cerevisiae.

Direct allelic variation scanning of the yeast genome.

As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).

Blue-fluorescent antibodies.

The forte of catalytic antibodies has resided in the control of the ground-state reaction coordinate. A principle and method are now described in which antibodies can direct the outcome of photophysical and photochemical events that take place on excited-state potential energy surfaces. The key component is a chemically reactive optical sensor that provides a direct report of the dynamic interplay between protein and ligand at the active site. To illustrate the concept, we used a trans-stilbene hapten to elicit a panel of monoclonal antibodies that displayed a range of fluorescent spectral behavior when bound to a trans-stilbene substrate. Several antibodies yielded a blue fluorescence indicative of an excited-state complex or "exciplex" between trans-stilbene and the antibody. The antibodies controlled the isomerization coordinate of trans-stilbene and dynamically coupled this manifold with an active-site residue. A step was taken toward the use of antibody-based photochemical sensors for diagnostic and clinical applications.

Mutations in Saccharomyces cerevisiae which confer resistance to several amino acid analogs.

Four new complementation groups of mutations which confer resistance to several amino acid analogs in Saccharomyces cerevisiae are described. These mutants were isolated on medium containing urea as the nitrogen source, in contrast to previous studies that had used medium containing proline. All four resistance to amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analog and amino acid uptake. In some genetic backgrounds, raa leu2 and raa thr4 double mutants are inviable, even on rich medium. The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are unable to use proline as a nitrogen source. raa4 is, however, unlinked to a previously described amino acid analog resistance and proline uptake mutant, aap1, or to the general amino acid permease mutant gap1. Both raa4 and gap1 prevent uptake of [3H]leucine in liquid cultures. The raa1, raa2, and raa3 mutants affect only a subset of the amino acid analogs and amino acids affected by raa4. The phenotypes of raa1, -2, and -3 mutants are readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured in liquid media.

Pleiotropic plasma membrane ATPase mutations of Saccharomyces cerevisiae.

We isolated a large number of mutations in the structural gene for the plasma membrane ATPase (PMA1) of Saccharomyces cerevisiae. These mutations were selected by their resistance to the aminoglycoside antibiotic hygromycin B. Biochemical analysis of purified membrane preparations showed that the plasma membrane ATPase activity of the mutants was reduced as much as 75%. Intragenic complementation of pma1 mutants suggested that the yeast plasma membrane ATPase was a multimeric enzyme. The pma1 mutants were apparently defective in maintaining internal pH; more than half of the mutants were unable to grow either at a low pH or in the presence of a weak acid. Most pma1 mutants were also osmotic pressure sensitive. At a very low temperature (5 degrees C) many pma1 mutants were unable to grow and were arrested as unbudded cells. The three most severely affected mutants were also unable to grow in the presence of NH4+. The most extreme mutant exhibited a severe defect in progression through the cell cycle; on synthetic medium, the cells progressively accumulated nucleus-containing small buds that generally failed to complete bud enlargement and cytokinesis. Most of the pleiotropic phenotypes of pma1 mutants could be suppressed by the addition of 50 mM KCl but not NaCl to the medium.

Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes.

Starting with two temperature-sensitive mutants (rpa190-1 and rpa190-5) of Saccharomyces cerevisiae, both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) of rpa190 mutations. All the SRP mutations were dominant over the corresponding wild-type genes. They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants. SRP mutations representing each of the seven loci were studied for their effects on other known rpa190 mutations. All of the SRP mutations were able to suppress both rpa190-1 and rpa190-5. In addition, one particular suppressor, SRP5, was found to suppress two other rpa190 mutations as well as an rpa190 deletion. Southern blot analysis combined with genetic crosses demonstrated that SRP5 maps to a region on chromosome XV loosely linked to rpa190 and represents a transposed mutant gene in two copies. Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases in rpa190-1 mutants after a shift to 37 degrees C and that in the mutant strain carrying SRP5 this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage. These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.

Cycloheximide-resistant temperature-sensitive lethal mutations of Saccharomyces cerevisiae.

We describe the isolation and preliminary characterization of a set of pleiotropic mutations resistant to the minimum inhibitory concentration of cycloheximide and screened for ts (temperature-sensitive) growth. These mutations fall into 22 complementation groups of cycloheximide resistant ts lethal mutations (crl). None of the crl mutations appears to be allelic with previously isolated mutations. Fifteen of the CRL loci have been mapped. At the nonpermissive temperature (37 degrees), these mutants arrest late in the cell cycle after several cell divisions. Half of these mutants are also unable to grow at very low temperatures (5 degrees). Although mutants from all of the 22 complementation groups exhibit similar temperature-sensitive phenotypes, an extragenic suppressor of the ts lethality of crl3 does not relieve the ts lethality of most other crl mutants. A second suppressor mutation allows crl10, crl12, and crl14 to grow at 37 degrees but does not suppress the ts lethality of the remaining crl mutants. We also describe two new methods for the enrichment of auxotrophic mutations from a wild-type yeast strain.

crl mutants of Saccharomyces cerevisiae resemble both mutants affecting general control of amino acid biosynthesis and omnipotent translational suppressor mutants.

Cyocloheximide resistant lethal (crl) mutants of Saccharomyces cerevisiae, defining 22 unlinked complementation groups, are unable to grow at 37 degrees. They are also highly pleiotropic at their permissive temperature of 25 degrees. The mutants are all unable to arrest at the G1 stage of the cell cycle when grown to stationary phase or when starved for a single amino acid, though they do arrest at G1 when deprived of all nitrogen. The crl mutants are also hypersensitive to various amino acid analogs and to 3-aminotriazole. These mutants also "tighten" leaky auxotrophic mutations that permit wild-type cells to grow in the absence of the appropriate amino acid. All of these phenotypes are also exhibited by gcn mutants affecting general control of amino acid biosynthesis. In addition, the crl mutants are all hypersensitive to hygromycin B, an aminoglycoside antibiotic that stimulates translational misreading. The crl mutations also suppress one nonsense mutation which is phenotypically suppressed by hygromycin B. Many crl mutants are also osmotically sensitive. These are phenotypes which the crl mutations have in common with previously isolated omnipotent suppressors. We suggest that the the crl mutations all affect the fidelity of protein translation.

Evidence of Chromosomal Breaks near the Mating-Type Locus of SACCHAROMYCES CEREVISIAE That Accompany MATalpha xMATalpha Matings.

In order for two heterothallic MATalpha haploids of Saccharomyces cerevisiae to mate, one parent must apparently become, at least transiently, an a-like cell. Only about 25% of the matings result from an actual transposition of MATa sequences to replace MATalpha, and about 1% result from a deletion joining MAT to the normally silent HMRa allele. The majority of matings occur after an apparent chromosome break that deletes MATalpha and all of the known markers more distal on the right arm of chromosome III.--The chromosome break occurs at or very near MAT, invariably leaving the distal marker tsm1 hemizygous, but the closely linked proximal marker cry1 usually is heterozygous. The resulting diploid containing the broken chromosome is mitotically unstable; about 10% of the colonies contain visible sectors in which the rest of the broken chromosome is lost. The region close to the breakpoint (i.e., cry1) is unusually active in recombination. About 20% of the intact homologues remaining after chromosome loss were gene-converted for cry1. In addition, the broken end participated in reciprocal recombination events that joined the chromosome to the distal portion of the intact homologous chromosome.--The unstable diploids may also become stable and no longer give rise to mitotic segregants. We have found two distinct ways in which stabilization occurs. Most often the diploid becomes euploid by a recombination event that yields a cell homozygous for all markers distal to (and sometimes including) cry1. In one of 9 cases so far analyzed, the stable diploid was still hemizygous for MATalpha and for other markers distal to MAT. This last case is similar to the healing of broken chromosomes in maize described by McClintock (1939, 1941, 1951).

Development of Saccharomyces cerevisiae as a model pathogen. A system for the genetic identification of gene products required for survival in the mammalian host environment.

Saccharomyces cerevisiae, a close relative of the pathogenic Candida species, is an emerging opportunistic pathogen. An isogenic series of S. cerevisiae strains, derived from a human clinical isolate, were used to examine the role of evolutionarily conserved pathways in fungal survival in a mouse host. As is the case for the corresponding Candida albicans and Cryptococcus neoformans mutants, S. cerevisiae purine and pyrimidine auxotrophs were severely deficient in survival, consistent with there being evolutionary conservation of survival traits. Resistance to the antifungal drug 5-fluorocytosine was not deleterious and appeared to be slightly advantageous in vivo. Of mutants in three amino acid biosynthetic pathways, only leu2 mutants were severely deficient in vivo. Unlike the glyoxylate cycle, respiration was very important for survival; however, the mitochondrial genome made a respiration-independent contribution to survival. Mutants deficient in pseudohyphal formation were tested in vivo; flo11Delta mutants were phenotypically neutral while flo8Delta, tec1Delta, and flo8Delta tec1Delta mutants were slightly deficient. Because of its ease of genetic manipulation and the immense S. cerevisiae database, which includes the best annotated eukaryotic genome sequence, S. cerevisiae is a superb model system for the identification of gene products important for fungal survival in the mammalian host environment.

Characterization of a mutation in yeast causing nonrandom chromosome loss during mitosis.

Diploid strains of the yeast Saccharomyces cerevisiae homozygous for a recessive chromosome loss mutation (chl) exhibit a high degree of mitotic instability. Cells become monosomic for chromosome III at a frequency of approximately one percent of all cell divisions. Chromosome loss at this high frequency is also found for chromosome I, and at lesser frequencies for chromosomes VIII and XVI. In contrast, little or no chromosome loss is found for six other linkage groups tested (II, V, VI, VII, XI and XVII). The chl mutation also induces a ten-fold increase in both intergenic and intragenic mitotic recombination on all ten linkage groups tested. The chl mutation does not cause an increase in spontaneous mutations, nor are mutant strains sensitive to UV or irradiation. The effects of chl during meiosis are observed primarily in reduced spore viability. A decrease in chromosome III linkage relationships is also found.

Genetic characterization of pathogenic Saccharomyces cerevisiae isolates.

Saccharomyces cerevisiae isolates from human patients have been genetically analyzed. Some of the characteristics of these isolates are very different from laboratory and industrial strains of S. cerevisiae and, for this reason, stringent genetic tests have been used to confirm their identity as S. cerevisiae. Most of these clinical isolates are able to grow at 42 degrees, a temperature that completely inhibits the growth of most other S. cerevisiae strains. This property can be considered a virulence trait and may help explain the presence of these isolates in human hosts. The ability to grow at 42 degrees is shown to be polygenic with primarily additive effects between loci. S. cerevisiae will be a useful model for the evolution and genetic analysis of fungal virulence and the study of polygenic traits.

Effectiveness of treatments of depression in older ambulatory patients.

OBJECTIVE: To determine the effectiveness of acute-phase pharmacological and psychological treatments of depression in older ambulatory patients by systematically reviewing original research relevant to this topic. METHODS: Searches in MEDLINE and PsycINFO and manual reviews of bibliographies located 233 articles. Of these, 40 (37 different studies) met our 8 inclusion criteria: original research, written in English or French, subjects 55 years and older, diagnosis of depression, outpatient or community setting, prospective controlled study design, acute-phase pharmacological or psychological treatment, and outcome measure of depression. Two independent reviewers assessed the methodological quality of each article using a standard form and a quality score was computed. Quantitative data on levels of depression at the end of treatment were abstracted. Results were grouped by specific treatment comparison (type of treatment and type of control group). For comparisons that used the Hamilton Depression Rating Scale, we computed mean posttreatment differences. Effect sizes were computed from the Hamilton Depression Rating Scale or an alternative scale. RESULTS: In studies that compared active drugs with placebo, the heterocyclic drugs significantly reduced the posttreatment Hamilton Depression Rating Scale score (mean difference, -5.78; 95% confidence interval, -8.31 to -3.25); other drugs had smaller effects. In studies that compared active drugs, there were no significant differences overall between different classes of drugs; selective serotonin reuptake inhibitors appeared to be as effective as heterocyclic drugs. Rational psychological treatments performed significantly better than no treatment (mean posttreatment Hamilton Depression Rating Scale difference, -7.25; 95% confidence interval, -10.10 to -4.40) but not significantly better than that for controls who received similar attention. Adjustment for the study quality score did not affect these results. CONCLUSIONS: Based on comparisons with untreated controls, heterocyclic antidepressants and rational psychological therapies appear to be the most effective treatments for older ambulatory patients with mild to moderate depression. Based on drug-drug comparisons, selective serotonin reuptake inhibitors appear to be as effective as heterocyclic drugs. However, overall, the magnitude of the treatment effects is modest. Limitations in the quantity and quality of appropriate studies suggest a sober approach to treatment in this population.

Use of medications with anticholinergic effect predicts clinical severity of delirium symptoms in older medical inpatients.

BACKGROUND: Use of anticholinergic (ACH) medications is a biologically plausible and potentially modifiable risk factor of delirium, but research findings are conflicting regarding its association with delirium. OBJECTIVES: To evaluate the longitudinal association between use of ACH medications and severity of delirium symptoms and to determine whether this association is modified by the presence of dementia. PATIENTS AND METHODS: A total of 278 medical inpatients 65 years and older with diagnosed incident or prevalent delirium were followed up with repeated assessments using the Delirium Index for up to 3 weeks. Exposure to ACH and other medications was measured daily. The association between change in medication exposure in the 24 hours preceding a Delirium Index assessment was assessed using a mixed linear regression model. RESULTS: During follow-up (mean +/- SD, 12.3 +/- 7.0 days), 47 medications with potential ACH effect were used in the population (mean, 1.4 medications per patient per day). Increase in delirium severity was significantly associated with several measures of ACH medication exposure on the previous day, adjusting for dementia, baseline delirium severity, length of follow-up, and number of non-ACH medications taken. Dementia did not modify the association between ACH medication use and delirium severity. CONCLUSION: Exposure to ACH medications is independently and specifically associated with a subsequent increase in delirium symptom severity in elderly medical inpatients with diagnosed delirium.

Effectiveness of psychosocial interventions in preventing HIV risk behaviour in injecting drug users.

OBJECTIVE: To consider evidence for the effectiveness of psychosocial interventions in reducing the risk of infection with HIV of injecting drug users. METHOD: We reviewed 19 published studies of the effectiveness of individual counselling, HIV testing, group interventions, street outreach, and a 'social' intervention designed to change norms for safer behaviours. RESULTS: Eight of 15 studies that examined behavioural outcomes provided evidence of the effectiveness of an experimental intervention, compared with a control or comparison group. In four of these studies, however, serious design limitations made results difficult to interpret. In the other four studies without design limitations, the success of the experimental interventions may have been due to their greater length and intensity as well as to having been conducted with stable and well-motivated populations. Nine of the 15 studies showed evidence of marked behaviorial changes in both experimental and comparison group(s), with the changes in many cases being sustained for upwards of 12 months. CONCLUSIONS: A close examination of the evidence and competing hypotheses for the pattern of results suggests that participating in evaluation research may itself be a valuable intervention. Implications for the development of interventions include the potential efficacy of health risk assessment. Implications for evaluation of interventions include the need for developing unobtrusive measures and for assessing the impact of behavioural assessments. Despite the large behavioural changes reported in most of the studies, a substantial proportion of subjects receiving interventions reported unacceptably high levels of risk behaviours. New, more potent interventions are needed, such as those designed to change the norms of entire communities of drug users concerning safer injection and safer sex.

Predictors of AIDS-preventive behavior among homosexually active men: a longitudinal study.

Predictors of adoption of safer sexual behaviors were examined in a cohort of 278 homosexually active men with stable HIV-antibody status followed over 12 months at a Boston community health center. The behaviors examined included: (1) restriction of partners to one monogamous or steady relationship and (2) among men who maintained multiple or non-steady partners, the avoidance of unprotected receptive and insertive anogenital contact. For each behavior, men who adopted consistently safer behavior were compared with those who remained unsafe, using bivariate analyses and multiple logistic regression modelling. The strongest predictor of all behaviors was the initial level of the unsafe behavior. After controlling for this, weak effects of several health beliefs were found, including perceived susceptibility and medical efficacy. Men who became aware of a positive HIV-antibody test result and who reported greater effort to change their behavior were more likely to adopt safer insertive anogenital contact. In this generally well-educated cohort with high levels of knowledge about AIDS, adoption of safer sexual behaviors is best predicted from previous levels of unsafe behavior.

Maintenance of behavioral change in a cohort of homosexually active men.

OBJECTIVES: To assess associations of perceptions of sexual behavior change with actual risk behaviors and psychosocial variables, and to determine whether perceptions of behavior change predict subsequent behavior. DESIGN: Cohort study of homosexually active men. SETTING: Community health center in Boston, Massachusetts, USA. PATIENTS, PARTICIPANTS: Two hundred and sixty-two cohort members who participated in follow-up in 1989 and who had at least one subsequent visit. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Composite risk behavior variable, based on unprotected anogenital contact and number of partners. RESULTS: Of the 96% who had effected behavior change, 47% perceived that they were able to maintain those changes consistently and 58% wanted to make more changes. Perceptions of consistent maintenance were associated with lower risk behavior, lower perceived riskiness of behaviors and susceptibility to AIDS, and fewer barriers to behavior change. Desire for more change was associated with increased behavioral effort, fewer barriers to condom use, and greater perceived riskiness of current behavior. Among those with lower risk behaviors, perceived inconsistent maintenance predicted relapse to more risky behavior at the following visit. CONCLUSIONS: Perceived maintenance of behavior change is potentially useful in identifying individuals at risk of relapse from safer sex.

PIP: Public health researchers followed a cohort of 322 homosexually active male clients enrolled at a community health center in Boston, Massachusetts between January 1985-April 1987 at 6-month intervals to examine cross-sectional and longitudinal data on perceptions of sexual behavior change among initially asymptomatic and homosexually active men. Only 262 completed the questionnaire addressing perceptions of behavior change. 40% of the 60 students who did not complete the questionnaire tested positive for HIV compared with 22% of those who did complete the questionnaire (p.01). High risk included unprotected anogenital contact with multiple partners. HIV status was not associated with behavior change. Behavior change was associated with alcohol or drug use during sex for men of low or modified high risk at baseline (p=.01). 96% of 262 men achieved sexual behavior change after learning about AIDS. 58% hoped to make additional changes. 47% thought that they could always sustain the changes. Men who were not consistent in practicing behavioral changes were more likely to want to make more changes (p.001). Men who perceived themselves to practice inconsistent behavior did indeed practice high risk behavior (p=.005). Those who perceived themselves to consistently practice lower risk behavior at follow up were 2.07 times more likely to practice safer behavior than those who did not see themselves as practicing consistent behavior. Follow up data showed that men were somewhat more likely to return to risky behavior than change to safe behavior (p=.07). In fact, men who at baseline participated in high risk behavior were 1.57 times likely to continue the high risk behavior. For men who at baseline participated in low risk behavior tended to continue that behavior (odds ratio=8.88). The researchers concluded that perceived maintenance of behavior change could help health professionals determine which people are at a risk of reverting to risky sex behavior.

The suppressor gene scl1+ of Saccharomyces cerevisiae is essential for growth.

In Saccharomyces cerevisiae, the SCL-1 mutation is a dominant suppressor of the cycloheximide-resistant, temperature-sensitive (ts) lethal mutation, crl3 [McCusker and Haber, Genetics 119 (1988a) 303-315]. The wild-type scl1+ gene was isolated by screening subclones of the 35-kb region between TRP5 and LEU1 for restoration of the ts phenotype in an SCL1-1 crl3-2 strain. The scl1+ mRNA is about 900 nt long and encodes an open reading frame of 810 bp. The polypeptide deduced from scl1+ possesses a putative secretory signal peptide. The 5'-noncoding region may be under multiple controls, since it contains significant homology to the consensus sequences for the DNA-binding proteins, GCN4, GFI and, possibly, TUF. Gene disruption of scl1+ demonstrates that it is an essential gene.