RAD55 belongs to a group of genes required for resistance to ionizing radiation, RAD50-RAD57, which are thought to define a pathway of recombinational repair. Since all four alleles of RAD55 are temperature conditional (cold sensitive) for their radiation phenotype, we investigated the phenotype produced by null mutations in the RAD55 gene, constructed in vitro and transplaced to the yeast chromosome. The X-ray sensitivity of these null mutant strains was surprisingly suppressed by increased temperature, osmotic strength of the growth medium and heterozygosity at the mating-type locus. These first two properties, temperature conditionality and osmotic remediability, are commonly associated with missense mutations; these rad55 null mutants are unique in that they exhibit these properties although the mutant gene cannot be expressed. X-ray-induced mitotic recombination was also cold sensitive in rad55 mutant diploids. Although mitotic growth was unaffected in these strains, meiosis was a lethal event at both high and low temperatures. Whereas the phenotype of rad55 null mutants is consistent with a role of RAD55 in recombination and recombinational repair, there is evidence for considerable RAD55-independent recombination, at least in mitotic cells, which is influenced by temperature and MAT. We discuss models for the role of RAD55 in recombination to explain the unusual properties of rad55 mutants.

Colonization of the intestinal epithelium by Vibrio cholerae was examined in two model systems, in ligated ileal loops of adult rabbits and in the patent gut of infant rabbits, using both scanning and transmission electron microscopy. Time studies in the adult model showed a lag period of up to 1 h before the attachment of significant numbers of the vibrios. The bacteria appeared initially in small patches on the sides of the villi, predominantly along the transverse furrows. The number of adherent bacteria steadily increased, reaching a maximum between 4 and 7 h, when a dense mat of bacteria several layers thick covered much of the villi. After this time there was a rapid decline in the number of V. cholerae bound. By 12 to 16 h only a few bacteria could be seen on the surface of the villi, which had a rough, patchy appearance at these later times. Globular protrusions, with vibrios attached, may play a role in the clearance of bacteria. Colonization and clearance in the patent intestine of the infant rabbit occurred much as in the adult model. However, the bacteria adhered more uniformly and there was no lag in attachment. In both models the majority of bacteria were aligned horizontally with the epithelial surface, but some were attached in an end-on manner, with their flagella extending into the lumen. The bacteria adhered via their surface coats directly to the tips of the microvilli, except for a few vibrios that were partly embedded into the brush border. Some changes in the microvilli occurred as a consequence of the bacterial attachment.

Microbial methanogenesis was examined in thermal waters, muds, and decomposing algal-bacterial mats associated with volcanic activity in Yellowstone National Park. Radioactive tracer studies with [(14)C]glucose, acetate, or carbonate and enrichment culture techniques demonstrated that methanogenesis occurred at temperatures near 70 degrees C but below 80 degrees C and correlated with hydrogen production from either geothermal processes or microbial fermentation. Three Methanobacterium thermoautotrophicum strains (YT1, YTA, and YTC) isolated from diverse volcanic habitats differed from the neotype sewage strain DeltaH in deoxyribonucleic acid guanosine-plus-cytosine content and immunological properties. Microbial methanogenesis was characterized in more detail at a 65 degrees C site in the Octopus Spring algal-bacterial mat ecosystem. Here methanogenesis was active, was associated with anaerobic microbial decomposition of biomass, occurred concomitantly with detectable microbial hydrogen formation, and displayed a temperature activity optimum near 65 degrees C. Enumeration studies estimated more than 10(9) chemoorganotrophic hydrolytic bacteria and 10(6) chemolithotrophic methanogenic bacteria per g (dry weight) of algal-bacterial mat. Enumeration, enrichment, and isolation studies revealed that the microbial population was predominantly rod shaped and asporogenous. A prevalent chemoorganotrophic organism in the mat that was isolated from an end dilution tube was a taxonomically undescribed gram-negative obligate anaerobe (strain HTB2), whereas a prevalent chemolithotrophic methanogen isolated from an end dilution tube was identified as M. thermoautotrophicum (strain YTB). Taxonomically recognizable obligate anaerobes that were isolated from glucose and xylose enrichment cultures included Thermoanaerobium brockii strain HTB and Clostridium thermohydrosulfuricum strain 39E. The nutritional properties, growth temperature optima, growth rates, and fermentation products of thermophilic bacterial strains 39E, HTB2, and YTB were determined.

The DNA sequence of the Ty1 activating region from the CYC7-H2 mutant of Saccharomyces cerevisiae is presented. Analysis of the data revealed the presence of four simian virus 40-type enhancer core sequences. Two of the Ty1 enhancer cores are contiguous with sequences also homologous to the diploid control site at MAT alpha. We postulate that these two Ty1 regions of approximately equal to 30 base pairs are regulatory blocks, and we have analyzed deletions to ascertain whether they are necessary for effects of Ty1 on adjacent gene expression. We found that activation is lost when a restriction fragment encompassing both postulated regulatory blocks is deleted. Deletion of restriction fragments that remove only one of the two regulatory blocks has little or no effect on Ty1 activating ability in haploid yeast cells or on repression of this function in diploid yeast cells. Because the most significant internal homologies in the restriction fragments analyzed are the putative regulatory blocks, these observations suggest that enhancer-like sequences are involved in cell-type control of Ty1 effects on gene expression.

BACKGROUND

We observed that some patients with clinical leptospirosis supported by positive results of rapid tests were negative for leptospirosis on the basis of our diagnostic gold standard, which involves isolation of Leptospira species from blood culture and/or a positive result of a microscopic agglutination test (MAT). We hypothesized that our reference standard was imperfect and used statistical modeling to investigate this hypothesis.

METHODS

Data for 1652 patients with suspected leptospirosis recruited during three observational studies and one randomized control trial that described the application of culture, MAT, immunofluorescence assay (IFA), lateral flow (LF) and/or PCR targeting the 16S rRNA gene were reevaluated using Bayesian latent class models and random-effects meta-analysis.

RESULTS

The estimated sensitivities of culture alone, MAT alone, and culture plus MAT (for which the result was considered positive if one or both tests had a positive result) were 10.5% (95% credible interval [CrI], 2.7%-27.5%), 49.8% (95% CrI, 37.6%-60.8%), and 55.5% (95% CrI, 42.9%-67.7%), respectively. These low sensitivities were present across all 4 studies. The estimated specificity of MAT alone (and of culture plus MAT) was 98.8% (95% CrI, 92.8%-100.0%). The estimated sensitivities and specificities of PCR (52.7% [95% CrI, 45.2%-60.6%] and 97.2% [95% CrI, 92.0%-99.8%], respectively), lateral flow test (85.6% [95% CrI, 77.5%-93.2%] and 96.2% [95% CrI, 87.7%-99.8%], respectively), and immunofluorescence assay (45.5% [95% CrI, 33.3%-60.9%] and 96.8% [95% CrI, 92.8%-99.8%], respectively) were considerably different from estimates in which culture plus MAT was considered a perfect gold standard test.

CONCLUSIONS

Our findings show that culture plus MAT is an imperfect gold standard against which to compare alterative tests for the diagnosis of leptospirosis. Rapid point-of-care tests for this infection would bring an important improvement in patient care, but their future evaluation will require careful consideration of the reference test(s) used and the inclusion of appropriate statistical models.

BACKGROUND

Recent investigations suggest that stress reactivity may play an important role in the relationship between stress and substance use. Important gender differences, such as reasons for using substances, have been well documented, and it is likely that men and women also differ in their stress response.

OBJECTIVE

In this study, gender differences in stress reactivity to two laboratory stress manipulations were examined among 18 men and 21 women with current cocaine dependence.

METHODS

Participants completed a psychological stress task, the Mental Arithmetic Task (MAT), and a physical stress task, the Cold Pressor Task (CPT). Subjective stress responses (i.e., feelings of stress, anxiety, nervousness, pain, mood), physiological stress responses (i.e., heart rate, skin conductance), and cocaine craving were assessed.

RESULTS

In response to both the MAT and the CPT, significant increases in subjective and physiological stress were observed among participants. Women, however, demonstrated greater subjective reactivity, as evidenced by significantly higher ratings of stress, nervousness, and pain as compared to men. Women also immersed their hand in the cold water bath for a significantly shorter period of time as compared to men.

CONCLUSIONS

The current findings are among the first to report on gender differences in stress reactivity among cocaine-dependent individuals. The findings suggest that the mechanisms linking stress and substance use may be gender-specific, and that stress reactivity may play a different role for women than for men. Future research and implications for clinical interventions are discussed.

Stromatolites are attached, lithified sedimentary growth structures, accretionary away from a point or limited surface of initiation. Though the accretion process is commonly regarded to result from the sediment trapping or precipitation-inducing activities of microbial mats, little evidence of this process is preserved in most Precambrian stromatolites. The successful study and interpretation of stromatolites requires a process-based approach, oriented toward deconvolving the replacement textures of ancient stromatolites. The effects of diagenetic recrystallization first must be accounted for, followed by analysis of lamination textures and deduction of possible accretion mechanisms. Accretion hypotheses can be tested using numerical simulations based on modem stromatolite growth processes. Application of this approach has shown that stromatolites were originally formed largely through in situ precipitation of laminae during Archean and older Proterozoic times, but that younger Proterozoic stromatolites grew largely through the accretion of carbonate sediments, most likely through the physical process of microbial trapping and binding. This trend most likely reflects long-term evolution of the earth's environment rather than microbial communities.

We have previously shown that each of four activated oncogenes (c-myc, neu, ras, and int-2) can serve as transgenic initiators of morphologically distinct adenocarcinomas of the murine mammary gland. Since abnormalities of these oncogenes are found frequently in human breast cancers, such differences are of particular interest. Thus, the distinctiveness of each murine tumor type might reflect a relationship between a specific oncogene and a susceptible target cell or might reflect distinctive changes brought about by the idiosyncratic action of each oncogene. We have identified six genes (two of them novel) expressed in tumors initiated by neu, but usually absent from tumors initiated by c-myc. The expression of these genes (kappa-casein, transferrin, cellular retinol binding protein I (CRBPI), WDNM1, and the two novel ones) cannot be induced in c-myc-initiated tumors by the introduction of an activated neu oncogene nor can their expression be inhibited in neu-initiated tumors by the introduction of c-myc. Therefore, these genes appear to represent markers of a cell type preferentially transformed by neu. Further analysis reveals that the six markers are also expressed by ras-initiated mammary tumors, but not by int-2-initiated tumors suggesting that neu/ras-initiated tumors share a common cellular lineage and/or a common signal transduction pathway. Interestingly, one of the novel marker genes (Mat-8) appears to encode a cell-surface chloride channel and the other, a secreted protein with homologies to glycosyl hydrolases, both of which might be useful for the diagnosis and treatment of specific mammary tumors.

Cultures of the yeast Saccharomyces cerevisiae that are heterozygous for the mating type (MATa/MAT alpha) undergo synchronous meiosis and spore formation when starved for nitrogen and supplied with a nonfermentable carbon source such as acetate. Haploid and homozygous MAT alpha/MAT alpha and MATa/MATa diploid cells incubated under the same conditions fail to undergo meiosis and are asporogenous. It has not yet been firmly established that gene expression during sporulation is controlled at the level of transcript accumulation. To examine this question, we used cloned genes that encode a variety of "housekeeping" functions to probe Northern blots to assay the appearance of specific transcripts in both sporulating and asporogenous S. cerevisiae. In sporulating cells, each transcript showed a characteristic pattern of accumulation, reaching a maximum relative abundance at one of several different periods. In contrast, in both asporogenous haploid MATa and diploid MAT alpha/MAT alpha cells, all transcripts accumulated with similar kinetics. These results suggest a sporulation-specific pattern for transcript appearance. During these studies, high levels of several different transcripts were observed at unexpected times in sporulating cells. Histone (H)2A and (H)2B1 transcripts, although most abundant during premeiotic DNA synthesis, remained at one-third to one-half maximal levels after its end and were found in mature ascospores. Their appearance at this time is in sharp contrast to vegetative cells in which these histone transcripts are only found just before and during the period of DNA synthesis. Furthermore, transcripts from GAL10 and CDC10 genes, which are believed to be dispensable for sporulation, were much more abundant in sporulating cells than in asporogenous cells and vegetative cells grown on glucose or acetate. The presence of these transcripts did not appear to be due to a general activation of transcription because each accumulated with different kinetics. In addition, the transcript for at least one gene, HO, that is also dispensable for sporulation was not detected. The increased abundance of transcripts from some genes not required for sporulation leads us to propose that genes preferentially expressed during sporulation need not be essential for this differentiation.

Currently, there is no consensus concerning the geographic distribution and extent of endemism in Antarctic cyanobacteria. In this paper we describe the phenotypic and genotypic diversity of cyanobacteria in a field microbial mat sample from Lake Fryxell and in an artificial cold-adapted sample cultured in a benthic gradient chamber (BGC) by using an inoculum from the same mat. Light microscopy and molecular tools, including 16S rRNA gene clone libraries, denaturing gradient gel electrophoresis, and sequencing, were used. For the first time in the study of cyanobacterial diversity of environmental samples, internal transcribed spacer (ITS) sequences were retrieved and analyzed to complement the information obtained from the 16S rRNA gene. Microscopy allowed eight morphotypes to be identified, only one of which is likely to be an Antarctic endemic morphotype. Molecular analysis, however, revealed an entirely different pattern. A much higher number of phylotypes (15 phylotypes) was found, but no sequences from Nodularia and Hydrocoryne, as observed by microscopy, were retrieved. The 16S rRNA gene sequences determined in this study were distributed in 11 phylogenetic lineages, 3 of which were exclusively Antarctic and 2 of which were novel. Collectively, these Antarctic sequences together with all the other polar sequences were distributed in 22 lineages, 9 of which were exclusively Antarctic, including the 2 novel lineages observed in this study. The cultured BGC mat had lower diversity than the field mat. However, the two samples shared three morphotypes and three phylotypes. Moreover, the BGC mat allowed enrichment of one additional phylotype. ITS sequence analysis revealed a complex signal that was difficult to interpret. Finally, this study provided evidence of molecular diversity of cyanobacteria in Antarctica that is much greater than the diversity currently known based on traditional microscopic analysis. Furthermore, Antarctic endemic species were more abundant than was estimated on the basis of morphological features. Decisive arguments concerning the global geographic distribution of cyanobacteria should therefore incorporate data obtained with the molecular tools described here.

MAT alpha cells of the yeast Saccharomyces cerevisiae produce a polypeptide mating pheromone, alpha factor. MATa cells respond to the pheromone by undergoing several inducible responses: the arrest of cell division, the production of a cell surface agglutinin, and the formation of one or more projections on the cell surface commonly termed the "shmoo" morphology. Dose-response curves were determined for each of these inducible responses as a function of alpha factor concentration. It is shown that under conditions commonly employed in previous studies, the dose-response for cell division arrest is determined by the rate at which cells inactivate the alpha factor. In order to achieve conditions where inactivation would not be the dominant parameter, the cell division response to alpha factor was monitored at low cell densities. Under conditions of essentially no alpha factor destruction, the dose of alpha factor at which cells exhibit a half-maximal response for cell division arrest (2.5 X 10(-10) M) is nearly the same as that at which cells exhibit a half-maximal response for agglutination induction (1.0 X 10(-10) M). On the contrary, the half-maximal response for projection formation was obtained at doses of alpha factor 2 orders of magnitude higher (1.4 X 10(-8) M). These results are consistent with the same high affinity alpha factor receptor mediating both cell division arrest and agglutination induction. A different system of lower affinity must mediate projection formation. Alternatively, if the same system and receptor are used, then a much higher occupancy is required for the induction of projections compared to division arrest and agglutination induction.

Two genes (<em>MAT</em>1A and <em>MAT</em>2A) encode for the essential enzyme methionine adenosyltransferase (<em>MAT</em>), which catalyzes the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and, in the liver, a precursor of glutathione. <em>MAT</em>1A is expressed mostly in the liver, whereas <em>MAT</em>2A is widely distributed. <em>MAT</em>2A is induced in the liver during periods of rapid growth and dedifferentiation. In human hepatocellular carcinoma (HCC) <em>MAT</em>1A is replaced by <em>MAT</em>2A. This is important pathogenetically because <em>MAT</em>2A expression is associated with lower SAMe levels and faster growth, whereas exogenous SAMe treatment inhibits growth. Rats fed ethanol intragastrically for 9 weeks also exhibit a relative switch in hepatic <em>MAT</em> expression, decreased SAMe levels, hypomethylation of c-myc, increased c-myc expression, and increased DNA strand break accumulation. Patients with alcoholic liver disease have decreased hepatic <em>MAT</em> activity owing to both decreased <em>MAT</em>1A expression and inactivation of the <em>MAT</em>1A-encoded isoenzymes, culminating in decreased SAMe biosynthesis. Consequences of chronic hepatic SAMe depletion have been examined in the <em>MAT</em>1A knockout mouse model. In this model, the liver is more susceptible to injury. In addition, spontaneous steatohepatitis develops by 8 months, and HCC develops by 18 months. Accumulating evidence shows that, in addition to being a methyl donor, SAMe controls hepatocyte growth response and death response. Whereas transient SAMe depletion is necessary for the liver to regenerate, chronic hepatic SAMe depletion may lead to malignant transformation. It is interesting that SAMe is antiapoptotic in normal hepatocytes, but proapoptotic in liver cancer cells. This should make SAMe an attractive agent for both chemoprevention and treatment of HCC.

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.

We studied 10 cutaneous telangiectatic lesions of hereditary hemorrhagic telangiectasia (HHT), ranging in size from pinpoint to 2 mm, by light and electron microscopy. Four representative lesions were reconstructed by computer from serial 1- or 2-mm plastic embedded sections. The earliest clinically detectable lesion of HHT is a focal dilatation of postcapillary venules, which continue to enlarge and eventually connect with dilated arterioles through capillaries. As the vascular lesion increases in size, the capillary segments disappear and a direct arterio-venous communication is formed. This entire sequence of morphologic events is associated with a perivascular mononuclear cell infiltrate in which the majority of cells are lymphocytes and the minority are monocytes/macrophages by ultrastructure. Comparison of these findings with the telangiectatic mats of scleroderma and cherry angiomas revealed that the former, previously shown to be composed of dilated postcapillary venules, are also associated with perivascular infiltrates, but the latter, which are produced by capillary loop aneurysms, are not.

A survey was conducted to identify reservoirs for urban leptospirosis in the city of Salvador, Brazil. Sampling protocols were performed in the vicinity of households of severe leptospirosis cases identified during active hospital-based surveillance. Among a total of 142 captured Rattus norvegicus (Norwegian brown rat), 80.3% had a positive culture isolate from urine or kidney specimens and 68.1% had a positive serum sample by microscopic agglutination test (MAT) titre of>> or = 1:100. Monoclonal antibody-based typing of isolates identified that the agent carried by rats was Leptospira interrogans serovar Copenhageni, which was the same serovar isolated from patients during hospital-based surveillance. Leptospira spp. were not isolated from 8 captured Didelphis marsupialis (Opossum), while 5/7 had a positive MAT titre against a saprophytic serogroup. R. rattus were not captured during the survey. The study findings indicate that the brown rat is a major rodent reservoir for leptospirosis in this urban setting. Furthermore, the high carriage rates of L. interrogans serovar Copenhageni in captured rats suggest that there is a significant degree of environmental contamination with this agent in the household environment of high risk areas, which in turn is a cause of transmission during urban epidemics.

Creatine synthesis is required in adult animals to replace creatine that is spontaneously converted to creatinine and excreted in the urine. Additionally, in growing animals it is necessary to provide creatine to the expanding tissue mass. Creatine synthesis requires three amino acids: glycine, methionine and arginine, and three enzymes: L-arginine:glycine amidinotransferase (AGAT), methionine adenosyltransferase (MAT) and guanidinoacetate methyltransferase (GAMT). The entire glycine molecule is consumed in creatine synthesis but only the methyl and amidino groups, respectively, from methionine and arginine. Creatinine loss averages approximately 2 g (14.6 mmol) for 70 kg males in the 20- to 39-year age group. Creatinine loss is lower in females and in older age groups because of lower muscle mass. Approximately half of this creatine lost to creatinine can be replaced, in omnivorous individuals, by dietary creatine. However, since dietary creatine is only provided in animal products, principally in meat and fish, virtually all of the creatine loss in vegetarians must be replaced via endogenous synthesis. Creatine synthesis does not appear to place a major burden on glycine metabolism in adults since this amino acid is readily synthesized. However, creatine synthesis does account for approximately 40% of all of the labile methyl groups provided by S-adenosylmethionine (SAM) and, as such, places an appreciable burden on the provision of such methyl groups, either from the diet or via de novo methylneogenesis. Creatine synthesis consumes some 20-30% of arginine's amidino groups, whether provided in the diet or synthesized within the body. Creatine synthesis is, therefore, a quantitatively major pathway in amino acid metabolism and imposes an appreciable burden on the metabolism of methionine and of arginine.

The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119,000 nearly full-length sequences and 28,000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.

Overexpression of the epithelial specific matrix metalloproteinase matrilysin (MAT) has been correlated with enhanced tumorigenicity and tumor cell invasion using in vitro model systems. We have determined the effects of MAT expression on the development of mammary tumorigenesis using transgenic mice that express human MAT under the control of the mouse mammary tumor virus (MMTV)-long terminal repeat promoter/enhancer. Examination of mammary glands from multiparous MMTV-MAT animals revealed the development of premalignant hyperplastic alveolar nodules in 50% of aged females. MMTV-MAT mice were mated with MMTV-neu transgenic mice to determine the effect of MAT on neu-induced mammary tumorigenesis. Bigenic MMTV-MAT/neu female offspring developed primary mammary tumors approximately 13 weeks earlier than did MMTV-neu controls. The mechanism of enhanced neu-induced tumorigenesis was explored. No discernible difference in Neu receptor dimerization or activation was detected in MMTV-MAT/neu tumors or mammary glands compared to MMTV-neu controls. A similar percentage of MMTV-MAT/neu and MMTV-neu tumors acquired deletions in the neu transgene, which have previously been shown to result in constitutive receptor activation. The presence of premalignant nodules and the accelerated development of oncogene-induced mammary tumors suggest that expression of MAT in the mammary epithelium contributes to early-stage mammary tumorigenesis.

Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59. A recessive cryR1 allele has also been cloned, using the integration excision method. The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval. The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo. The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed.

The mating-type a and alpha alleles of the yeast Saccharomyces cerevisiae interconvert by a transposition-substitution reaction where replicas of the silent mating loci, at HML and HMR, are transmitted to the expressed mating-type locus (MAT). HML is on the left arm and HMR on the right arm, while MAT is in the middle of chromosome III. Cells with the genotype HML alpha HMRa switch mating type efficiently at a frequency of about 86%. Since well over 50% of the cells switch, it is thought that switches do not occur randomly, but are directed to occur to the opposite mating-type allele. In contrast, we report that strains possessing the reverse HMLa HMR alpha arrangement switch (phenotype) inefficiently at a maximum of about 6%. The basis for this apparent reduced frequency of switching is that these strains preferentially yield futile homologous MAT locus switches--that is, MATa to MATa and MAT alpha to MAT alpha--and consequently, most of these events are undetected. We used genetically marked HM loci to demonstrate that alpha cells preferentially choose HMR as donor and a cells preferentially choose HML as donor, irrespective of the genetic content of the silent loci. Because of this feature, HML alpha HMRa strains generate predominantly heterologous while HMLa HMR alpha strains produce predominantly homologous MAT switches. The control for directionality of switching therefore is not at the level of transposing heterologous mating-type information, but only at the level of choosing HML versus HMR as the donor. In strains where the preferred donor locus is deleted, the inefficient donor becomes capable of donating efficiently. Thus the preference seems to be mediated by competition between the HM loci for donating information to MAT.

Sex determination in fungi is controlled by a small, specialized region of the genome in contrast to the large sex-specific chromosomes of animals and some plants. Different gene combinations reside at these mating-type (MAT) loci and confer sexual identity; invariably they encode homeodomain, alpha-box, or high mobility group (HMG)-domain transcription factors. So far, MAT loci have been characterized from a single monophyletic clade of fungi, the Dikarya (the ascomycetes and basidiomycetes), and the ancestral state and evolutionary history of these loci have remained a mystery. Mating in the basal members of the kingdom has been less well studied, and even their precise taxonomic inter-relationships are still obscure. Here we apply bioinformatic and genetic mapping to identify the sex-determining (sex) region in Phycomyces blakesleeanus (Zygomycota), which represents an early branch within the fungi. Each sex allele contains a single gene that encodes an HMG-domain protein, implicating the HMG-domain proteins as an earlier form of fungal MAT loci. Additionally, one allele also contains a copy of a unique, chromosome-specific repetitive element, suggesting a generalized mechanism for the earliest steps in the evolution of sex determination and sex chromosome structure in eukaryotes.

Pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time-of-day-specific fashion, supporting a role for the clock in mating.

Filamentous fungi are indispensable biotechnological tools for the production of organic chemicals, enzymes, and antibiotics. Most of the strains used for industrial applications have been--and still are--screened and improved by classical mutagenesis. Sexual crossing approaches would yield considerable advantages for research and industrial strain improvement, but interestingly, industrially applied filamentous fungal species have so far been considered to be largely asexual. This is also true for the ascomycete Trichoderma reesei (anamorph of Hypocrea jecorina), which is used for production of cellulolytic and hemicellulolytic enzymes. In this study, we report that T. reesei QM6a has a MATMATMAT-loci and sexual crossing techniques will facilitate research with other Trichoderma spp. relevant for agriculture and human health.

The amygdala has been repeatedly implicated in emotional processing of both positive and negative-valence stimuli. Previous studies suggest that the amygdala response to emotional stimuli is lower when the subject is in a meditative state of mindful-attention, both in beginner meditators after an 8-week meditation intervention and in expert meditators. However, the longitudinal effects of meditation training on amygdala responses have not been reported when participants are in an ordinary, non-meditative state. In this study, we investigated how 8 weeks of training in meditation affects amygdala responses to emotional stimuli in subjects when in a non-meditative state. Healthy adults with no prior meditation experience took part in 8 weeks of either Mindful Attention Training (MAT), Cognitively-Based Compassion Training (CBCT; a program based on Tibetan Buddhist compassion meditation practices), or an active control intervention. Before and after the intervention, participants underwent an fMRI experiment during which they were presented images with positive, negative, and neutral emotional valences from the IAPS database while remaining in an ordinary, non-meditative state. Using a region-of-interest analysis, we found a longitudinal decrease in right amygdala activation in the Mindful Attention group in response to positive images, and in response to images of all valences overall. In the CBCT group, we found a trend increase in right amygdala response to negative images, which was significantly correlated with a decrease in depression score. No effects or trends were observed in the control group. This finding suggests that the effects of meditation training on emotional processing might transfer to non-meditative states. This is consistent with the hypothesis that meditation training may induce learning that is not stimulus- or task-specific, but process-specific, and thereby may result in enduring changes in mental function.