Regulatory intricacies that confer temporal control of conjugation and antibiotic production functions in Streptomyces: new variations on old themes

The complex life style of streptomycetes requires that the appropriate program of genes be expressed at the appropriate times. For example, conjugation occurs solely within the substrate mycelium stage of development while antibiotic production occurs in the aerial mycelium. Thus, complex regulatory mechanisms involving numerous modes of control have evolved that confer temporal regulation of functions governing these expression programs. Conjugation of the Streptomyces lividans plasmid pIJ101 utilizes only seven plasmid functions in its transmission. Two of these, tra and clt, are essential while three additional functions, spdA, spdB and kilB augment the process, with KorA and KorB regulating the transfer and spread functions. The transmission operon of pIJ101 is unique, in that multiple layers of transcriptional and posttranscriptional control converging to implement tight control of the transfer and spread functions, specifically the potentially lethal kilB gene product. When expressed unregulated, as a chromosomally integrated gene, kilB transcription decreases during the later stages of development, in sharp contrast to the temporally increasing pattern of KilB protein. However, when expressed on pIJ101, the kilB promoter is largely, if not completely repressed, and expression of kilB requires transcription readthrough from upstream. Furthermore, readthrough transcription terminates within a 105 base-pair intergenic region prior to kilB. Interestingly, kilB-operator-bound KorB repressor appears to act, at least in part, as an attenuator of operon transcription, perhaps by physically barring transcription elongation. Finally, the formation of a stem-and-loop in the readthrough transcript within the intercistronic region appears to promote antitermination of operon transcription to counteract the effects of the KorB roadblock. Regulation of antibiotic production in Streptomyces typically involves the streptomycete global regulatory mechanism bldA. The sweet potato pathogen Streptomyces ipomoeae strain 91-03 produces a bacteriocin-like antibiotic, ipomicin. In liquid culture, ipomicin is produced in low concentrations throughout exponential phase followed by a dramatic 10-fold increase as the culture enters stationary phase. In contrast, transcription of the ipomicin structural gene ipoA decreases as the culture ages. The contrasting patterns of ipoA transcription and ipomicin production, coupled to the fact that ipoA contains a TTA codon in its leader sequence, makes ipomicin a strong candidate for bldA regulation

The evolution of prezygotic reproductive isolation in the Drosophila pseudoobscura subgroup

Newly forming species that have differentiated in allopatry may evolve numerous barriers that prevent the interbreeding when they come back into contact with each other. The objective of this dissertation is to evaluate some mechanisms of prezygotic reproductive isolation in the D. pseudoobscura subgroup. I begin by evaluating how the evolution of female preferences and male sexual characters lead to reinforcement between Drosophila pseudoobscura and its congener D. persimils. In particular, I will evaluate two alternative hypotheses; Preference Evolution and Discrimination Enhancement, to determine how selection reduces hybridization between these sister species. Both hypotheses predict a reduction in the overlap of male traits and female preferences in hybridizing populations; however, the target of selection differs between the two. Next, I will discuss reproductive isolation as a result of competiton between gametes, in particular conspecific sperm precedence. Until this study, patterns of sperm precedence had rarely been examined between divergent populations or subspecies within a species. I will evaluate conspecific sperm precedence and its role in reproductive isolation between two subspecies: Drosophila pseudoobscura pseudoobscura and D. p. bogotana. The final portion of this dissertation examines the rapid evolution of some proteins potentially tied to the evolution of reproductive isolation. I focus on some seminal fluid proteins that may play a role in the reproductive isolation of Drsosphila species. In particular, I examine the rapid evolution of accessory gland proteins in the D. pseudoobscura subgroup by looking for the signature of positive selection in the genes that encode them. I will also evaluate the roles of insertion / deletion mutations in the evolution of these proteins. Together, the chapters of this dissertation contribute to the understanding of three forms of prezygotic reproductive isolation and their roles in speciation

Positive selection on nucleotide substitutions and indels in accessory gland proteins of the Drosophila pseudoobscura subgroup

Genes encoding reproductive proteins often diverge rapidly due to positive selection on nucleotide substitutions. While this general pattern is well established, the extent to which specific reproductive genes experience similar selection in different clades has been little explored, nor have possible targets of positive selection other than nucleotide substitutions, such as indels, received much attention. Here, we inspect for the signature of positive selection in the genes encoding five accessory gland proteins (Acps) (Acp26Aa, Acp32CD, Acp53Ea, Acp62F, and Acp70A) originally described from Drosophila melanogaster but with recognizable orthologues in the D. pseudoobscura subgroup. We compare patterns of selection within the D. psuedoobscura subgroup to those in the D. melanogaster subgroup. Similar patterns of positive selection were found in Acp26Aa and Acp62F in the two subgroups, while Acp53Ea and Acp70A experienced purifying selection in both subgroups. These proteins have thus remained targets for similar types of selection over long (\u3e21-MY) periods of time. We also found several indel substitutions and polymorphisms in Acp26Aa and Acp32CD. These indels occur in the same regions as positively selected nucleotide substitutions for Acp26Aa in the D. pseudoobscura subgroup but not in the D. melanogaster subgroup. Rates of indel substitution within Acp26Aa in the D. pseudoobscura subgroup were up to several times those in noncoding regions of the Drosophila genome. This suggests that indel substitutions may be under positive selection and may play a key role in the divergence of some Acps. © Springer Science+Business Media, Inc. 2006

A microsatellite linkage map of Drosophila mojavensis

BACKGROUND: Drosophila mojavensis has been a model system for genetic studies of ecological adaptation and speciation. However, despite its use for over half a century, no linkage map has been produced for this species or its close relatives. RESULTS: We have developed and mapped 90 microsatellites in D. mojavensis, and we present a detailed recombinational linkage map of 34 of these microsatellites. A slight excess of repetitive sequence was observed on the X-chromosome relative to the autosomes, and the linkage groups have a greater recombinational length than the homologous D. melanogaster chromosome arms. We also confirmed the conservation of Muller's elements in 23 sequences between D. melanogaster and D. mojavensis. CONCLUSIONS: The microsatellite primer sequences and localizations are presented here and made available to the public. This map will facilitate future quantitative trait locus mapping studies of phenotypes involved in adaptation or reproductive isolation using this species

Expression characteristics of the transfer-related kilB gene product of Streptomyces plasmid pIJ101: Implications for the plasmid spread function

Intermycelial transfer of Streptomyces plasmid pIJ101 occurs prior to cellular differentiation and is mediated by plasmid functions that are also required for production of zones of growth-inhibited recipient cells (i.e., pocks) that develop around individual donors during mating on agar medium. Several other pIJ101 functions, including that of the kilB gene, whose unregulated expression on pIJ101 is lethal, are required for normal pock size and so have been postulated to mediate intramycelial spread of the plasmid throughout recipient cells. Using antibodies raised against a KilB fusion protein expressed in Escherichia coli, native KilB protein was detected throughout development of pIJ101-containing Streptomyces lividans cells, with the concentration of KilB increasing dramatically and reaching a maximum during the final stages (i.e., sporulation and secondary metabolism) of cellular differentiation. Insertion of the kilB gene of pIJ101 into the S. lividans chromosome in cells lacking the pIJ101 KorB protein, which normally represses kilB gene transcription, resulted in elevated but still temporally increasing amounts of KilB. The increased expression or accumulation of the KilB spread protein throughout cellular differentiation of S. lividans, which leads to maximum KilB concentrations during developmental stages that occur far later than when intermycelial transfer of pIJ101 is mediated, supports the existence of a subsequent intramycelial component to the pIJ101 spread function. The results also suggest that intramycelial spread of pIJ101 molecules within the recipient extends beyond intercompartmental movements within the mycelia and includes undetermined steps within the spore-yielding aerial hyphae as well

Evaluating the discriminating capacity of cell death (apoptotic) biomarkers in sepsis.

Background: Sepsis biomarker panels that provide diagnostic and prognostic discrimination in sepsis patients would be transformative to patient care. We assessed the mortality prediction and diagnostic discriminatory accuracy of two biomarkers reflective of cell death (apoptosis), circulating cell-free DNA (cfDNA), and nucleosomes. Methods: The cfDNA and nucleosome levels were assayed in plasma samples acquired in patients admitted from four emergency departments with suspected sepsis. Subjects with non-infectious systemic inflammatory response syndrome (SIRS) served as controls. Samples were acquired at enrollment (T0) and 24 h later (T24). We assessed diagnostic (differentiating SIRS from sepsis) and prognostic (28-day mortality) predictive power. Models incorporating procalcitonin (diagnostic prediction) and APACHE II scores (mortality prediction) were generated. Results: Two hundred three subjects were included (107 provided procalcitonin measurements). Four subjects exhibited uncomplicated sepsis, 127 severe sepsis, 35 septic shock, and 24 had non-infectious SIRS. There were 190-survivors and 13 non-survivors. Mortality prediction models using cfDNA, nucleosomes, or APACHEII yielded AUC values of 0.61, 0.75, and 0.81, respectively. A model combining nucleosomes with the APACHE II score improved the AUC to 0.84. Diagnostic models distinguishing sepsis from SIRS using procalcitonin, cfDNA(T0), or nucleosomes(T0) yielded AUC values of 0.64, 0.65, and 0.63, respectively. The three parameter model yielded an AUC of 0.74. Conclusions: To our knowledge, this is the first head-to-head comparison of cfDNA and nucleosomes in diagnosing sepsis and predicting sepsis-related mortality. Both cfDNA and nucleosome concentrations demonstrated a modest ability to distinguish sepsis survivors and non-survivors and provided additive diagnostic predictive accuracy in differentiating sepsis from non-infectious SIRS when integrated into a diagnostic prediction model including PCT and APACHE II. A sepsis biomarker strategy incorporating measures of the apoptotic pathway may serve as an important component of a sepsis diagnostic and mortality prediction tool

Examining sociodemographic correlates of opioid use, misuse, and use disorders in the All of Us Research Program

BACKGROUND: The All of Us Research Program enrolls diverse US participants which provide a unique opportunity to better understand the problem of opioid use. This study aims to estimate the prevalence of opioid use and its association with sociodemographic characteristics from survey data and electronic health record (EHR). METHODS: A total of 214,206 participants were included in this study who competed survey modules and shared EHR data. Adjusted logistic regressions were used to explore the associations between sociodemographic characteristics and opioid use. RESULTS: The lifetime prevalence of street opioids was 4%, and the nonmedical use of prescription opioids was 9%. Men had higher odds of lifetime opioid use (aOR: 1.4 to 3.1) but reduced odds of current nonmedical use of prescription opioids (aOR: 0.6). Participants from other racial and ethnic groups were at reduced odds of lifetime use (aOR: 0.2 to 0.9) but increased odds of current use (aOR: 1.9 to 9.9) compared with non-Hispanic White participants. Foreign-born participants were at reduced risks of opioid use and diagnosed with opioid use disorders (OUD) compared with US-born participants (aOR: 0.36 to 0.67). Men, Younger, White, and US-born participants are more likely to have OUD. CONCLUSIONS: All of Us research data can be used as an indicator of national trends for monitoring the prevalence of receiving prescription opioids, diagnosis of OUD, and non-medical use of opioids in the US. The program employs a longitudinal design for routinely collecting health-related data including EHR data, that will contribute to the literature by providing important clinical information related to opioids over time. Additionally, this data will enhance the estimates of the prevalence of OUD among diverse populations, including groups that are underrepresented in the national survey data

Scanning for the Signatures of Positive Selection for Human-Specific Insertions and Deletions

Human-specific small insertions and deletions (HS indels, with lengths <100 bp) are reported to be ubiquitous in the human genome. However, whether these indels contribute to human-specific traits remains unclear. Here we employ a modified McDonald–Kreitman (MK) test and a combinatorial population genetics approach to infer, respectively, the occurrence of positive selection and recent selective sweep events associated with HS indels. We first extract 625,890 HS indels from the human–chimpanzee–macaque–mouse multiple alignments and classify them into nonpolymorphic (41%) and polymorphic (59%) indels with reference to the human indel polymorphism data. The modified MK test is then applied to 100-kb partially overlapped sliding windows across the human genome to scan for the signs of positive selection. After excluding the possibility of biased gene conversion and controlling for false discovery rate, we show that HS indels are potentially positively selected in about 10 Mb of the human genome. Furthermore, the indel-associated positively selected regions overlap with genes more often than expected. However, our result suggests that the potential targets of positive selection are located in noncoding regions. Meanwhile, we also demonstrate that the genomic regions surrounding HS indels are more frequently involved in recent selective sweep than the other regions. In addition, HS indels are associated with distinct recent selective sweep events in different human subpopulations. Our results suggest that HS indels may have been associated with human adaptive changes at both the species level and the subpopulation level