The post-transcriptional gene silencing pathway in Eucalyptus

Post-transcriptional gene silencing (PTGS) is a conserved surveillance mechanism that identifies and cleaves double-stranded RNA molecules and their cellular cognate transcripts. The RNA silencing response is actually used as a powerful technique (named RNA interference) for potent and specific inhibition of gene expression in several organisms. To identify gene products in Eucalyptus sharing similarities with enzymes involved in the PTGS pathway, we queried the expressed sequence tag database of the Brazilian Eucalyptus Genome Sequence Project Consortium (FORESTs) with the amino acid sequences of known PTGS-related proteins. Among twenty-six prospected genes, our search detected fifteen assembled sequences encoding products presenting high level of similarity (E value < 10 -40) to proteins involved in PTGS in plants and other organisms. We conclude that most of the genes known to be involved in the PTGS pathway are represented in the FORESTs database. Copyright by the Brazilian Society of Genetics.

Molecular analysis of three FUT3 gene single nucleotide polymorphisms and their relationship with the lewis erythrocytary phenotype in a human population of japanese-ancestry living in Tomé Açu, a town in the Brazilian Amazon

The Lewis blood group system involves two major antigens, Le^ª and Le^b. Their antigenic determinants are not primary gene products but are synthesized by the transfer of sugar subunits to a precursory chain by a specific enzyme which is the product of the FUT3 gene (Lewis gene). The presence of three FUT3 gene single nucleotide polymorphisms (SNPs) (59T > G; 508G > A and 1067T > A) was related to the Lewis phenotype of erythrocytes from 185 individuals of Japanese ancestry living in the town of Tomé-Açu in the Brazilian Amazon region. This relationship was detected using a serological hemagglutination test and the Dot-ELISA assay along with the molecular technique PCR-RFLP. We found that the three SNPs investigated in this study only accounted for a proportion of the Lewis-negative phenotype of the erythrocytes.

Molecular analysis of three FUT3 gene single nucleotide polymorphisms and their relationship with the lewis erythrocytary phenotype in a human population of japanese-ancestry living in Tomé Açu, a town in the Brazilian Amazon

The Lewis blood group system involves two major antigens, Le^a and Le^b. Their antigenic determinants are not primary gene products but are synthesized by the transfer of sugar subunits to a precursory chain by a specific enzyme which is the product of the FUT3 gene (Lewis gene). The presence of three FUT3 gene single nucleotide polymorphisms (SNPs) (59T > G; 508G > A and 1067T > A) was related to the Lewis phenotype of erythrocytes from 185 individuals of Japanese ancestry living in the town of Tomé-Açu in the Brazilian Amazon region. This relationship was detected using a serological hemagglutination test and the Dot-ELISA assay along with the molecular technique PCR-RFLP. We found that the three SNPs investigated in this study only accounted for a proportion of the Lewis-negative phenotype of the erythrocytes.

Modeling gene expression regulatory networks with the sparse vector autoregressive model

Abstract Background To understand the molecular mechanisms underlying important biological processes, a detailed description of the gene products networks involved is required. In order to define and understand such molecular networks, some statistical methods are proposed in the literature to estimate gene regulatory networks from time-series microarray data. However, several problems still need to be overcome. Firstly, information flow need to be inferred, in addition to the correlation between genes. Secondly, we usually try to identify large networks from a large number of genes (parameters) originating from a smaller number of microarray experiments (samples). Due to this situation, which is rather frequent in Bioinformatics, it is difficult to perform statistical tests using methods that model large gene-gene networks. In addition, most of the models are based on dimension reduction using clustering techniques, therefore, the resulting network is not a gene-gene network but a module-module network. Here, we present the Sparse Vector Autoregressive model as a solution to these problems. Results We have applied the Sparse Vector Autoregressive model to estimate gene regulatory networks based on gene expression profiles obtained from time-series microarray experiments. Through extensive simulations, by applying the SVAR method to artificial regulatory networks, we show that SVAR can infer true positive edges even under conditions in which the number of samples is smaller than the number of genes. Moreover, it is possible to control for false positives, a significant advantage when compared to other methods described in the literature, which are based on ranks or score functions. By applying SVAR to actual HeLa cell cycle gene expression data, we were able to identify well known transcription factor targets. Conclusion The proposed SVAR method is able to model gene regulatory networks in frequent situations in which the number of samples is lower than the number of genes, making it possible to naturally infer partial Granger causalities without any a priori information. In addition, we present a statistical test to control the false discovery rate, which was not previously possible using other gene regulatory network models.

Multimodality and flexibility of stochastic gene expression

We consider a general class of mathematical models for stochastic gene expression where the transcription rate is allowed to depend on a promoter state variable that can take an arbitrary (finite) number of values. We provide the solution of the master equations in the stationary limit, based on a factorization of the stochastic transition matrix that separates timescales and relative interaction strengths, and we express its entries in terms of parameters that have a natural physical and/or biological interpretation. The solution illustrates the capacity of multiple states promoters to generate multimodal distributions of gene products, without the need for feedback. Furthermore, using the example of a three states promoter operating at low, high, and intermediate expression levels, we show that using multiple states operons will typically lead to a significant reduction of noise in the system. The underlying mechanism is that a three-states promoter can change its level of expression from low to high by passing through an intermediate state with a much smaller increase of fluctuations than by means of a direct transition.

Gene expression responses to acute and chronic heat stress in the common reef-building coral Pocillopora verrucosa

Global climate change is impacting coral reefs worldwide, with approximately 19% of reefs being permanently degraded, 15% showing symptoms of imminent collapse, and 20% at risk of becoming critically affected in the next few decades. This alarming level of reef degradation is mainly due to an increase in frequency and intensity of natural and anthropogenic disturbances. Recent evidence has called into question whether corals have the capacity to acclimatize or adapt to climate changes and some groups of corals showed inherent physiological tolerance to environmental stressors. The aim of the present study was to evaluate mRNA expression patterns underlying differences in thermal tolerance in specimen of the common reef-building coral Pocillopora verrucosa collected at different locations in Bangka Island waters (North Sulawesi, Indonesia). Part of the experimental work was carried out at the CoralEye Reef Research Outpost (Bangka Island). This includes sampling of corals at selected sites and at different depths (3 and 12 m) as well as their experimental exposure to an increased water temperature under controlled conditions for 3 and 7 days. Levels of mRNAs encoding ATP synthase (ATPs) NADH dehydrogenase (NDH) and a 70kDa Heat Shock Protein (HSP70) were evaluated by quantitative real time PCR. Transcriptional profiles evaluated under field conditions suggested an adaptation to peculiar local environmental conditions in corals collected at different sites and at the low depth. Nevertheless, high–depth collected corals showed a less pronounced site-to-site separation suggesting more homogenous environmental conditions. Exposure to an elevated temperature under controlled conditions pointed out that corals adapted to the high depth are more sensitive to the effects of thermal stress, so that reacted to thermal challenge by significantly over-expressing the selected gene products. Being continuously exposed to fluctuating environmental conditions, low-depth adapted corals are more resilient to the stress stimulus, and indeed showed unaffected or down-regulated mRNA expression profiles. Overall these results highlight that transcriptional profiles of selected genes involved in cellular stress response are modulated by natural seasonal temperature changes in P. verrucosa. Moreover, specimens living in more variable habitats (low-depth) exhibit higher basal HSP70 mRNA levels, possibly enhancing physiological tolerance to environmental stressors.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral

OBJECTIVES The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

Heart failure gene therapy: the path to clinical practice

Gene therapy, aimed at the correction of key pathologies being out of reach for conventional drugs, bears the potential to alter the treatment of cardiovascular diseases radically and thereby of heart failure. Heart failure gene therapy refers to a therapeutic system of targeted drug delivery to the heart that uses formulations of DNA and RNA, whose products determine the therapeutic classification through their biological actions. Among resident cardiac cells, cardiomyocytes have been the therapeutic target of numerous attempts to regenerate systolic and diastolic performance, to reverse remodeling and restore electric stability and metabolism. Although the concept to intervene directly within the genetic and molecular foundation of cardiac cells is simple and elegant, the path to clinical reality has been arduous because of the challenge on delivery technologies and vectors, expression regulation, and complex mechanisms of action of therapeutic gene products. Nonetheless, since the first demonstration of in vivo gene transfer into myocardium, there have been a series of advancements that have driven the evolution of heart failure gene therapy from an experimental tool to the threshold of becoming a viable clinical option. The objective of this review is to discuss the current state of the art in the field and point out inevitable innovations on which the future evolution of heart failure gene therapy into an effective and safe clinical treatment relies.

The cloning and expression of myogenin, a gene regulating myogenesis

Expression of the differentiated skeletal muscle phenotype is a process that appears to occur in at least two stages. First, pluripotent stem cells become committed to the myogenic lineage. Although undifferentiated and capable of continued proliferation, determined myoblasts are restricted to a single developmental fate. Upon receiving the appropriate environmental signals, these determined myoblasts withdraw from the cell cycle, fuse to form multi-nucleated myotubes, and begin to express a battery of muscle-specific gene products that make up the functional and contractile apparatus of the muscle. This project is aimed at the identification and characterization of factors that control the determination and differentiation of myogenic cells. We have cloned a cDNA, called myogenin, that plays an important role in these processes. Myogenin is expressed exclusively in skeletal muscle in vivo and myogenic cell lines in vitro. Its expression is sharply upregulated during differentiation. When constitutively expressed in fibroblasts, myogenin converts these cells to the myogenic lineage. Transfected cells behave as myogenic tissue culture cells with respect to the genes they express, the way they respond to environmental cues, and are capable of fusing to form multinucleated myotubes. Sequence analysis showed that this cDNA has homology to a family of transcription factors in a region of 72 amino acids known as the basic helix-loop-helix motif. This domain appears to mediate binding to a DNA sequence element known as an E-box (CANNTG) essential for the activity of the enhancers of many muscle-specific genes.^ Analysis of myogenin in tissue culture cells showed that its expression is responsive to many of the environmental cues, such as the presence of growth factors and oncogenes, that modulate myogenesis. In an attempt to identify the cis- and trans-elements that control myogenin expression and thereby understand what factors are responsible for the establishment of the myogenic lineage, we have cloned the myogenin gene. After analysis of the gene structure, we constructed a series of reporter constructs from the 5$\prime$ upstream sequence of the myogenin gene to determine which cis-acting sequences might be important in myogenin regulation. We found that 184 nucleotides of the 5$\prime$ sequence was sufficient to direct high-level muscle-specific expression of the reporter gene. Two sequence elements present in the 184 fragment, an E-box and a MEF-2 site, have been shown previously to be important in muscle-specific transcription. Mutagenesis of these sites revealed that both sites are necessary for full activity of the myogenin promoter, and suggests that a complex hierarchy of transcription factors control myogenic differentiation. ^

Analysis of differential gene expression in nontumorigenic glioblastoma multiforme

Loss of chromosome 10 represents the most common cytogenetic abnormality in high grade gliomas (glioblastoma multiforme). To identify genes involved in the malignant progression of human gliomas, a subtractive hybridization was performed between a tumorigenic glioblastoma cell line (LG11) and a nontumorgenic hybrid cell (LG11.3) containing an introduced chromosome 10. LG11 mRNA was subtracted from LG11.3 cDNA to produce cDNA probes enriched for sequences whose expression differs quantitatively from the parental tumorigenic cells. Both known and novel sequences were identified as a result of the subtraction. Northern blot analysis was then used to confirm differential expression of several subtracted clones. One novel clone, clone 17, identified a 2.6 kb message that showed a consistent two to four fold increase in expression in the LG11.3 nontumorigenic cells. Clone 17 (340 bp) was used successfully to screen for a near full-length version, RIG (regulated in glioma), which was 2,569 bp in size. The RIG cDNA sequence showed homology to clone 17 and to an anonymous EST (IB666), but to no previously identified genes. This screening effort also identified several independent clones representing novel sequences, most of which failed to show increased expression in the nontumorigenic GBM cells. Tissue distribution studies of RIG indicated highest levels of expression in human brain with appreciably lower levels in heart and lung. In vitro transcription and translation experiments demonstrated the ability of RIG to direct the synthesis of a 13 kD protein product. However, open reading frame analysis revealed no identify with previously described motifs or any known proteins. Using a combination of somatic cell hybrid panels and in situ hybridization, the RIG gene was mapped to chromosome 11p14-11p15. Further study of RIG and related gene products may provide insight into the negative regulation of glial oncogenesis. ^

Identification of two vitamin D response elements in the rat osteopontin gene

Studies to elucidate the function of vitamin D have demonstrated an important role in regulating bone-related cells, including osteoblasts and osteoclasts. A seemingly paradoxical observation is that 1,25(OH)$\sb2$D$\sb3$, the active metabolite of vitamin D, stimulates bone resorption, yet regulates transcription of genes expressed by osteoblasts. One mechanism that could explain these actions is the upregulation of transcription of osteoblast-specific genes. These gene products could then act as effectors to influence osteoclastic activity. We hypothesized that molecular signals could be deposited directly into the mineralized matrix in the form of noncollagenous proteins, such as osteopontin (OPN). The structure, biosynthesis and localization of OPN suggest that it could function to mediate the molecular "cross talk" between osteoblasts and osteoclasts in response to 1,25(OH)$\sb2$D$\sb3$. To begin to address this hypothesis, elucidation of the molecular mechanisms of action involved in the transactivation of OPN by 1,25(OH)$\sb2$D$\sb3$ is essential.^ In the present study, the rat opn gene was isolated and characterized. Functional analysis by transient transfection of the 5$\sp\prime$ flanking sequences of the rat opn gene fused to the luciferase gene demonstrated that OPN is transcriptionally upregulated by 1,25(OH)$\sb2$D$\sb3$, mediated through two vitamin D response elements (VDRE). Both proximal and distal VDREs are structurally similar (two imperfect direct repeats separated by a 3 nucleotide spacer) and bind protein complexes that include the VDR and retinoid-X receptor (RXR). Isolated VDRE expression constructs produce functional activity of equivalent magnitude of responsiveness to 1,25(OH)$\sb2$D$\sb3$. However, expression constructs containing either VDRE and at least 200 bp of 5$\sp\prime$ and 3$\sp\prime$ flanking sequence demonstrated that the distal VDRE produces an amplitude of response significantly higher than the proximal VDRE. We conclude that the transcriptional upregulation of the opn gene by 1,25(OH)$\sb2$D$\sb3$ involves the transactivation of two VDREs, while maximal responsiveness requires interaction of the VDREs with additional cis-elements contained in the 5$\sp\prime$ sequence. ^

THE BIOSYNTHESIS AND CHARACTERIZATION OF MURINE LEUKEMIA VIRAL PROTEINS

Two murine leukemia viruses (MuLVs), Rauscher (R-MuLV) and Moloney (Mo-MuLV) MuLVs, were studied to identify the biosynthetic pathways leading to the generation of mature virion proteins. Emphasis was placed on the examination of the clone 1 Mo-MuLV infected cell system.^ At least three genetic loci vital to virion replication exist on the MuLV genome. The 'gag' gene encodes information for the virion core proteins. The 'pol' gene specifies information for the RNA-dependent-DNA-polymerase (pol), or reverse transcriptase (RT). The 'env' gene contains information for the virion envelope proteins.^ MuLV specified proteins were synthesized by way of precursor polyproteins, which were processed to yield mature virion proteins. Pulse-chase kinetic studies, radioimmunoprecipitation, and peptide mapping were the techniques used to identify and characterize the MuLV viral precursor polyproteins and mature virion proteins.^ The 'gag' gene of Mo-MuLV coded for two primary gene products. One 'gag' gene product was found to be a polyprotein of 65,000 daltons M(,r) (Pr65('gag)). Pr65('gag) contained the antigenic and structural determinants of all four viral core proteins--p30, p15, pp12 and p10. Pr65('gag) was the major intracellular precursor polyprotein in the generation of mature viral core proteins. The second 'gag' gene product was a glycosylated gene product (gPr('gag)). An 85,000 dalton M(,r) polyprotein (gPr85('gag)) and an 80,000 dalton M(,r) (gPr80('gag)) polyprotein were the products of the 'gag' genes of Mo-MuLV and R-MuLV, respectively. gPr('gag) contained the antigenic and structural determinants of the four virion core proteins. In addition, gPr('gag) contained peptide information over and above that of Pr65('gag). Pulse-chase kinetic studies in the presence of tunicamycin revealed a separate processing pathway of gPr('gag) that did not seem to involve the generation of mature virion core proteins. Subglycosylated gPr('gag) was found to have a molecular weight of 75,000 daltons (Pr75('gag)) for both Mo-MuLV and R-MuLV.^ The Mo-MuLV 'pol' gene product was initially synthesized as a read-through 'gag-pol' intracellular polyprotein containing both antigenic and structural determinants of both the 'gag' and 'pol' genes. This read-through polyprotein was found to be a closely spaced doublet of two similarly sized proteins at 220-200,000 daltons M(,r) (Pr220/200('gag-pol)). Pulse-chase kinetic studies revealed processing of Pr220/200('gag-pol) to unstable intermediate intracellular proteins of 145,000 (Pr145('pol)), 135,000 (Pr135('pol)), and 125,000 (Pr125('pol)) daltons M(,r). Further chase incubations demonstrated the appearance of an 80,000 dalton M(,r) protein, which represented the mature polymerase (p80('pol)).^ The primary intracellular Mo-MuLV 'env' gene product was found to be a glycosylated polyprotein of 83,000 daltons M(,r) (gPr83('env)). gPr83('env) contained the antigenic and structural determinants of both mature virion envelope proteins, gp70 and p15E. In addition, gPr83('env) contained unique peptide sequences not present in either gp70 or p15E. The subglycosylated form of gPr83('env) had a molecular weight of 62,000 daltons (Pr62('env)).^ Virion core proteins of R-MuLV and Mo-MuLV were examined. Structural homology was observed betwen p30s and p10s. Significant structural non-homology was demonstrated between p15s and pp12s. ^

A TEMPERATURE-SENSITIVE DEFECT IN THE EXPRESSION OF A SARCOMA VIRUS TRANSFORMING GENE

The viral proteins synthesized by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in a function required for the maintenance of the transformed state (ts110) were examined. Normal rat kidney cells (NRK) were infected with the ts110 virus and a non-virus-producing cell clone, termed 6m2, was isolated. This cell clone had a malignant phenotype at 33(DEGREES), the permissive temperature, but changed to a normal phenotype at 39(DEGREES).^ Two viral proteins were detected in 6m2 cells. A 58,000 dalton protein (P58) was detected at both 33(DEGREES) and 39(DEGREES) and contained only core protein (gag) coded sequences. An 85,000 dalton protein (P85) was detected only at 33(DEGREES) and contained sequences of viral core proteins p15, pp12, and part of p30 as well as protein sequences attributed by peptide mapping to P23 and P38, two candidate viral mouse src (v-mos) gene products. These results provide good evidence that P85 is a gag-mos polyprotein. As expected for a functional mos-gene product, P85 synthesis preceded parameters characteristic of the transformed state, including changes in cell morphology, in the cytoplasmic microtubule complex (CMTC) and in the rate of hexose uptake.^ Other studies were conducted to ascertain the defect which prohibited the synthesis of P85 at 39(DEGREES), the non-permissive temperature. When 6m2 cells were treated with actinomycin D at 39(DEGREES) and shifted to 33(DEGREES), the cells were unable to synthesize P85, but P58 continued to be made. P85 mRNA, active at 33(DEGREES), continued to be translated for two to three hours after shifting to 39(DEGREES) as judged by pulse-labeling experiments. Virus harvested at 33(DEGREES) from ts110 MuSV producer cells packaged both P85 and P58 coding RNAs while virus harvested at 39(DEGREES) was deficient in the amount of P85 coding RNA. Agarose gel electrophoresis of 6m2 cellular RNA showed that RNA harvested at 33(DEGREES) contained the 4.0 and 3.5 kb RNAs. Similar experiments on cells maintained at 39(DEGREES) have detected only the 4.0 kb RNA, suggesting that the 3.5 kb RNA codes for P85. The defect appeared to be in the long term stability of the P85 coding RNA at 39(DEGREES), since, in shift-up experiments (33(DEGREES) (--->) 39(DEGREES)), P85 was translated for only three hours at 39(DEGREES), while P58 was synthesized for at least eight hours. However, at 33(DEGREES) in the presence of actinomycin D, the ratio of P85 and P58 synthesis at hourly intervals was similar throughout a 12 hour period. ^

The molecular basis for beta-lactamase gene expression in B. anthracis, B. cereus and B. thuringiensis

The susceptibility of most Bacillus anthracis strains to β-lactam antibiotics is intriguing considering that the B. anthracis genome harbors two β-lactamase genes, bla1 and bla2, and closely-related species, Bacillus cereus and Bacillus thuringiensis, typically produce β-lactamases. This work demonstrates that B. anthracis bla expression is affected by two genes, sigP and rsp, predicted to encode an extracytoplasmic function sigma factor and an antisigma factor, respectively. Deletion of the sigP/rsp locus abolished bla expression in a penicillin-resistant clinical isolate and had no effect on bla expression in a prototypical penicillin-susceptible strain. Complementation with sigP/rsp from the penicillin-resistant strain, but not the penicillin-susceptible strain, conferred β-lactamase activity upon both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsp in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsp homologues are required for inducible penicillin resistance in those species. Expression of the B. cereus or B. thuringiensis sigP and rsp genes in a B. anthracis sigP/rsp-null mutant confers resistance to β-lactam antibiotics, suggesting that while B. anthracis contains the genes necessary for sensing β-lactam antibiotics, the B. anthracis sigP/rsp gene products are insufficient for bla induction. ^ Because alternative sigma factors recognize unique promoter sequence, direct targets can be elucidated by comparing transcriptional profiling results with an in silico search using the sigma factor binding sequence. Potential σP -10 and -35 promoter elements were identified upstream from bla1 bla2 and sigP. Results obtained from searching the B. anthracis genome with the conserved sequences were evaluated against transcriptional profiling results comparing B. anthracis 32 and an isogenic sigP/rsp -null strain. Results from these analyses indicate that while the absence of the sigP gene significantly affects the transcript levels of 16 genes, only bla1, bla2 and sigP are directly regulated by σP. The genomes of B. cereus and B. thuringiensis strains were also analyzed for the potential σP binding elements. The sequence was located upstream from the sigP and bla genes, and previously unidentified genes predicted to encode a penicillin-binding protein (PBP) and a D-alanyl-D-alanine carboxypeptidase, indicating that the σ P regulon in these species responds to cell-wall stress caused by β-lactam antibiotics. ^ β-lactam antibiotics prevent attachment of new peptidoglycan to the cell wall by blocking the active site of PBPs. A B. cereus and B. thuringiensis pbp-encoding gene located near bla1 contains a potential σP recognition sequence upstream from the annotated translational start. Deletion of this gene abolished β-lactam resistance in both strains. Mutations in the active site of the PBP were detrimental to β-lactam resistance in B. cereus, but not B. thuringiensis, indicating that the transpeptidase activity is only important in B. cereus. I also found that transcript levels of the PBP-encoding gene are not significantly affected by the presence of β-lactam antibiotic. Based on these data I hypothesize that the gene product acts a sensor of β-lactam antibiotic. ^

DETERMINING THE GENOTYPE-PHENOTYPE CONNECTION IN SYNTHETIC INDUCIBLE GENE EXPRESSION SYSTEMS

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise. Methods I created probabilistic biochemical reaction models from known behaviors of the tetR and rtTA genes, gene products, and their gene architectures. I then simplified these models to account for essential behaviors of gene expression systems. Finally, I used these models to predict behaviors of modified gene expression systems, which were experimentally verified. Results Cell growth, which is often ignored when formulating chemical kinetics models, was essential for understanding gene expression behavior. Models incorporating growth effects were used to explain unexpected reductions in gene expression noise, design a set of gene expression systems with “linear” dose-responses, and quantify the speed with which cells explored their fitness landscapes due to noisy gene expression. Conclusions Models incorporating noisy gene expression and cell division were necessary to design, understand, and predict the behaviors of synthetic gene expression systems. The methods and models developed here will allow investigators to more efficiently design new gene expression systems, and infer gene expression properties of TetR based systems.