Involvement of a region of 23S ribosomal RNA of {\it E. coli\/} in decoding of termination codons

Involvement of E. coli 23S ribosomal RNA (rRNA) in decoding of termination codons was first indicated by the characterization of a 23S rRNA mutant that causes UGA-specific nonsense suppression. The work described here was begun to test the hypothesis that more 23S rRNA suppressors of specific nonsense mutations can be isolated and that they would occur non-randomly in the rRNA genes and be clustered in specific, functionally significant regions of rRNA.^ Approximately 2 kilobases of the gene for 23S rRNA were subjected to PCR random mutagenesis and the amplified products screened for suppression of nonsense mutations in trpA. All of the suppressor mutations obtained were located in a thirty-nucleotide part of the GTPase center, a conserved rRNA sequence and structure, and they and others made in that region by site-directed mutagenesis were shown to be UGA-specific in their suppression of termination codon mutations. These results proved the initial hypothesis and demonstrated that a group of nucleotides in this region are involved in decoding of the UGA termination codon. Further, it was shown that limitation of cellular availability or synthesis of L11, a ribosomal protein that binds to the GTPase center rRNA, resulted in suppression of termination codon mutations, suggesting the direct involvement of L11 in termination in vivo.^ Finally, in vivo analysis of certain site-specific mutations made in the GTPase center RNA demonstrated that (a) the G$\cdot$A base pair closing the hexanucleotide hairpin loop was not essential for normal termination, (b) the "U-turn" structure in the 1093 to 1098 hexaloop is critical for normal termination, (c) nucleotides A1095 and A1067, necessary for the binding to ribosomes of thiostrepton, an antibiotic that inhibits polypeptide release factor binding to ribosomes in vitro, are also necessary for normal peptide chain termination in vivo, and (d) involvement of this region of rRNA in termination is determined by some unique subset structure that includes particular nucleotides rather than merely by a general structural feature of the GTPase center.^ This work advances the understanding of peptide chain termination by demonstrating that the GTPase region of 23S rRNA participates in recognition of termination codons, through an associated ribosomal protein and specific conserved nucleotides and structural motifs in its RNA. ^

Functions and intramolecular interactions of ribosomal RNA in decoding of termination codons

Two regions in the 3$\prime$ domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3$\prime$ domain of 16S rRNA. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.^ With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484$\Delta$) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The $\Delta$2484 and A1093/$\Delta$2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.^ Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned the genes for EF-G and all three release factors into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay. (Abstract shortened by UMI.) ^

Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, σH is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

Role of the glutathione S -transferase P1 (GSTP1) electrophile binding site (H-site) in protection from doxorubicin -mediated cytotoxicity

The hypothesis addressed in this project was that novel variants of naturally occurring human glutathione S-transferase P1 (GSTP1) can be created by random mutagenesis of the GSTP1 active site to yield polypeptides with increased enzymatic activity against electrophilic substrates. Specifically, the mutant proteins would metabolize and inactivate selected electrophiles more efficiently than wild-type GSTP1 and confer significant cytoprotection, as measured by reduced apoptosis and increased clonogenic survival. Glutathione S-transferase P1, a major electrophile metabolizing and detoxifying enzyme, is encoded by a polymorphic genetic locus. This locus contains nucleotide transitions in the region encoding the active site of the peptide that yields proteins with significant structural and functional differences. The method of Degenerate Oligonucleotide Mediated Random Mutagenesis (DOMRM) was used to generate cDNAs encoding unique GSTP1 polypeptides with mutations within electrophile binding site (H-site) while leaving the glutathione binding site unaffected. A prokaryotic expression library of the mutant GSTP1 polypeptides was created and screened for increased resistance to cisplatin. This screen resulted in the isolation of 96 clones representing 22 distinct mutant cDNA sequences. To investigate the effects of the changes in the H-site on the biological activity of GSTP1, the cDNA of wild-type GSTP1c and two of the identified mutants were stably transfected into human LNCaP-Pro5 prostate cancer cells that do not endogenously express GSTP1. Wild-type transfectants were resistant to doxorubicin-induced apoptosis and displayed increased clonogenic survival compared to vector controls. However, contrary to the hypothesis, in both assays the mutant transfectants were no more resistant to doxorubicin than the wild-type transfectants. To elucidate the mechanisms underlying GSTP1-mediated survival, an in-vitro assay was developed to determine whether active GSTP1 protein directly metabolizes doxorubicin by conjugation to reduced glutathione (GSH). Although GSH did promote the appearance of a unique doxorubicin conjugate, conjugate formation was not substantially increased by the addition of GSTP1 in a variety of reaction conditions. ^

Mechanisms of perceptual learning of depth discrimination in random dot stereograms.

Perceptual learning is a training induced improvement in performance. Mechanisms underlying the perceptual learning of depth discrimination in dynamic random dot stereograms were examined by assessing stereothresholds as a function of decorrelation. The inflection point of the decorrelation function was defined as the level of decorrelation corresponding to 1.4 times the threshold when decorrelation is 0%. In general, stereothresholds increased with increasing decorrelation. Following training, stereothresholds and standard errors of measurement decreased systematically for all tested decorrelation values. Post training decorrelation functions were reduced by a multiplicative constant (approximately 5), exhibiting changes in stereothresholds without changes in the inflection points. Disparity energy model simulations indicate that a post-training reduction in neuronal noise can sufficiently account for the perceptual learning effects. In two subjects, learning effects were retained over a period of six months, which may have application for training stereo deficient subjects.

Effect of nonnormal random components on level and power in group-randomized trials

The use of group-randomized trials is particularly widespread in the evaluation of health care, educational, and screening strategies. Group-randomized trials represent a subset of a larger class of designs often labeled nested, hierarchical, or multilevel and are characterized by the randomization of intact social units or groups, rather than individuals. The application of random effects models to group-randomized trials requires the specification of fixed and random components of the model. The underlying assumption is usually that these random components are normally distributed. This research is intended to determine if the Type I error rate and power are affected when the assumption of normality for the random component representing the group effect is violated. ^ In this study, simulated data are used to examine the Type I error rate, power, bias and mean squared error of the estimates of the fixed effect and the observed intraclass correlation coefficient (ICC) when the random component representing the group effect possess distributions with non-normal characteristics, such as heavy tails or severe skewness. The simulated data are generated with various characteristics (e.g. number of schools per condition, number of students per school, and several within school ICCs) observed in most small, school-based, group-randomized trials. The analysis is carried out using SAS PROC MIXED, Version 6.12, with random effects specified in a random statement and restricted maximum likelihood (REML) estimation specified. The results from the non-normally distributed data are compared to the results obtained from the analysis of data with similar design characteristics but normally distributed random effects. ^ The results suggest that the violation of the normality assumption for the group component by a skewed or heavy-tailed distribution does not appear to influence the estimation of the fixed effect, Type I error, and power. Negative biases were detected when estimating the sample ICC and dramatically increased in magnitude as the true ICC increased. These biases were not as pronounced when the true ICC was within the range observed in most group-randomized trials (i.e. 0.00 to 0.05). The normally distributed group effect also resulted in bias ICC estimates when the true ICC was greater than 0.05. However, this may be a result of higher correlation within the data. ^

TUMOR PROGRESSION, METASTATIC POTENTIAL AND GENOMIC INSTABILITY

To investigate the hypothesis that increased malignant potential correlates with increased levels of genetic instability, the following parameters of instability were measured: (1) spontaneous mutation rates for ouabain resistance in murine cell lines of different malignant potentials, (2) the background prevalence of 6-thioguanine (6-TG) resistance in clone 4 (highly metastatic) and clone 19 (poorly metastatic) of the K1735 murine melanoma, (3) the prevalence of ouabain resistant variants in three murine cell lines and their variants after exposure to the mutagen MNNG, (4) the rate of generation of major karyotypic abnormalities in B16 F1 (poorly metastatic) and B16 F10 (highly metastatic) murine melanoma, and (5) analysis of the G-banded karyotypes of cloned B16 F1 and B16 F10 melanoma.^ No correlation of increased spontaneous mutation rates with increased malignant potential was found in repeated experiments with three murine cell lines and their variants of different malignant potential. The background prevalence of g-TG resistance was not significantly different for the poorly and highly metastatic clones of K1735 melanoma. The studies with MNNG-induced mutation showed no increased sensitivity of the highly metastatic variants of the three murine cell lines to mutagenesis. Neither did the rate of generation of major karyotypic abnormalities correlate with malignant potential. However, certain karyotypic differences were demonstrated after G-banding of the B16 F1 and F10 melanomas.^ One hypothesis which is consistent with these results is that the rate of generation of genetic abnormalities need not be strongly related to the degree of malignant potential. An increased prevalence of genetic changes may merely reflect the accumulation of abnormalities while their rate of production remains constant. The presence of specific nonrandom changes likely is the main determinant of malignant potential rather than the rate of production of random changes. ^

The evidence behind the level of a random urine protein:creatinine in the diagnosis of preeclampsia: A systematic review

Objective. To determine the accuracy of the urine protein:creatinine ratio (pr:cr) in predicting 300 mg of protein in 24-hour urine collection in pregnant patients with suspected preeclampsia. ^ Methods. A systematic review was performed. Articles were identified through electronic databases and the relevant citations were hand searching of textbooks and review articles. Included studies evaluated patients for suspected preeclampsia with a 24-hour urine sample and a pr:cr. Only English language articles were included. The studies that had patients with chronic illness such as chronic hypertension, diabetes mellitus or renal impairment were excluded from the review. Two researchers extracted accuracy data for pr:cr relative to a gold standard of 300 mg of protein in 24-hour sample as well as population and study characteristics. The data was analyzed and summarized in tabular and graphical form. ^ Results. Sixteen studies were identified and only three studies met our inclusion criteria with 510 total patients. The studies evaluated different cut-points for positivity of pr:cr from 130 mg/g to 700 mg/g. Sensitivities and specificities for pr:cr of 130mg/g -150 mg/g were 90-93% and 33-65%, respectively; for a pr:cr of 300 mg/g were 81-95% and 52-80%, respectively; for a pr:cr of 600-700mg/g were 85-87% and 96-97%, respectively. ^ Conclusion. The value of a random pr:cr to exclude pre-eclampsia is limited because even low levels of pr:cr (130-150 mg/g) may miss up to 10% of patients with significant proteinuria. A pr:cr of more than 600 mg/g may obviate a 24-hour collection.^

An empirical evaluation of the Random Forests classifier models for variable selection in a large-scale lung cancer case-control study

Random Forests™ is reported to be one of the most accurate classification algorithms in complex data analysis. It shows excellent performance even when most predictors are noisy and the number of variables is much larger than the number of observations. In this thesis Random Forests was applied to a large-scale lung cancer case-control study. A novel way of automatically selecting prognostic factors was proposed. Also, synthetic positive control was used to validate Random Forests method. Throughout this study we showed that Random Forests can deal with large number of weak input variables without overfitting. It can account for non-additive interactions between these input variables. Random Forests can also be used for variable selection without being adversely affected by collinearities. ^ Random Forests can deal with the large-scale data sets without rigorous data preprocessing. It has robust variable importance ranking measure. Proposed is a novel variable selection method in context of Random Forests that uses the data noise level as the cut-off value to determine the subset of the important predictors. This new approach enhanced the ability of the Random Forests algorithm to automatically identify important predictors for complex data. The cut-off value can also be adjusted based on the results of the synthetic positive control experiments. ^ When the data set had high variables to observations ratio, Random Forests complemented the established logistic regression. This study suggested that Random Forests is recommended for such high dimensionality data. One can use Random Forests to select the important variables and then use logistic regression or Random Forests itself to estimate the effect size of the predictors and to classify new observations. ^ We also found that the mean decrease of accuracy is a more reliable variable ranking measurement than mean decrease of Gini. ^

Prevalence of gastro esophageal reflux disease following the Montreal Consensus and its association with Helicobacter pylori infection and obesity in a random sample of adults of Ciudad Juarez, Mexico, 2004.

Gastroesophageal reflux disease is a common condition affecting 25 to 40% of the population and causes significant morbidity in the U.S., accounting for at least 9 million office visits to physicians with estimated annual costs of $10 billion. Previous research has not clearly established whether infection with Helicobacter pylori, a known cause of peptic ulcer, atrophic gastritis and non cardia adenocarcinoma of the stomach, is associated with gastroesophageal reflux disease. This study is a secondary analysis of data collected in a cross-sectional study of a random sample of adult residents of Ciudad Juarez, Mexico, that was conducted in 2004 (Prevalence and Determinants of Chronic Atrophic Gastritis Study or CAG study, Dr. Victor M. Cardenas, Principal Investigator). In this study, the presence of gastroesophageal reflux disease was based on responses to the previously validated Spanish Language Dyspepsia Questionnaire. Responses to this questionnaire indicating the presence of gastroesophageal reflux symptoms and disease were compared with the presence of H. pylori infection as measured by culture, histology and rapid urease test, and with findings of upper endoscopy (i.e., hiatus hernia and erosive and atrophic esophagitis). The prevalence ratio was calculated using bivariate, stratified and multivariate negative binomial logistic regression analyses in order to assess the relation between active H. pylori infection and the prevalence of gastroesophageal reflux typical syndrome and disease, while controlling for known risk factors of gastroesophageal reflux disease such as obesity. In a random sample of 174 adults 48 (27.6%) of the study participants had typical reflux syndrome and only 5% (or 9/174) had gastroesophageal reflux disease per se according to the Montreal consensus, which defines reflux syndromes and disease based on whether the symptoms are perceived as troublesome by the subject. There was no association between H. pylori infection and typical reflux syndrome or gastroesophageal reflux disease. However, we found that in this Northern Mexican population, there was a moderate association (Prevalence Ratio=2.5; 95% CI=1.3, 4.7) between obesity (≥30 kg/m2) and typical reflux syndrome. Management and prevention of obesity will significantly curb the growing numbers of persons affected by gastroesophageal reflux symptoms and disease in Northern Mexico. ^