Transcriptional analysis of pqqD and study of the regulation of pyrroloquinoline quinone biosynthesis in Methylobacterium extorquens AM1

Methanol dehydrogenase, the enzyme that oxidizes methanol to formaldehyde in gram-negative methylotrophs, contains the prosthetic group pyrroloquinoline quinone (PQQ). To begin to analyze how the synthesis of PQQ is coordinated with the production of other methanol dehydrogenase components, the transcription of one of the key PQQ synthesis genes has been studied. This gene (pqqD) encodes a 29-amino- acid peptide that is thought to be the precursor for PQQ biosynthesis. A unique transcription start site was mapped to a guanidine nucleotide 95 bp upstream of the pqqD initiator codon. RNA blot analysis identified two transcripts, a major one of 240 bases encoding pqqD and a minor one of 1,300 bases encoding pqqD and the gene immediately downstream, pqqG. Both transcripts are present at similar levels in cells grown on methanol and on succinate, but the levels of PQQ are about fivefold higher in cells grown on methanol than in cells grown on succinate. These results suggest that PQQ production is regulated at a level different from the transcription of pqqD. The genes mxbM, mxbD, mxcQ, mxcE, and mxaB are required for transcription of the genes encoding the methanol dehydrogenase subunits and were assessed for their role in PQQ production. PQQ levels were measured in mutants defective in each of these regulatory genes and compared with levels of pqqD transcription, measured with a transcriptional fusion between the pqqD promoter and xylE. The results showed that only a subset of these regulatory genes (mxbM, mxbD, and mxaB) is required for transcription of pqqD, and only mxbM and mxbD mutants affected the final levels of PQQ significantly

Analyzing Breast Tumor Response To Neoadjuvant Therapy By Pk Models Of Dynamic-­contrast Enhanced Mr

Introduction: Pharmacokinetic modeling of contrast uptake by Dynamic-­‐Contrast Enhanced Magnetic Resonance Imaging studies has shown potential to predict the pathologic response to neoadjuvant therapy in breast cancer patients via several small studies. We will attempt to prospectively validate the performance of several previously published criteria in women undergoing neoadjuvant therapy with bevacizumab or trastuzumab. Methods: 11 patients underwent dynamic contrast enhanced magnetic resonance imaging both before and after receiving one cycle of trastuzumab or bevacizumab neoadjuvant chemotherapy for a primary breast lesion of greater than two centimeters. By abstracting pharmacokinetic parameters (Ktrans) from each study, predictions for therapeutic response based on previously published criteria (Ah–See and Yu utilize a threshold for percentage change in median Ktrans; Padhani, a percentage change in Ktrans range) were compared with the response by pathology acquired after completion of neoadjuvant therapy. Results: 7 patients were able to successfully complete imaging at the two requisite time points. All utilized criteria correctly identified 5/5 non–responders; the Ah­–See and Padhani criteria were able to identify 1/2 positive responders; and the Yu criterion identified 0/2 positive responders. Discussion: The efficacy of the Ah–See and Padhani criteria identify responders and non–responders equally well. Due to the Padhani criterion\u27s susceptibility to noise, however, it is likely that Ah–See would outperform Padhani on a larger cohort

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from Methylococcus capsulatus bath and Methylomonas albus BG8

An open-reading-frame fragment of a Methylobacterium sp. strain AM1 gene (moxF) encoding a portion of the methanol dehydrogenase structural protein has been used as a hybridization probe to detect similar sequences in a variety of methylotrophic bacteria. This hybridization was used to isolate clones containing putative moxF genes from two obligate methanotrophic bacteria, Methylococcus capsulatus Bath and Methylomonas albus BG8. The identity of these genes was confirmed in two ways. A T7 expression vector was used to produce methanol dehydrogenase protein in Escherichia coli from the cloned genes, and in each case the protein was identified by immunoblotting with antiserum against the Methylomonas albus methanol dehydrogenase. In addition, a moxF mutant of Methylobacterium strain AM1 was complemented to a methanol-positive phenotype that partially restored methanol dehydrogenase activity, using broad-host-range plasmids containing the moxF genes from each methanotroph. The partial complementation of a moxF mutant in a facultative serine pathway methanol utilizer by moxF genes from type I and type X obligate methane utilizers suggests broad functional conservation of the methanol oxidation system among gram-negative methylotrophs

Identification and nucleotide sequences of mxaA, mxaC, mxaK, mxaL, and mxaD genes from Methylobacterium extorquens AM1

The DNA sequence for a 4.4-kb HindIII-XhoI Methylobacterium extorquens AM1 DNA fragment that is known to contain three genes (mxaAKL) involved in incorporation of calcium into methanol dehydrogenase (I. W. Richardson and C. Anthony, Biochem. J. 287:709-7115, 1992) was determined. Five complete open reading frames and two partial open reading frames were found, suggesting that this region contains previously unidentified genes. A combination of sequence analysis, mutant complementation data, and gene expression studies showed that these genes correspond to mxaSACKLDorf1. Of the three previously unidentified genes (mxaC, mxaD, and orf1), mutant complementation studies showed that mxaC is required for methanol oxidation, while the function of the other two genes is still unknown

Cloning, Mutagenesis, and Physiological Effect of a Hydroxypyruvate Reductase Gene from Methylobacterium extorquens AM1

The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase

Towards a Neuroscience of Computer Programming & Education:A thesis submitted in partial fulfilment of the requirements of the University of East Anglia for the degree of Doctor of Philosophy. Research undertaken in the School of Psychology, University of East Anglia.

Computer programming is fast becoming a required part of School curricula, but students find the topic challenging and university dropout rates are high. Observations suggest that hands-on keyboard typing improves learning, but quantitative evidence for this is lacking and the mechanisms are still unclear. Here we study neural and behavioral processes of programming in general, and Hands-on in particular. In project 1, we taught naïve teenagers programming in a classroom-like session, where one student in a pair typed code (Hands-on) while the other participated by discussion (Hands-off). They were scanned with fMRI 1-2 days later while evaluating written code, and their knowledge was tested again after a week. We find confidence and math grades to be important for learning, and easing of intrinsic inhibitions of parietal, temporal, and superior frontal activation to be a typical neural mechanism during programming, more so in stronger learners. Moreover, left inferior frontal cortex plays a central role; operculum integrates information from the dorsal and ventral streams and its intrinsic connectivity predicts confidence and long-term memory, while activity in Broca’s area also reflects deductive reasoning. Hands-on led to greater confidence and memory retention. In project 2, we investigated the impact of feedback on motivation and reaction time in a rule-switching task. We find that feedback targeting personal traits increasingly impair performance and motivation over the experiment, and we find that activity in precentral gyrus and anterior insula decrease linearly over time during the personal feedback condition, implicating these areas in this effect. These findings promote hands-on learning and emphasize possibilities for feedback interventions on motivation. Future studies should investigate interventions for increasing Need for Cognition, the relationship between computer programming and second language learning (L2), and the role of explicit verbalization of knowledge for successful coding, given the language-like processing of code

Development of an expression system for eukarytoic proteins in methylotropic bacteria

The objective of this project was to develop an expression vector for methylotrophic bacteria for use in the production of C{sup 13} and H{sup 2} labelled eukaryotic proteins by growing methylotrophic bacteria on labelled methanol or methylamine. The eukaryotic proteins calmodulin and troponin C were chosen as test cases. Genes encoding both proteins were cloned into different constructions and tested for expression. Moderate amounts of troponin C were found with one of the constructions

Genetic Engineering of a Radiation-Resistant Bacterium for Biodegradation of Mixed Wastes

The mixture of toxic chemicals, heavy metals, halogenated solvents and radionuclides in many DOE waste materials presents a challenging problem for separating the different species and disposing of individual contaminants. One approach for dealing with mixed wastes is to genetically engineer the radiation-resistant bacterium, Deinococcus radiodurans to survive in and detoxify DOE's mixed waste streams, and to develop process parameters for treating mixed wastes with such constructed strains. The goal for this project is to develop a suite of genetic tools for Deinococcus radiodurans and to use these tools to construct and test stable strains for detoxification of haloorganics in mixed wastes

Flux Analysis Uncovers Key Role of Functional Redundancy in Formaldehyde Metabolism

Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and (14)C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop