Supervisors: Phillip A. Rice and Carl R. Woese Phone: 713-743-8363 Address: Dept. Biology and Biochemistry University of Houston 3455 Cullen Blvd. Suite 342 Houston, TX 77204-5001
Introduction: To understand the extent to which complexity can emerge in an RNA World and how it ... more Introduction: To understand the extent to which complexity can emerge in an RNA World and how it might be effected by peptides or amino acids, we are pursuing a novel experimental approach based on dynamic combinatorial chemistry (DCC)[1,2]. It is hypothesized that when subject to a persistent equilibrium of ligation and cleavage, RNAs will naturally increase in complexity while gaining resistance to degradation over time. It will be of immense interest to see if this equilibrium or the pathways towards increasing complexity are strongly affected by the presence of amino acids or peptides. To obtain such equilibrium, we are using a two enzyme system. The cleavage enzyme is Benzonase [3], which is the commercial name for an extracellular endonuclease secreted by Serratia marcescens. This enzyme cleaves RNA, including circular forms, to produce products with a 3’ hydroxyl and 5’ phosphate. This is ideal for ligation by T4 RNA ligase [4], which requires these exact ends and utilizes AT...
Motivation: The phylogenetic structure of the bacterial world has been intensively studied by com... more Motivation: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. Results: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S ...
Human space travelers experience a unique environment that affects homeostasis and physio-logic a... more Human space travelers experience a unique environment that affects homeostasis and physio-logic adaptation. One of the important regulatory biology interactions affected by space flight is the alteration of the immune response. As such, the impairment of the immune system may lead to higher risk of bacterial and/or viral infection during human space flight missions. Mi-crobiological contaminants have been a source
Microorganisms are unavoidable in space environments and their presence has, at times, been a sou... more Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.
ABSTRACT Previous space flight experience has demonstrated that microorganisms are just as ubiqui... more ABSTRACT Previous space flight experience has demonstrated that microorganisms are just as ubiquitous in space habitats as they are on Earth. Numerous incidences of biofilm formation within space habitats have been reported; some of which were identified only after damage to spacecraft structures and irritation to astronaut's skin occurred. As we increase the duration of spaceflight missions, it becomes legitimate to question the long-term effects of microgravity on bacteria. To begin this assessment, Escherichia coli K-12 strain MG1655 was grown for one thousand generations (1000G) under low shear modeled microgravity. Subsequently, growth kinetics and the presence of biofilm were assessed in the 1000G strain as compared to a strain (1G) briefly exposed to LSMMG. Overall, the analysis revealed that (i) there was no obvious difference in growth kinetics between the 1G and 1000G strains, and (ii) although biofilm formation was not seen in the 1G strain it did in fact occur as exposure time increased. The results suggest that long-term exposure to the space environment likely favors biofilm formation in many organisms.
Reliable detection and identification of pathogens in complex biological samples, in the presence... more Reliable detection and identification of pathogens in complex biological samples, in the presence of contaminating DNA from a variety of sources, is an important and challenging diagnostic problem for the development of field tests. The problem is compounded by the difficulty of finding a single, unique genomic sequence that is present simultaneously in all genomes of a species of closely related pathogens and absent in the genomes of the host or the organisms that contribute to the sample background. Here we describe 'host-blind probe design'- a novel strategy of designing probes based on highly frequent genomic signatures found in the pathogen genomes of interest but absent from the host genome. Upon hybridization, an array of such informative probes will produce a unique pattern that is a genetic fingerprint for each pathogen strain. This multiprobe approach was applied to 83 dengue virus genome sequences, available in public databases, to design and perform in silico microarray experiments. The resulting patterns allow one to unequivocally distinguish the four major serotypes, and within each serotype to identify the most similar strain among those that have been completely sequenced. In an environment where dengue is indigenous, this would allow investigators to determine if a particular isolate belongs to an ongoing outbreak or is a previously circulating version. Using our probe set, the probability that misdiagnosis at the serotype level would occur is approximately 1 : 10(150).
Introduction: To understand the extent to which complexity can emerge in an RNA World and how it ... more Introduction: To understand the extent to which complexity can emerge in an RNA World and how it might be effected by peptides or amino acids, we are pursuing a novel experimental approach based on dynamic combinatorial chemistry (DCC)[1,2]. It is hypothesized that when subject to a persistent equilibrium of ligation and cleavage, RNAs will naturally increase in complexity while gaining resistance to degradation over time. It will be of immense interest to see if this equilibrium or the pathways towards increasing complexity are strongly affected by the presence of amino acids or peptides. To obtain such equilibrium, we are using a two enzyme system. The cleavage enzyme is Benzonase [3], which is the commercial name for an extracellular endonuclease secreted by Serratia marcescens. This enzyme cleaves RNA, including circular forms, to produce products with a 3’ hydroxyl and 5’ phosphate. This is ideal for ligation by T4 RNA ligase [4], which requires these exact ends and utilizes AT...
Motivation: The phylogenetic structure of the bacterial world has been intensively studied by com... more Motivation: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. Results: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S ...
Human space travelers experience a unique environment that affects homeostasis and physio-logic a... more Human space travelers experience a unique environment that affects homeostasis and physio-logic adaptation. One of the important regulatory biology interactions affected by space flight is the alteration of the immune response. As such, the impairment of the immune system may lead to higher risk of bacterial and/or viral infection during human space flight missions. Mi-crobiological contaminants have been a source
Microorganisms are unavoidable in space environments and their presence has, at times, been a sou... more Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.
ABSTRACT Previous space flight experience has demonstrated that microorganisms are just as ubiqui... more ABSTRACT Previous space flight experience has demonstrated that microorganisms are just as ubiquitous in space habitats as they are on Earth. Numerous incidences of biofilm formation within space habitats have been reported; some of which were identified only after damage to spacecraft structures and irritation to astronaut's skin occurred. As we increase the duration of spaceflight missions, it becomes legitimate to question the long-term effects of microgravity on bacteria. To begin this assessment, Escherichia coli K-12 strain MG1655 was grown for one thousand generations (1000G) under low shear modeled microgravity. Subsequently, growth kinetics and the presence of biofilm were assessed in the 1000G strain as compared to a strain (1G) briefly exposed to LSMMG. Overall, the analysis revealed that (i) there was no obvious difference in growth kinetics between the 1G and 1000G strains, and (ii) although biofilm formation was not seen in the 1G strain it did in fact occur as exposure time increased. The results suggest that long-term exposure to the space environment likely favors biofilm formation in many organisms.
Reliable detection and identification of pathogens in complex biological samples, in the presence... more Reliable detection and identification of pathogens in complex biological samples, in the presence of contaminating DNA from a variety of sources, is an important and challenging diagnostic problem for the development of field tests. The problem is compounded by the difficulty of finding a single, unique genomic sequence that is present simultaneously in all genomes of a species of closely related pathogens and absent in the genomes of the host or the organisms that contribute to the sample background. Here we describe 'host-blind probe design'- a novel strategy of designing probes based on highly frequent genomic signatures found in the pathogen genomes of interest but absent from the host genome. Upon hybridization, an array of such informative probes will produce a unique pattern that is a genetic fingerprint for each pathogen strain. This multiprobe approach was applied to 83 dengue virus genome sequences, available in public databases, to design and perform in silico microarray experiments. The resulting patterns allow one to unequivocally distinguish the four major serotypes, and within each serotype to identify the most similar strain among those that have been completely sequenced. In an environment where dengue is indigenous, this would allow investigators to determine if a particular isolate belongs to an ongoing outbreak or is a previously circulating version. Using our probe set, the probability that misdiagnosis at the serotype level would occur is approximately 1 : 10(150).
The modern ribosomal machinery is very complex and its core subsystems and many of its individual... more The modern ribosomal machinery is very complex and its core subsystems and many of its individual components are universally found in all three Domains of life. This indicates that much of the story of ribosome origins and its subsequent evolution predates the last universal common ancestor (LUCA). Thus, ribosome history relates to other early life issues such as the possibility and nature of an RNA World, the early history of chirality, and always most hopefully the origins of the genetic code. However, this is not the end of the story. As discussed elsewhere in this volume, important events have also occurred after LUCA, especially in eukaryotic ribosomes that have served to integrate the machinery with other cellular systems. Ribosome origins and subsequent evolution are in reality somewhat separate problems. In addressing the former, this chapter initially examines the source and nature of the peptidyl transferase center, (PTC), including where and how the peptide bond is made. This is followed by efforts to understand the subsequent evolution of the ribosome, which led to the addition and refinement of various other functional centers including the decoding center. This is being accomplished using what is in essence is a reverse engineering approach to develop a timeline of major events in the ribosome history. Finally, significant events on the time line are discussed in detail.
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Papers by George E Fox
Ribosome origins and subsequent evolution are in reality somewhat separate problems. In addressing the former, this chapter initially examines the source and nature of the peptidyl transferase center, (PTC), including where and how the peptide bond is made. This is followed by efforts to understand the subsequent evolution of the ribosome, which led to the addition and refinement of various other functional centers including the decoding center. This is being accomplished using what is in essence is a reverse engineering approach to develop a timeline of major events in the ribosome history. Finally, significant events on the time line are discussed in detail.