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The Genome of the Western Clawed Frog Xenopus tropicalis

Description: The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes over 20,000 protein-coding genes, including orthologs of at least 1,700 human disease genes. Over a million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like other tetrapods, the genome contains gene deserts enriched for conserved non-coding elements. The genome exhibits remarkable shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.
Date: October 1, 2009
Creator: Hellsten, Uffe; Harland, Richard M.; Gilchrist, Michael J.; Hendrix, David; Jurka, Jerzy; Kapitonov, Vladimir et al.
Partner: UNT Libraries Government Documents Department

Invariability of Central Metabolic Flux Distribution in Shewanella oneidensis MR-1 Under Environmental or Genetic Perturbations

Description: An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time {approx} 3 hrs), salt stressed minimal medium (doubling time {approx} 6 hrs), and minimal medium with amino acid supplementation (doubling time {approx}1.5 hrs). {sup 13}C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, ten transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism.
Date: April 21, 2009
Creator: Tang, Yinjie; Martin, Hector Garcia; Deutschbauer, Adam; Feng, Xueyang; Huang, Rick; Llora, Xavier et al.
Partner: UNT Libraries Government Documents Department

Drosophila by the dozen

Description: This year's conference on Drosophila research illustratedwell the current focus of Drosophila genomics on the comprehensiveidentification of functional elements in the genome sequence, includingmRNA transcripts arising from multiple alternative start sites and splicesites, a multiplicity of noncoding transcripts and small RNAs,identification of binding sites for transcription factors, sequenceconservation in related species and sequence variation within species.Resources and technologies for genetics and functional genomics aresteadily being improved, including the building of collections oftransposon insertion mutants and hairpin constructs for RNA interference(RNAi). The conference also highlighted progress in the use of genomicinformation by many laboratories to study diverse aspects of biology andmodels of human disease. Here we will review a few highlights of especialinterest to readers of Genome Biology.
Date: July 13, 2007
Creator: Celniker, Susan E. & Hoskins, Roger A.
Partner: UNT Libraries Government Documents Department

A probe-based mapping strategy for DNA sequencing with mobile primers. Progress report

Description: Research on DNA sequencing continued. The specific areas of research targeted for the period of this Progress Report included three general phases: (1) optimization of probe-mapping by both the development of new transposons and the design of stream-lined methods for mapping; (2) application of transposon-based methods to larger plasmids and cosmids; and (3) initiation of PCR-based applications of transposons.
Date: December 31, 1991
Creator: Strausbaugh, L. D. & Berg, C. M.
Partner: UNT Libraries Government Documents Department

A probe-based mapping strategy for DNA sequencing with mobile primers

Description: Research on DNA sequencing continued. The specific areas of research targeted for the period of this Progress Report included three general phases: (1) optimization of probe-mapping by both the development of new transposons and the design of stream-lined methods for mapping; (2) application of transposon-based methods to larger plasmids and cosmids; and (3) initiation of PCR-based applications of transposons.
Date: January 1, 1991
Creator: Strausbaugh, L.D. & Berg, C.M.
Partner: UNT Libraries Government Documents Department

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, June 1, 1989--September 1, 1990

Description: The most exciting discovery made over the past year derives from an analysis of the interaction between DNA repair and P-element transposition. A powerful new system was developed for analyzing the repair of DNA double-strand breaks. A screen was completed of mutagenized autosomes obtained from two San Francisco laboratories with the recovery of several mutants that will provide the foundation for future efforts to clone repair related genes. At the same time, strong progress has been made in the cloning and characterization of the repair-related genes mei-41 and mus209. Finally, the efforts to clone the mei-9 gene have uncovered the existence of a unsuspected feature of the system used for transposon-tagging in Drosophila. This new knowledge will aid future cloning efforts as well as those of others in the field.
Date: December 31, 1990
Partner: UNT Libraries Government Documents Department

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, September 1, 1990--July 1, 1991

Description: Research is focused on the following areas: characterization of DNA double-strand break repair; using injected oligonucleotides as templates to repair double-strand DNA breaks; analysis of a gene required for postreplication repair; cloning of a gene required for resistance to DNA cross-linking agents; cloning of a gene required for excision repair; cloning of a gene required for X-ray resistance; and transposon tagging DNA repair genes.
Date: December 31, 1991
Partner: UNT Libraries Government Documents Department

Growth and development of maize that contains mutant tubulin genes

Description: Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.
Date: July 23, 2004
Creator: Wick, Susan M.
Partner: UNT Libraries Government Documents Department

Transposon tagging of disease resistance genes. Final report, May 1, 1988--April 30, 1993

Description: The goal of this project was to develop a transposon mutagenesis system for lettuce and to clone and characterize disease resistance genes by transposon tagging. The majority of studies were conducted with the Ac/Ds System. Researchers made and tested several constructs as well as utilized constructions shown to be functional in other plant species. Researchers demonstrated movement of Ac and DS in lettuce; however, they transposed at much lower frequencies in lettuce than in other plant species. Therefore, further manipulation of the system, particularly for flower specific expression of transposase, is required before a routine transposon system is available for lettuce. Populations of lettuce were generated and screened to test for the stability of resistance genes and several spontaneous mutations were isolated. Researchers also identified a resistance gene mutant in plants transformed with a Ds element and chimeric transposase gene. This is currently being characterized in detail.
Date: September 1, 1994
Creator: Michelmore, R.
Partner: UNT Libraries Government Documents Department

Transposon tagging of disease resistance genes. Progress report, May 1, 1988--1992

Description: Our goal is to clone genes in lettuce determining resistance to downy mildew. One approach involves the mobilization of transposons into resistance genes to mutate and tag the target gene. Because transposons have yet to be isolated and characterized from lettuce, the majority of our experiments have involved Ac from corn as this is increasingly the best characterized transposon. Over the past several years, various labs have contributed to a detailed understanding of the biology of Ac in corn and heterologous plant species. We have collaborated closely with several of these labs, exchanged materials and incorporated their advances into our analysis of transposition in lettuce. The original proposal described the development of a transposon mutagenesis system for lettuce and its subsequent use to tag disease resistance genes. The development phase involved characterization and manipulation of Ac transposition, identification of suitable whole plant selectable markers for the construction of chimeric non-autonomous elements, and investigation of the stability of resistance genes. Investigation of Ac transposition in lettuce has received the majority of our attention. Initially, we made a simple construct with wildtype Ac and introduced it into lettuce. No transposition was observed; although other labs demonstrated that the same construct was functional in tomato. We then focused on assaying for Ac transposition with constructs of increasing sophistication that had been demonstrated by others to be functional in other species. The latest constructs for transposon mutagenesis clearly demonstrated transposition in lettuce. This allowed us to generate seed stocks that we will start to screen for insertional inactivation of resistance genes this year.
Date: June 1, 1994
Creator: Michelmore, R.
Partner: UNT Libraries Government Documents Department

Detection of Cell Wall Chemical Variation in Zea Mays Mutants Using Near-Infrared Spectroscopy

Description: Corn stover is regarded as the prime candidate feedstock material for commercial biomass conversion in the United States. Variations in chemical composition of Zea mays cell walls can affect biomass conversion process yields and economics. Mutant lines were constructed by activating a Mu transposon system. The cell wall chemical composition of 48 mutant families was characterized using near-infrared (NIR) spectroscopy. NIR data were analyzed using a multivariate statistical analysis technique called Principal Component Analysis (PCA). PCA of the NIR data from 349 maize leaf samples reveals 57 individuals as outliers on one or more of six Principal Components (PCs) at the 95% confidence interval. Of these, 19 individuals from 16 families are outliers on either PC3 (9% of the variation) or PC6 (1% of the variation), the two PCs that contain information about cell wall polymers. Those individuals for which altered cell wall chemistry is confirmed with wet chemical analysis will then be subjected to fermentation analysis to determine whether or not biomass conversion process kinetics, yields and/or economics are significantly affected. Those mutants that provide indications for a decrease in process cost will be pursued further to identify the gene(s) responsible for the observed changes in cell wall composition and associated changes in process economics. These genes will eventually be incorporated into maize breeding programs directed at the development of a truly dual use crop.
Date: January 1, 2001
Creator: Buyck, N. & Thomas, S.
Partner: UNT Libraries Government Documents Department

Bio-Engineering High Performance Microbial Strains for MEOR

Description: The main objectives of this three-year research project are: (1) to employ the latest advances in genetics and bioengineering, especially Directed Protein Evolution technology, to improve the effectiveness of the microbial enhanced oil recovery (MEOR) process. (2) to improve the surfactant activity and the thermal stability of bio-surfactant systems for MEOR; and (3) to develop improved laboratory methods and tools that screen quickly candidate bio-systems for EOR. Biosurfactants have been receiving increasing attention as Enhanced Oil Recovery (EOR) agents because of their unique properties (i.e., mild production conditions, lower toxicity, and higher biodegradability) compared to their synthetic chemical counterparts. Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including EOR and bioremediation. During the three-year of the project period, we have successfully cloned the genes involved in the rhamnolipid bio-synthesis. And by using the Transposon containing Rhamnosyltransferase gene rhlAB, we engineered the new mutant strains P. aeruginosa PEER02 and E. coli TnERAB so they can produce rhamnolipid biosurfactans. We were able to produce rhamnolipds in both P. aeroginosa PAO1-RhlA- strain and P. fluorescens ATCC15453 strain, with the increase of 55 to 175 fold in rhamnolipid production comparing with wild type bacteria strain. We have also completed the first round direct evolution studies using Error-prone PCR technique and have constructed the library of RhlAB-containing Transposon to express mutant gene in heterologous hosts. Several methods, such as colorimetric agar plate assay, colorimetric spectrophotometer assay, bioactive assay and oil spreading assay have been established to detect and screen rhamnolipid production. Our engineered P. aeruginosa PEER02 strain can produce rhamnolipids with different carbon sources as substrate. Interfacial tension analysis (IFT) showed that different rhamnolipids from different substrates gave different performance. Those rhamnolipids with plant oil as substrate showed as low an IFT as 0.05mN/m in the buffer solution with pH5.0 ...
Date: December 30, 2007
Creator: Fang, Xiangdong; Wang, Qinghong & Shuler, Patrick
Partner: UNT Libraries Government Documents Department

Final report

Description: The originally funded project was geared to pursue research on regulation of photosystem II (PSII) in the cyanobacterium Synechococcus elongatus PCC 7942. We characterized a locus, psfR, (psbA stimulating factor) that affects expression of the psbAI gene, which encodes the PSII protein D1. Over-expression of psfR, which encodes a protein with receiver and pseudo-receiver domains, acts at the promoter region to elevate expression of psbAI and a subset of other loci. We reoriented the remainder of the funding to make a greater impact through completion of a functional genomics project that had been initiated with funding from another agency. The goal is inactivation of each gene individually in the S. elongatus genome, and completion of the entire genome sequence. At the end of the project we will have screened all loci for involvement in circadian rhythms of gene expression and assembled an archived set of clones that can be used to create the mutations to screen for any other phenotype. During the project period we: (1) prepared a functional genomics website for S. elongatus PCC 7942 that posts sequences prior to GenBank release, and presents the strategy and progress for the genomics project (http://www.bio.tamu.edu/synecho/); (2) determined the sequence of and annotated the S. elongatus 46 kb plasmid, pANL; (3) submitted assembled sequences with annotation of 8 cosmid inserts to GenBank (313 kb), with sites of transposon insertions indicated; (4) mutagenized approximately an additional 600 kb of the genome (16 cosmids) and identified sequences flanking the mutations; (5) recombined mutagenesis substrates into the S. elongatus genome to produce gene inactivations (at the sites of transposon insertions) for approximately 415 kb of mutagenized sequence (85% of these have already been screened for circadian phenotypes) (6) identified the clpPIIclpX locus as important in determining circadian period; and (7) demonstrated effectiveness of antisense RNA ...
Date: March 31, 2005
Creator: Golden, Susan S.
Partner: UNT Libraries Government Documents Department

Global Regulatory Pathways in the Alphaproteobacteria

Description: A major goal for microbiologists in the twenty-first century is to develop an understanding of the microbial cell in all its complexity. In addition to understanding the function of individual gene products we need to focus on how the cell regulates gene expression at a global level to respond to different environmental parameters. Development of genomic technologies such as complete genome sequencing, proteomics, and global comparisons of mRNA expression patterns allows us to begin to address this issue. This proposal focuses on a number of phylogenetically related bacteria that are involved in environmentally important processes such as carbon sequestration and bioremediation. Genome sequencing projects of a number of these bacteria have revealed the presence of a small family of regulatory genes found thus far only in the alpha-proteobacteria. These genes encode proteins that are related to the global regulatory protein RosR in Rhizobium etli, which is involved in determining nodulation competitiveness in this bacterium. Our goal is to examine the function of the proteins encoded by this gene family in several of the bacteria containing homologs to RosR. We will construct gene disruption mutations in a number of these bacteria and characterize the resulting mutant strains using two-dimensional gel electrophoresis and genetic and biochemical techniques. We will thus determine if the other proteins also function as global regulators of gene expression. Using proteomics methods we will identify the specific proteins whose expression varies depending on the presence or absence of the RosR homolog. Over fifty loci regulated by RosR have been identified in R. etli using transposon mutagenesis; this will serve as out benchmark to which we will compare the other regulons. We expect to identify genes regulated by RosR homologs in several bacterial species, including, but not limited to Rhodopseudomonas palustris and Sphingomonas aromaticivorans. In this way we will ...
Date: April 27, 2007
Partner: UNT Libraries Government Documents Department

The BDGP gene disruption project: Single transposon insertions associated with 40 percent of Drosophila genes

Description: The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in more than 30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6,300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7,140 lines. It now includes individual lines predicted to disrupt 5,362 of the 13,666 currently annotated Drosophila genes (39 percent). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene mis-expression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.
Date: January 13, 2004
Creator: Bellen, Hugo J.; Levis, Robert W.; Liao, Guochun; He, Yuchun; Carlson, Joseph W.; Tsang, Garson et al.
Partner: UNT Libraries Government Documents Department

A genetic analysis of Adhl regulation. Progress report, June 1991--May 1993

Description: Several separate but related studies are reported on the mechanism of alcohol dehydrogenase (Adh-1) are reported. A study of a deletion mutation in the TATA box region which resulted in an increase from 6--60% of wildtype Adh-1 expression in the revertant has led to a focus on trans-acting protein factors that bind the TATA box. Analysis of another revertant has led to study of cis-acting sequences in Adh-1 expression. Screening efforts aimed at defining different mutants affecting Adh-1 expression are reported.
Date: December 1, 1992
Creator: Freeling, M.
Partner: UNT Libraries Government Documents Department

[Genetics and biochemistry of surfactant synthesis in Rhodococcus sp. H13-A]. Final report

Description: The rationale for these studies resides is that biosurfactant synthesis occurs only when cells are grown with alkanes as sole source of carbon and energy. It is reasoned that biosurfactant synthesis is linked genetically to alkane oxidation and that the identification and characterization of the alk genes would provide information about the structural and regulator genes involved, in biosurfactant synthesis. Rhodococcus H13-A chromosomal DNA was isolated and digested with the restriction endonuclease Sau3A. The shuttle vector, pMVS301, was single site cleaved with Bgl II, phosphorylated, and the chromosomal DNA fragments agated into linearized pMVS301. The ligation mixture was used to transform competent E. coli HB101. The chromosomal DNA fragment has been cloned into pMVS301 which appears to contain genes encoding the initial oxidation of alkane. Electrophoration of Rhodococaus indicates transformation by this technique. Further studies are required to optimize conditions.
Date: December 31, 1989
Partner: UNT Libraries Government Documents Department

Transposon-induced nuclear mutations that alter chloroplast gene expression. Annual report, September 1, 1992--April 15, 1993

Description: The goal of this project is to use mutant phenotypes as a guide to nuclear genes that control the timing and cell-type specificity of chloroplast gene expression. Studies are being conducted with nuclear mutants of maize that are defective in the biogenesis or translation of chloroplast mRNAs. Currently studies are focused on two nuclear mutants with specific and unique lesions in chloroplast RNA processing (crp mutants). Crp1 mutants (formerly called hcf136) fail to accumulate the cytochrome f/b6 complex. The protein loss is due to a defect in the metabolism of transcripts encoding the petB and petD gene products, two subunits of the missing complex. Mutant seedlings lack the monocistronic petB and petD MRNAS, which both arise in nominal plants by endonucleolytic cleavage of the polycistronic primary transcript of the psbB gene cluster. Precursor mRNAs accumulate normally in crp1, indicating that its defect is due either to a failure to cleave the precursors, or a failure to stabilize the fully processed mRNAs. We are interested in both the biochemistry of this site-specific RNA processing and in the role of the processing in generating translatable mRNAs. To address the latter, we are quantifying the rates of synthesis of the petB and petD gene products with the goal of determining whether the missing transcripts are more efficiently translated than their precursors. To address the biochemistry of the defect in RNA metabolism, the crp1 gene is being cloned via the transposon tag. crp2 (formerly called hcf142) lacks the predominant mRNA encoding petA, but appears to be otherwise unimpaired in chloroplast RNA metabolism. The precise role of crp2 in synthesizing or stabilizing the petA mRNA is being investigated through biochemical studies.
Date: April 20, 1993
Creator: Barkan, A.
Partner: UNT Libraries Government Documents Department

In-Situ Survival Mechanisms of U and Tc Reducing Bacteria in Contaminated Sediments

Description: Desulfovibrio desulfuricans G20 and Shewanella oneidensis MR-1 are model subsurface organisms for studying genes involving in situ radionuclide transformation and sediment survival. Our research objective for this project has been to develop a signature-tagged mutagenesis (STM) procedure and use it to identify mutants in genes of these subsurface bacteria involved in sediment survival and radionuclide reduction. The mutant genes identified in these studies allow us for the first time to describe at the genetic level microbial processes that are actually being used by environmental bacteria while growing in their natural ecosystems. Identification of these genes revealed facets of microbial physiology and ecology that are not accessible through laboratory studies. Ultimately, this information may be used to optimize bioremediation or other engineered microbial processes. Furthermore, the identification of a mutant in a gene conferring multidrug resistance in strain MR-1 shows that this widespread mechanism of antibiotic resistance, likely has its origins as a mechanism of bacterial defense against naturally occurring toxins. Studies with D. desulfuricans G20: The STM procedure first involved generating a library of 5760 G20 mutants and screening for potential non-survivors in subsurface sediment microcosms. After two rounds of screening, a total of 117 mutants were confirmed to be true non-survivors. 97 transposon insertion regions have been sequenced to date. Upon further analysis of these mutants, we classified the sediment survival genes into COG functional categories. STM mutant insertions were located in genes encoding proteins related to metabolism (33%), cellular processes (42%), and information storage and processing (17%). We also noted 8% of STM mutants identified had insertions in genes for hypothetical proteins or unknown functions. Interestingly, at least 64 of these genes encode cytoplasmic proteins, 46 encode inner membrane proteins, and only 7 encode periplasmic space and outer membrane associated proteins. Through blast search analysis, we also showed ...
Date: June 1, 2005
Creator: Krumholz, Lee R.
Partner: UNT Libraries Government Documents Department

Transposon facilitated DNA sequencing

Description: The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses, and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.
Date: January 1, 1990
Creator: Berg, D. E.; Berg, C. M. & Huang, H. V.
Partner: UNT Libraries Government Documents Department

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, September 1, 1990--July 1, 1991

Description: At this time last year, we had identified two genes in Drosophila that are required for repair of double strand breaks. These genes (mei-41 and mus302) have now been completely analyzed. We have developed an efficient system for site-directed mutagenesis using injected oligonucleotides as a template for the repair of double strand breaks. mus308, a gene responsible for resistance to DNA cross-linking, is being recovered through chromosome walking. It is believed this gene may be the Drosophila analog of the human Fanconi anemia A gene. A collaborative effort to clone the excision repair gene, mei-9, is under way. The X-ray resistance gene mus209 has been cloned. Finally, we are analyzing a group of mus mutations from other labs which we have tagged with a single transposon inserted randomly into one of the two major autosomes. 4 refs.
Date: December 1, 1991
Partner: UNT Libraries Government Documents Department