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[Developing a physical map of human chromosome 22 using Pace electrophoresis and large fragment cloning]. Annual report, October 1, 1989--September 30, 1990

Description: In the past year the authors have made significant progress in the development of a bacterial based cloning system for large fragments of mammalian DNA. They have completed construction of several recombination deficient bacterial host strains designed to minimize homologous recombination arising with repeats within cloned DNA. Despite the multiple mutations, these strains are viable and grow readily on standard media (LB). One of the chief attractions of a bacterial system is the promise of high transformation efficiencies. The author have pursued two separate strategies with the vector. The first makes use of the cos sites in the vector to package cloned DNA as phage particles for infection. By maintaining the vector as a single copy in the recombination minus host, they believe that the recombination that affects conventional cosmid libraries will be eliminated. They encountered no difficulties in preparing such a ``Fosmid`` (F factor based cosmid) library of human DNA.
Date: December 31, 1990
Creator: Simon, M.I.
Partner: UNT Libraries Government Documents Department

Cloning and sequencing of the trpE gene from Arthrobacter globiformis ATCC 8010 and several related subsurface Arthrobacter isolates

Description: Tryptophan dependent mutants of Arthrobacter globiformis ATCC 8010 were isolated and trp genes were cloned by complementation and marker rescue of the auxotrophic strains. Rescue studies and preliminary sequence analysis reveal that at least the genes trpE, trpC, and trpB are clustered together in this organism. In addition, sequence analysis of the entire trpE gene, which encodes component I of anthranilate synthase, is described. Segments of the trpE gene from 17 subsurface isolates of Arthrobacter sp. were amplified by PCR and sequenced. The partial trpE sequences from the various strains were aligned and subjected to phylogenetic analysis. The data suggest that in addition to single base changes, recombination and genetic exchange play a major role in the evolution of the Arthrobacter genome.
Date: September 1, 1998
Creator: Chernova, T.; Viswanathan, V.K.; Austria, N. & Nichols, B.P.
Partner: UNT Libraries Government Documents Department

A bacteriophage T4 in vitro system to clone long DNA molecules. Final report, June 1, 1990--January 31, 1996

Description: A summary is presented of the following objectives: development of a bacteriophage T4 in vitro system, and techniques to clone long segments of foreign DNA; development of a giant prohead DNA packaging system that could potentially be used to clone even a megabase size DNA; and development of techniques to rapidly map the cloned DNA inserts.
Date: September 1, 1997
Creator: Rao, V.B.
Partner: UNT Libraries Government Documents Department

Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

Description: Two Genes for DNA polymerase delta were identified from the wild type Chinese hamster ovary cells. These genes were cloned via RT-PCR from mRNA prepared the Chinese hamster ovary cells using primers specific to conserved sequences of the DNA polymerase {delta} gene. The first gene encodes a PCNA dependent DNA polymerase {delta} gene whereas the second gene encodes a PCNA independent DNA polymerase {delta} gene. Methods were developed to clone these genes in expression vector and host systems. The role of the two genes in DNA replication and repair was determined.
Date: January 1, 1995
Creator: Mishra, N.C.
Partner: UNT Libraries Government Documents Department

9. international mouse genome conference

Description: This conference was held November 12--16, 1995 in Ann Arbor, Michigan. The purpose of this conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on genetic mapping in mice. This report contains abstracts of presentations, focusing on the following areas: mutation identification; comparative mapping; informatics and complex traits; mutagenesis; gene identification and new technology; and genetic and physical mapping.
Date: December 31, 1995
Partner: UNT Libraries Government Documents Department

Reporting of the Third International Workshop on Human Chromosome 22 Mapping

Description: The third international workshop on the mapping of human chromosome 22 was held at the Sugarloaf Conference Center in Philadelphia Pennsylvania USA from September 17--20, 1992. It was organized by Beverly S. Emanuel of Children`s Hospital of Philadelphia and the Human Genome Center for Chromosome 22. The highlights of the conference included the discussion of the chromosome 22 gene at the Ewings Sarcoma breakpoint, the identification of a polymorphic TG/CA repeat containing locus tightly linked to the NF2 gene, the isolation of a candidate tumor suppressor locus for meningioma, the isolation of numerous as yet uncharacterized new cDNAs for chromosome 22 and the progress which has been made on generating physical and genetic maps of the chromosome. There is a new genetic map comprised of 16 short tandem repeat polymorphism (STRP) markers of which 12 have greater than 70% heterozygosity (Fig. 1). It was decided that the next meeting should be held in 18 months and it will be organized by Peter Scambler in the United Kingdom.
Date: December 31, 1992
Creator: Emanuel, Beverly S.; Buetow, Ken; Nussbaum, Robert; Scambler, Peter; Lipinski, Marc & Overton, Chris
Partner: UNT Libraries Government Documents Department

Requirements in screening cDNA libraries for new genes and solutions offered by SBH technology

Description: Under different assumptions about the total number of genes, the number of housekeeping and tissue-specific genes, and the difference in the number of mRNAs per cell for functional and nonfunctional genes, significantly different results can be expected from screening random cDNA clones. We have developed gene expression models as a guide for interpretation of experimental results. For statistical, biological, and technical reasons, the search for 100,000 plus genes and discrimination between nonfunctional, housekeeping, and tissue-specific genes requires the analysis of up to 10 million clones from 20 to 50 tissues. Oligonucleotide hybridization of dense clone blots is an inexpensive and fast way to screen such large clone sets. Our preliminary results on control clones and thousands of cDNA clones from an infant brain library demonstrate the feasibility of the method. We present several models of gene expression and analyze the main factors which can influence the hunt for new genes via the screening of random cDNA libraries. The basic steps in the preparation and use of dense DNA dot arrays are described, and some results that demonstrate the feasibility and efficiency of gene inventorying by oligonucleotide hybridization are presented. Furthermore, partial SBH and single-pass gel sequencing are compared and a gene analysis scheme that combines the two approaches is discussed.
Date: December 31, 1993
Creator: Drmanac, R.; Drmanac, S.; Labat, I. & Stavropoulos, N.
Partner: UNT Libraries Government Documents Department

Sequential cloning of chromosomes

Description: A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.
Date: December 31, 1991
Creator: Lacks, S.A.
Partner: UNT Libraries Government Documents Department

A report from the Sixth International Mouse Genome Conference

Description: The Sixth Annual Mouse Genome Conference was held in October, 1992 at Buffalo, USA. The mouse is one of the primary model organisms in the Human Genome Project. Through the use of gene targeting studies the mouse has become a powerful biological model for the study of gene function and, in addition, the comparison of the many homologous mutations identified in human and mouse have widened our understanding of the biology of these two organisms. A primary goal in the mouse genome program has been to create a genetic map of STSs of high resolution (<1cM) that would form the basis for the physical mapping of the whole mouse genome. Buffalo saw substantial new progress towards the goal of a very high density genetic map and the beginnings of substantive efforts towards physical mapping in chromosome regions with a high density of genetic markers.
Date: December 31, 1992
Creator: Brown, S.
Partner: UNT Libraries Government Documents Department

International workshop on Chromosome 12 held at St. Catherine`s College, Oxford, England, September 18--20, 1992. Final report

Description: The First International Mapping Workshop on Human Chromosome 12 was held on Sept. 18--20, 1992, at St Catherine`s College, Oxford, UK. The meeting was hosted by Ian Craig and organized by Drs. Craig, Gemmill and Kutcherlapati. It was attended by 50 participants primarily from Europe and the USA. The workshop was highly successful and achieved the major goal of fostering direct and personal interactions between investigators with strong research interests in this chromosome. Participants reviewed the status of several critical aspects of chromosome mapping and prepared consensus views of the current state of knowledge. In addition, lists of resources available for this chromosome including somatic cell hybrids, individual DNA clones and libraries and genetic markers were prepared.
Date: December 31, 1992
Creator: Gemmill, R.
Partner: UNT Libraries Government Documents Department

Physical mapping of human chromosome 16. Annual progress report

Description: We aim to isolate cDNAs mapping to human chromosome 16 and localise such cDNAs on the high resolution physical map. In collaboration with LANL, PCR primers will be synthesised from cDNA sequences mapped to chromosome 16 and used as ESTs in the generation of mega-YAC contigs for this chromosome. Probing of high density cosmid grids will enable integration of the ESTs into cosmid contigs and location of the cosmid contigs on the YAC contig. A hn-cDNA library has been constructed from the hybrid CY18 which contains chromosome 16 as the only human chromosome. A modified screening protocol has been successfully developed and 15 hn-cDNA clones have been sequenced and localised on the hybrid map. Sequence analysis of four of these revealed that they were known cDNAs, which are now mapped to chromosome 16. Development of techniques to allow the isolation of longer cDNAs from the identified exons is in progress. This will depend on PCR amplification of cDNAs from a total human CDNA library.
Date: August 1, 1993
Creator: Sutherland, G. R.
Partner: UNT Libraries Government Documents Department

Mapping and ordered cloning of the human X chromosome. Final progress report, March 1991--February 1995

Description: A reciprocal probing method is described which uses pooled cDNA probes to order chromosome specific libraries in order to identify cosmids containing sequences capable to hybridizing to the pool. In this pilot study, placental DNA clones were used to identify cosmids from both chromosomes X and 17. Sixty unique cDNA`s were identified of which 22 were novel.
Date: September 1, 1995
Creator: Caskey, C.T.
Partner: UNT Libraries Government Documents Department

Development of an expression system for eukarytoic proteins in methylotropic bacteria

Description: 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.
Date: September 1, 1996
Creator: Lidstrom, M.E.
Partner: UNT Libraries Government Documents Department

Starch synthesis in the maize endosperm as affected by starch-synthesizing mutants. Final technical report, June 15, 1988--December 31, 1996

Description: The goal of this project was the elucidation of the pathway of starch biosynthesis in the developing maize endosperm with mutants affecting the process constituting the experimental probes. Studies involving a total of seven different loci were undertaken, with a concentration on four of these. The four studies focus on the following: brittle endosperm1 (bt1); brittle endosperm2 (bt2); phosphoglucomutase (pgm); and sugary3 (su3).
Date: June 1, 1998
Creator: Nelson, O.E.
Partner: UNT Libraries Government Documents Department

Development of BAC libraries and integrated physical mapping of human chromosome 22 using BACs. Annual report, July 1994--June 1995

Description: BACs and fosmids are stable, nonchimeric, and highly representative cloning systems. BACs maintain large-fragment genomic inserts (100 to 300 kb) that are easily prepared for most types of experiments, including DNA sequencing. The authors have improved the methods for generating BACs and developed extensive BAC libraries. They have constructed human BAC libraries with more than 175,000 clones from male fibroblast and sperm, and a mouse BAC library with more than 200,000 clones. The authors are currently expanding human library with the aim of achieving total 50X coverage human genomic library using sperm samples from anonymous donors.
Date: December 31, 1995
Creator: Kim, U.J.; Shizuya, Hiroaki & Simon, M.I.
Partner: UNT Libraries Government Documents Department

DOE project on genome mapping and sequencing. Progress report, 1992

Description: These efforts on the human genome project were initiated in September, 1990, to contribute towards completion of the human genome project physical mapping effort. In the original application, the authors proposed a novel strategy for constructing a physical map of human chromosome 11, based upon techniques derived in this group and by others. The original goals were to (1) produce a set of cosmid reference clones mapped to specific sites by high resolution fluorescence in situ hybridization, (2) produce a set of associated STS sequences and PCR primers for each site, (3) isolate YAC clones corresponding to each STS and, (4) construct YAC contigs such that > 90% of the chromosome would be covered by contigs of 2 mb or greater. Since that time, and with the advent of new technology and reagents, the strategy has been modified slightly but still retains the same goals as originally proposed. The authors have added a project to produce chromosome 11-specific cDNAs and determine the map location and DNA sequence of a selected portion of them.
Date: December 31, 1992
Creator: Evans, G.A.
Partner: UNT Libraries Government Documents Department

Cloning, characterization, and regulation of the human type II IMP dehydrogenase gene

Description: Human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. Regulated IMPDH activity is associated with cellular proliferation, transformation, and differentiation. The authors cloned and sequenced the entire gene for type II IMPDH and here provide details regarding the organization of the gene and the characterization of its promoter. The gene spans approximately 5 kb and is disrupted by 12 introns. The transcriptional start sites were determined by S1 nuclease mapping to be somewhat heterogeneous but predominated at 102 and 85 nucleotides from the translational initiation codon. Through the use of heterologous gene constructs and transient transfection assays, a minimal promoter from {minus}206 to {minus}85 was defined. This promoter is TATA-less and contains several transcription factor motifs including four potential Sp 1 binding sites. The minimal promoter is GC-rich (69%) and resembles a CpG island. Through the use of gel mobility shift assays, nuclear proteins were shown to specifically interact with this minimal promoter. Stable transfectants were used to demonstrate that the down-regulation of IMPDH gene expression in response to reduced cellular proliferation occurs by a transcriptional mechanism.
Date: January 1, 1997
Creator: Glesne, D.A. & Huberman, E.
Partner: UNT Libraries Government Documents Department

[Developing a physical map of human chromosome 22 using Pace electrophoresis and large fragment cloning]. Annual report, October 1, 1990--September 30, 1991

Description: Recent technical progress in molecular biology has made the mapping of entire mammalian chromosomes an attainable goal. However, a number of problems must still be overcome before genome mapping becomes rapid, efficient, and reliable. The limited size of cosmid inserts, as well as their tendency to rearrange, necessitates construction of very large libraries for mapping, due to the many gaps encountered in aligning cosmid contigs. Larger fragments can be cloned using the phage P1, but the maximum size of cloned inserts is fixed at only twice that of cosmids. The power of YACs has been demonstrated in isolating large regions of human DNA, recombining them to build up even larger regions and closing gaps in cosmid based maps. However, existing YAC libraries contain a high proportion of chimeric clones, and YACs are difficult to use for detailed mapping, often requiring recloning into cosmid sized pieces. The work has addressed some of these issues by creating an alternative and complementary approach to cloning and mapping large DNA.
Date: December 31, 1991
Creator: Simon, M.I.
Partner: UNT Libraries Government Documents Department

[Developing a physical map of human chromosome 22 using Pace electrophoresis and large fragment cloning]. Annual report, October 1, 1991--July 1, 1994

Description: In the past two years, the authors have made a great deal of progress in establishing Fosmid and BAC libraries and in using large BAC libraries for gene mapping. In addition, they initiated work on the application of BAC clones to long range genome sequencing. They continue to increase the ability to rapidly generate large BAC libraries and to efficiently apply these libraries to genome mapping. The BACs provide a very effective means of developing physical maps. The current work suggests that BAC contigs will be extremely useful as source material for genome sequencing.
Date: December 31, 1994
Creator: Simon, M.I.
Partner: UNT Libraries Government Documents Department

Inflammatory bowel disease gene discovery. CRADA final report

Description: The ultimate goal of this project is to identify the human gene(s) responsible for the disorder known as IBD. The work was planned in two phases. The desired products resulting from Phase 1 were BAC clone(s) containing the genetic marker(s) identified by gene/Networks, Inc. as potentially linked to IBD, plasmid subclones of those BAC(s), and new genetic markers developed from these plasmid subclones. The newly developed markers would be genotyped by gene/Networks, Inc. to ascertain evidence for linkage or non-linkage of IBD to this region. If non-linkage was indicated, the project would move to investigation of other candidate chromosomal regions. Where linkage was indicated, the project would move to Phase 2, in which a physical map of the candidate region(s) would be developed. The products of this phase would be contig(s) of BAC clones in the region exhibiting linkage to IBD, as well as plasmic subclones of the BACs and further genetic marker development. There would also be continued genotyping with new polymorphic markers during this phase. It was anticipated that clones identified and developed during these two phases would provide the physical resources for eventual disease gene discovery.
Date: September 9, 1997
Partner: UNT Libraries Government Documents Department

Effect of nutrient limitation on genomic rearrangements in prokaryotes. Final report

Description: During the total agreement period, more than 200 unique heterotrophic aerobic bacteria were isolated from samples of deep subsurface sediments retrieved from depths of 149.2 and 178.9 m of a vertical bore hole at Cerro Negro (CNV1). All the isolates were characterized phylogenetically and deposited to SMCC. Several clones belonging to a family currently believed to be of strictly marine origin (Microscilla) were found among the isolates. The bacteria might be the descendants of those present in marine environment at the time of deposition. This is an important conclusion for the entire deep subsurface Microbial Origins project. The authors determined diversity of populations of prokaryotic genomes of independent bacterial clones isolated from both surface soil, and subsurface sediments. The degree of diversity for deep subsurface isolates was exceedingly high, much higher than that observed for surface soil isolates. The authors have found that the genomes of some subsurface isolates are significantly more plastic than those for strains isolated from surface environments. The most intriguing discovery was isolation and cultivation of populations of nonplateable novel ultra small bacteria, which are phylogenetically more close to chloroplasts of higher plants, than any other existing bacteria including Cyanobacteria.
Date: March 1, 1998
Creator: Zlatkin, I.V. & Forney, L.J.
Partner: UNT Libraries Government Documents Department

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, October 1, 1988--June 1, 1989

Description: The primary goal of this study is to achieve a more thorough understanding of the mechanisms employed by higher organisms to repair DNA damage induced by both ionizing and nonionizing radiation. These studies are also contributing to an improved understanding of the processes of mutagenesis and carcinogenesis in higher eukaryotes. The studies employ Drosophila as a model organism for investigating repair functions that are common to all higher eukaryotes. Drosophila was chosen in the early phases of this study primarily because of the ease with which one can isolate and characterize repair-deficient mutants in a metazoan organism. The laboratory has gone on to investigate the metabolic defects of such mutants while others have performed complementary genetic and cytogenetic studies which relate DNA repair processes to mutagenesis and chromosome stability. The repair studies have exploited the capacity to introduce mutant Drosophila cells into tissue culture and thereby compare repair defects directly with those of homologous human disorders. Researchers are currently employing recombinant DNA technology to investigate the mechanisms of the DNA repair pathways defined by those mutants.
Date: December 31, 1989
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

[Genetics in methylotrophic bacteria]. Final progress report, July 1, 1987--June 30, 1995

Description: This project is related to development of genetic techniques for methylotrophic bacteria and the use of these to study genes involved in methanol oxidation. During the first two years of the project the authors defined the genetic organization of these Mox (methanol oxidation) genes and started to define their regulatory regions. During the next three year period, work involved defining genetic organization of key methylotrophic genes, studying upstream regions involved in transcription, and defining key regulatory genes for these systems. In the last three years of support, the project focused on identifying methylotrophy genes and the regions involved in their expression for comparative purposes, and began the process of analyzing the genes involved in transcriptional regulation of the Mox system in the strain for which they have ther most information, Methylobacter extorquens AM1.
Date: September 1, 1998
Partner: UNT Libraries Government Documents Department