ISSN:0173-0835    

DOI:10.1002/elps.1150190402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThis paper consists of two components: the use of gene encyclopedias in genomic studies andRhodobacter capsulatusgenome project. A survey of vectors used for encyclopedia construction includes a brief discussion of their relative advantages and limitations. Projects employing various methods of encyclopedia assembly including the comparison of restriction patterns, restriction maps, linking by hybridization, oligonucleotide fingerprinting, sequence tagged site (STS) fingerprinting and encyclopedias derived from genetic maps are listed and briefly described. The R.capsulatusSB 1003 genome project started with the construction of its cosmid encyclopedia, which comprises 192 cosmids covering the chromosome and the 134 kbp plasmid in strain SB 1003, with the exact map coordinates of each cosmid. In a pilot sequencing study, several cosmids were individually subcloned using the vector M13mp18 and merged into one 189 kbp contig. About 160 open reading frames (ORFs) identified by the CodonUse program were subjected to similarity searches. The biological functions of eighty ORFs could be assigned reliably using the WIT (what is there) genome investigation environment. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Recently, another 1.2 Mbp genome fragment of theRhodobactergenome was sequenced using a slightly modified approach. These results together with some genome investigation tools, have been placed at our web site (http://capsulapedia.uchicago.edu). The sequence of R.capsulatusis expected to be completed by summer 1998. A project to construct a systematic set of deletion strains of R.capsulatusin order to assign functions to unknown ORFs has been started. Preliminary data demonstrate the extreme convenience of the unique gene transfer agent (GTA) system to perform such work.

ISSN:0173-0835    

DOI:10.1002/elps.1150190403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractFrom a total genomic cosmid library of the pathogenTrypanosoma cruzi, specific sublibraries of the smallest four chromosomes were isolated by hybridization of the respective chromosomal bands obtained from pulsed‐field gels. These libraries form the basis for initial mapping analyses that should provide information useful for both the ongoing physical mapping of the entire genome and eventual sequence analyses. Selectivity of the procedure was high with 75% to 92%, although cross‐hybridization had to be expected from ubiquitous DNA features, such as centromeric and telomeric sequences, and other regions homologous between individual chromosomes. Overall, the number of identified clones was slightly higher than expected but well within the intrinsic experimental variation considering the uncertainty about the exact genome size, the variability in clonability and the higher frequency of repeat sequences in larger chromosomes. Chromosome III‐ and IV‐specific cosmids were analyzed on Southern blots of chromosomal separations. For strain CL Brener, all clones tested exhibited cross‐hybridization to a homologous chromosome larger than 1 Mbp, supporting the assumption of the respective chromosome couple being dipl

ISSN:0173-0835    

DOI:10.1002/elps.1150190404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractAs part of theTrypanosomaGenome Initiative launched by the World Health Organization (WHO), a physical clone map ofTrypanosoma cruzichromosomes III and IV was generated to facilitate both DNA sequence analysis of the parasite's genome and the investigation of chromosome organization. Apart from a few genetic markers, anonymous cosmids were taken from chromosomal sublibraries and individually hybridized to filter arrays of the relevant cosmid library. The probe order was determined from the hybridization fingerprint results and used to define a fitting clone order, with few gaps remaining. The results were independently verified by hybridizations to a bacterial artificial chromosome (BAC) library and, in case of chromosome III, restriction mapping. For gap closure, additional experiments on a total cosmid library were carried out. The possible tiling paths consist of 26 clones for chromosome III (610 kbp) and 28 clones for chromosome IV (680 kbp).

ISSN:0173-0835    

DOI:10.1002/elps.1150190405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractYeast artificial chromosomes (YACs) can accommodate large inserts and hence should be attractive tools for intra‐ and interspecies comparisons of bacterial genomes. YAC libraries were constructed from size‐selected partial digests of human andPseudomonas aeruginosaPAO DNA andSpeI‐restricted PAO DNA. Whereas YACs from human DNA had an average size of 350 kilobase pairs (kbp), a P.aeruginosasequence larger than 120 kbp was absent or truncated in the eukaryotic host. Coligation occurred for YACs smaller than 40 kbp, but stable YACs with 40–120 kbp large inserts of P.aeruginosaDNA were obtained in high yield.SpeI‐restricted chromosomes from 97 P.aeruginosastrains representing 47 genotypes were hybridized with stable YACs from three equidistant regions of the PAO genome. The low complexity of hybridizing bands demonstrated that the analyzed 100 kbp sequence contigs were stably maintained in most P.aeruginosaisolates from both disease and environmental

ISSN:0173-0835    

DOI:10.1002/elps.1150190406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractA novel application of the Smith/Birnstiel technique is presented for the analysis of intraspecies genomic diversity in small genomes. Rare‐cutter total/Nfrequent‐cutter partial restriction digestions are separated inNseparate lanes by pulsed‐field gel electrophoresis, blotted and hybridized with rare‐cutter fragment ends as probes. The evaluation of the autoradiogram results in high‐resolution restriction maps of 10–200 kbp regions. The technique was applied to the analysis of genome rearrangements inPseudomonas aeruginosastrains. Comparison of the region encoding the tryptophan biosynthesis genes in the PAO and the IATS serotype 5 strains revealed that shared sequence characterized by almost identical restriction fingerprints was interrupted in the serotype 5 strain by small islands displaying strain‐specific restriction site signatures. A multistep rearrangement in a hypervariable chromosome region downstream of thephnlocus was detected in serial airway isolates from a patient with cy

ISSN:0173-0835    

DOI:10.1002/elps.1150190407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractA high‐throughput robotic workstation system was used for double‐stranded plasmid DNA template preparation and sequencing reaction setup to streamline the sequencing process in genome projects. All 96‐well miniprep kits that were tested provided high quality plasmid DNA suitable for fluorescent DNA sequencing. After quantitation in a 96‐well UV spectrophotometer, the plasmid DNA was used as template to automatically set up sequencing reactions. The setup was controlled by spread sheets that were imported into the robotic system. We utilized this integrated system to prepare all necessary shotgun templates for our contributions to a number of large‐scale genome projects as well as a full‐length cDNA sequenc

ISSN:0173-0835    

DOI:10.1002/elps.1150190408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractLarge‐scale hybridization‐based genome mapping projects, such as the production of sequence‐ready physical clone maps, call for robust and cheap DNA labeling techniques that are amenable to automation. We routinely use a high‐throughput protocol based on fluorescence detection. DNA probes are labeledviapolymerase chain reaction (PCR) amplification with primers that are digoxigenin‐modified at their 5′ ends. Alternatively, digoxigenin‐labeled dUTP is incorporated in a random hexamer priming reaction. Hybridization takes place in small volumes by sandwiching the probe between filters and plastic sheets. A fluorescence signal is produced by the activity of alkaline phosphatase attached to an anti‐digoxigenin antibody upon the addition of AttoPhosTMsubstrate. Signals are directly detected with a charge‐coupled device (CCD) camera and scored by an image data analysis system. DNA filters can be reused at least 40 times without loss of data quality. Significant advantages compared to radioactive techniques are the reduced health risk, enabling highly parallel processing; the production of spot signals uniform in size and intensity, which is essential for efficient image analysis; and a cost reduc

ISSN:0173-0835    

DOI:10.1002/elps.1150190409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractA comprehensive analysis of the differences between the genomes of two closely related bacterial strains should give insight into the molecular basis of their individual phenotypic and genotypic characteristics. Here we present an integrative approach including two different strategies for the thorough investigation of genomic divergence. We have combined two techniques including genomic subtractive hybridization and comparative genome mapping by pulsed field gel electrophoresis (PFGE) techniques. The subtractive method for which a protocol is given herein results in the production of a library of specific DNA sequence tags present only in one strain, while the construction of macrorestriction maps of the bacterial chromosomes yields data about the overall genome organization and the arrangement and distance of gene loci. Comparison of the physical and genetic maps and determination of the map positions of the strain‐specific DNA sequences reveals gross chromosomal modifications, insertions or deletions of additional genetic material, and transpositional events. The further investigation of the strain‐specific regions yields information about the nature and origin of the acquired DNA and their influence on the evolution of the individual bacterial genome. The two methods were applied to differential genome analysis of clonal divergence inPseudomonas aeruginosachoosing two clone C isolates from diverse habit

ISSN:0173-0835    

DOI:10.1002/elps.1150190410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractPresent availability of the genomic text of bacteria allows assignment of biological known functions to many genes (typically, half of the genome's gene content). It is now time to try and predict new unexpected functions, using inductive procedures that allow correlating the content of the genomic text to possible biological functions. We show here that analysis of the genomes ofEscherichia coliandBacillus subtilisfor the distribution of AGCT motifs predicts that genes exist for which the mRNA molecule can be translated as several different proteins synthesized after ribosomal frameshifting or hopping. Among these genes we found that several coded for the same function inE. coliand B.subtilis. We analyzed in depth the situation of theinfB gene (experimentally known to specify synthesis of several proteins differing in their translation starts), theaceF/pdhC gene, theenogene, and therplI gene. In addition, genes specific toE. coliwere also studied:ompA,ompFandtolA(predicting epigenetic variation that could help escape infection by phages or colicins).

ISSN:0173-0835    

DOI:10.1002/elps.1150190411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY