A bacterial artificial chromosome based physical map of the Ustilago maydis genome

Genome ◽  
2005 ◽  
Vol 48 (2) ◽  
pp. 207-216 ◽  
Author(s):  
Khalid Meksem ◽  
Jeffry Shultz ◽  
Faiza Tebbji ◽  
Aziz Jamai ◽  
Jürgen Henrich ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Gel Electrophoresis ◽  
Physical Map ◽  
Artificial Chromosome ◽  
Ustilago Maydis ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field ◽  
Artificial Chromosomes ◽  
Bac Clones ◽  
Copy Dna

Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and codigested with HaeIII to reduce fragments to 50–750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P ≤ 1 x 10–6 to 1 x 10–24). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P ≤ 1 x 10–7 to 1 x 10–12. The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P ≤ 1 × 10–7. The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18–20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.Key words: Ustilago maydis, physical map, bacterial artificial chromosomes, whole-genome sequencing.

Construction of a genomic map ofMoraxella (Branhamella) catarrhalisATCC 25238 and physical mapping of virulence-associated genes

1999 ◽  
Vol 45 (4) ◽  
pp. 299-303 ◽  
Author(s):  
K T Nguyen ◽  
E J Hansen ◽  
M A Farinha
Keyword(s):  
Gel Electrophoresis ◽  
Moraxella Catarrhalis ◽  
Southern Hybridization ◽  
Physical Map ◽  
Outer Membrane Proteins ◽  
Restriction Enzymes ◽  
Pulsed Field Gel Electrophoresis ◽  
Respiratory Pathogens ◽  
Circular Chromosome ◽  
Pulsed Field

A physical genome map of the Moraxella catarrhalis type strain (ATCC 25238) has been constructed using pulsed field gel electrophoresis. Macrorestriction analyses of the genome of M. catarrhalis were performed by digestion with the restriction enzymes SmaI, NotI, and RsrII, which cleave the single circular chromosome into 9, 10, and 6 fragments, respectively. The chromosomal fragments generated by pulsed field gel electrophoresis were converted to a linkage map utilizing a combination of partial digestions, and cross-hybridizations. Moraxella catarrhalis, like a number of other respiratory pathogens, has a relatively small genome estimated at 1750 kilobase pairs or about 40% of the size of the Escherichia coli genome. The locations of the four ribosomal RNA operons (rrnLS) were determined by Southern hybridization and by digestion with I-CeuI endonuclease. A number of genes involved in virulence have been placed onto the physical map by Southern hybridization including those encoding the predominant outer-membrane proteins and the chromosomal gene encoding beta-lactamase.Key words: Moraxella catarrhalis, physical map, genome analysis, pulsed-field gel electrophoresis, virulence.

Construction of a MluI-YAC library from barley (Hordeum vulgare L.) and analysis of YAC insert terminal regions

Genome ◽  
1997 ◽  
Vol 40 (6) ◽  
pp. 896-902 ◽  
Author(s):  
Michael Kleine ◽  
Christian Jung ◽  
Wolfgang Michalek ◽  
Thomas Diefenthal ◽  
Harald Dargatz
Keyword(s):  
Hordeum Vulgare ◽  
Gel Electrophoresis ◽  
Yeast Artificial Chromosome ◽  
Single Copy ◽  
Pulsed Field Gel Electrophoresis ◽  
Hordeum Vulgare L ◽  
High Molecular Weight Dna ◽  
Pulsed Field ◽  
Artificial Chromosomes ◽  
Yac Library

We describe the construction of a specific yeast artificial chromosome (YAC) library from barley (Hordeum vulgare L.) using the vector pYAC-RC. The library was generated by total digestion of high molecular weight DNA with the infrequently cutting restriction enzyme MluI. Only 10–30% of the colonies were recombinant, as visualized by red–white selection and subsequent pulsed-field gel electrophoresis analysis. About 17 000 individual recombinant YAC clones with insert sizes ranging from 50 to 700 kb, with a mean of 170 kb, were selected. No chloroplast sequences were detected and the proportion of YAC clones containing BARE–1 copia–like retroelements is about 5%. Screening of the library with a single-copy RFLP marker closely linked to the Mla locus yielded three identical clones of the same size. Insert termini of randomly chosen YAC clones were investigated with respect to their redundancy in the barley genome and compared with termini of YAC clones from an EcoRI-based YAC library, resulting in a fourfold enrichment of single-copy sequences at the MluI vector–insert junctions.Key words: yeast artificial chromosomes, YAC, Hordeum vulgare, pulsed-field gel electrophoresis.

scholarly journals A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP.

1988 ◽  
Vol 167 (2) ◽  
pp. 664-669 ◽  
Author(s):  
J Rey-Campos ◽  
P Rubinstein ◽  
S Rodriguez de Cordoba
Keyword(s):  
Gene Cluster ◽  
Complement Activation ◽  
Gel Electrophoresis ◽  
Physical Map ◽  
Pulsed Field Gel Electrophoresis ◽  
Complement Components ◽  
Pulsed Field ◽  
Human Genes ◽  
Tight Linkage ◽  
Genes Encoding

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster.

scholarly journals Mapping of Escherichia coli chromosomal Tn5 and F insertions by pulsed field gel electrophoresis.

Genetics ◽  
1988 ◽  
Vol 119 (2) ◽  
pp. 227-236
Author(s):  
C L Smith ◽  
R D Kolodner
Keyword(s):  
Escherichia Coli ◽  
Physical Mapping ◽  
Gel Electrophoresis ◽  
Physical Map ◽  
Pulsed Field Gel Electrophoresis ◽  
Genetic Maps ◽  
Low Resolution ◽  
Transposon Tn5 ◽  
Pulsed Field ◽  
E Coli

Abstract A low resolution Not I physical map of Escherichia coli was recently constructed. In this report we demonstrated that this map can be used to map Tn5 and F insertions physically. The transposon, Tn5, contains Not I recognition sequences in its IS50 sequences. F plasmid contains an unmapped Not I site. Hence, the location of Tn5 and F in the chromosome can be mapped by identifying the location of the introduced Not I sites using pulsed field gel electrophoresis. The physical mapping of genetically mapped Tn5 insertions confirm the previously constructed Not I map and helps align the E. coli physical and genetic maps. The use of Tn5 can assist the construction of both physical and genetic maps for microorganisms lacking such maps. Variations on this approach will facilitate physical mapping with a wide variety of organisms, enzymes, and genetic elements.

Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research

Genome ◽  
2000 ◽  
Vol 43 (1) ◽  
pp. 199-204 ◽  
Author(s):  
Junqi Song ◽  
Fenggao Dong ◽  
Jiming Jiang
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Physical Map ◽  
Molecular Cytogenetics ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Average Insert Size ◽  
Chromosome Identification ◽  
Insert Size ◽  
Potato Genome ◽  
Bac Clones

Lack of reliable techniques for chromosome identification is the major obstacle for cytogenetics research in plant species with large numbers of small chromosomes. To promote molecular cytogenetics research of potato (Solanum tuberosum, 2n = 4x = 48) we developed a bacterial artificial chromosome (BAC) library of a diploid potato species S. bulbocastanum. The library consists of 23 808 clones with an average insert size of 155 kb, and represents approximately 3.7 equivalents to the potato genome. The majority of the clones in the BAC library generated distinct signals on specific potato chromosomes using fluorescence in situ hybridization (FISH). The hybridization signals provide excellent cytological markers to tag individual potato chromosomes. We also demonstrated that the BAC clones can be mapped to specific positions on meiotic pachytene chromosomes. The excellent resolution of pachytene FISH can be used to construct a physical map of potato by mapping molecular marker-targeted BAC clones on pachytene chromosomes. Key words: potato, BAC library, chromosome identification, physical mapping, molecular cytogenetics.

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