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1.
Microb Cell Fact ; 23(1): 18, 2024 Jan 11.
Article in English | MEDLINE | ID: mdl-38212806

ABSTRACT

BACKGROUND: Antibiotic-based plasmid selection and maintenance is a core tool in molecular biology; however, while convenient, this strategy has numerous drawbacks for biological manufacturing. Overuse of antibiotics and antibiotic resistance genes (ARG) contributes to the development of antimicrobial resistance, which is a growing threat to modern medicine. Antibiotics themselves are costly and therefore often omitted in fermentations, leading to plasmid loss and a corresponding loss in product yield. Furthermore, constitutive expression of a plasmid-encoded antibiotic resistance gene imposes a significant metabolic burden on the cells. For many fermentation products (e.g., in nutrition and medicine), the use of antibiotic resistance genes is subject to strict regulations and should be avoided. We present a method for plasmid selection and maintenance with stringent selection pressure that is independent of antibiotics and ARG. Furthermore, it can be used without any restrictions regarding culture medium and temperature. RESULTS: The developed method involves modification of a bacterial strain such that an essential gene is expressed genomically under the control of an inducible promoter. A copy of the same essential gene with the endogenous promoter is supplied on a plasmid for selection. In the absence of the inducer for the genomic copy of the essential gene, cells rely on expression of the plasmid-encoded gene copy, leading to tight selection for plasmid maintenance. Induction of the genomic copy of the essential gene enables the engineered strain to be propagated in the absence of a plasmid. Here, we describe the genetic setup and demonstrate long-term, tight selection for plasmid maintenance with a variety of different plasmids and E. coli strains. CONCLUSIONS: This method facilitates plasmid-based fermentations by eliminating the need for antibiotic selection and improving plasmid maintenance.


Subject(s)
Anti-Bacterial Agents , Escherichia coli , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Fermentation , Escherichia coli/metabolism , Plasmids/genetics , Promoter Regions, Genetic
2.
Mol Microbiol ; 116(3): 909-925, 2021 09.
Article in English | MEDLINE | ID: mdl-34181784

ABSTRACT

The Escherichia coli dnaE gene encodes the α-catalytic subunit (pol IIIα) of DNA polymerase III, the cell's main replicase. Like all high-fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so-called "steric gate" residue in the active site of the polymerase that physically clashes with the 2'-OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α-θ-ε subunits). Of the possible 38 mutants, only nine were successfully sub-cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid-encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature.


Subject(s)
Catalytic Domain , DNA Polymerase III/chemistry , DNA Polymerase III/genetics , DNA/chemistry , DNA/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Alleles , Amino Acid Substitution , DNA Mismatch Repair , DNA Polymerase III/metabolism , DNA Replication , Deoxyribonucleotides/chemistry , Escherichia coli/enzymology , Escherichia coli/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Genomic Instability , Models, Molecular , Mutation , Phenotype , Ribonucleotides/chemistry
3.
Microb Cell Fact ; 19(1): 14, 2020 Jan 28.
Article in English | MEDLINE | ID: mdl-31992282

ABSTRACT

BACKGROUND: Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed. RESULTS: Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response. CONCLUSIONS: Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.


Subject(s)
Batch Cell Culture Techniques/methods , Escherichia coli , Genes, Reporter , Genetic Heterogeneity , Single-Cell Analysis/methods , Bioreactors/microbiology , Biotechnology/methods , Escherichia coli/genetics , Escherichia coli/growth & development , Glucose/metabolism , Oxygen/metabolism , Stress, Physiological/physiology
4.
Eng Life Sci ; 23(1): e2100162, 2023 Jan.
Article in English | MEDLINE | ID: mdl-36619877

ABSTRACT

Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.

5.
Nat Commun ; 11(1): 5581, 2020 11 04.
Article in English | MEDLINE | ID: mdl-33149216

ABSTRACT

The chromatin landscape of human brain cells encompasses key information to understanding brain function. Here we use ATAC-seq to profile the chromatin structure in four distinct populations of cells (glutamatergic neurons, GABAergic neurons, oligodendrocytes, and microglia/astrocytes) from three different brain regions (anterior cingulate cortex, dorsolateral prefrontal cortex, and primary visual cortex) in human postmortem brain samples. We find that chromatin accessibility varies greatly by cell type and, more moderately, by brain region, with glutamatergic neurons showing the largest regional variability. Transcription factor footprinting implicates cell-specific transcriptional regulators and infers cell-specific regulation of protein-coding genes, long intergenic noncoding RNAs and microRNAs. In vivo transgenic mouse experiments validate the cell type specificity of several of these human-derived regulatory sequences. We find that open chromatin regions in glutamatergic neurons are enriched for neuropsychiatric risk variants, particularly those associated with schizophrenia. Integration of cell-specific chromatin data with a bulk tissue study of schizophrenia brains increases statistical power and confirms that glutamatergic neurons are most affected. These findings illustrate the utility of studying the cell-type-specific epigenome in complex tissues like the human brain, and the potential of such approaches to better understand the genetic basis of human brain function.


Subject(s)
Astrocytes/metabolism , Chromatin/metabolism , GABAergic Neurons/metabolism , Microglia/metabolism , Neurons/metabolism , Oligodendroglia/metabolism , Schizophrenia/metabolism , Animals , Chromatin/genetics , Epigenesis, Genetic , Gene Expression Regulation/genetics , Gyrus Cinguli/cytology , Gyrus Cinguli/metabolism , Humans , Mice , Mice, Transgenic , MicroRNAs/metabolism , Prefrontal Cortex/cytology , Prefrontal Cortex/metabolism , Promoter Regions, Genetic , RNA, Long Noncoding/metabolism , Risk Factors , Schizophrenia/genetics , Transcription Factors/metabolism , Visual Cortex/cytology , Visual Cortex/metabolism
6.
Sci Rep ; 9(1): 4486, 2019 03 14.
Article in English | MEDLINE | ID: mdl-30872616

ABSTRACT

Temperature shifts trigger genome-wide changes in Escherichia coli's gene expression. We studied if chromosome integration impacts on a gene's sensitivity to these shifts, by comparing the single-RNA production kinetics of a PLacO3O1 promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar. At critically low temperatures, the chromosome-integrated promoter becomes weaker and noisier. Dissection of its initiation kinetics reveals longer lasting states preceding open complex formation, suggesting enhanced supercoiling buildup. Measurements with Gyrase and Topoisomerase I inhibitors suggest hindrance to escape supercoiling buildup at low temperatures. Consistently, similar phenomena occur in energy-depleted cells by DNP at 30 °C. Transient, critically-low temperatures have no long-term consequences, as raising temperature quickly restores transcription rates. We conclude that the chromosomally-integrated PLacO3O1 has higher sensitivity to low temperatures, due to longer-lasting super-coiled states. A lesser active, chromosome-integrated native lac is shown to be insensitive to Gyrase overexpression, even at critically low temperatures, indicating that the rate of escaping positive supercoiling buildup is temperature and transcription rate dependent. A genome-wide analysis supports this, since cold-shock genes exhibit atypical supercoiling-sensitivities. This phenomenon might partially explain the temperature-sensitivity of some transcriptional programs of E. coli.


Subject(s)
Chromosomes, Bacterial/genetics , Escherichia coli/growth & development , Gene Expression Profiling/methods , Plasmids/genetics , Cold Temperature , DNA, Superhelical/drug effects , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Single Molecule Imaging , Stress, Physiological , Topoisomerase I Inhibitors/pharmacology , Topoisomerase II Inhibitors/pharmacology , Whole Genome Sequencing
7.
Biotechniques ; 45(3): 335-7, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18778259

ABSTRACT

System-oriented applications of genetic engineering, such as metabolic engineering, often require the serial optimization of enzymatic reaction steps, which can be achieved by transcriptional fine-tuning. However, approaches to changing gene expression are usually limited to deletion and/or strong overexpression and rarely match the desired optimal transcript levels. A solution to this all-or-nothing approach has been the use of a synthetic promoter library (SPL) that is based on randomized sequences flanking the consensus -10 and -35 promoter regions and allows for fine-tuning of bacterial gene expression. Red/ET recombination perfectly complements SPL technology, since it enables easy modification of the Escherichia coli genome and can be accomplished with linear DNA (i.e., the SPL). To demonstrate the synergistic use of Red/ET and SPL for metabolic engineering applications, we replaced the native promoter of a genomic localized phosphoglucose isomerase (pgi)-lacZ reporter construct by an SPL. Using these technologies together, we were able to rapidly identify synthetic promoter sequences that resulted in activity range of 25% to 570% of the native pgi-promoter.


Subject(s)
Escherichia coli/genetics , Gene Library , Genetic Engineering/methods , Promoter Regions, Genetic/genetics , Transcription, Genetic , Base Sequence , Consensus Sequence , Escherichia coli Proteins/genetics , Molecular Sequence Data , Recombination, Genetic
8.
Mol Cell Biol ; 23(3): 864-72, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12529392

ABSTRACT

Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRbeta as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRbeta is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRalpha and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRbeta. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRbeta as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.


Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Lanosterol/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cholesterol/metabolism , Cloning, Molecular , DNA, Complementary/genetics , DNA-Binding Proteins/chemistry , Dimerization , Dogs , Humans , Ligands , Male , Mice , Molecular Sequence Data , Primates , Protein Structure, Quaternary , Pseudogenes , Rabbits , Rats , Transcription Factors/chemistry
9.
Mol Biotechnol ; 32(1): 43-53, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16382181

ABSTRACT

Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.


Subject(s)
Bacteriophage lambda/genetics , Genetic Engineering/methods , Operon/genetics , Rec A Recombinases/genetics , Recombination, Genetic/genetics , Animals , Arabinose/pharmacology , Chromosomes, Artificial, Bacterial/genetics , Chromosomes, Bacterial/genetics , DNA Repair/genetics , Escherichia coli/genetics , Gene Expression/drug effects , Gene Expression/genetics , Gene Expression Regulation, Bacterial/genetics , Histone-Lysine N-Methyltransferase , Mice , Mutation/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Plasmids/genetics , Transformation, Bacterial/genetics
10.
Biotechniques ; 57(4): 179, 181-7, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25312087

ABSTRACT

Despite the pressing need for novel cancer treatments, our improved understanding of tumor biology is not being successfully translated into better therapies. Here we present a lentiviral vector that enables in vivo validation of cancer therapeutic targets when combined with existing cancer animal models that faithfully reproduce the natural history of human disease. Unlike the conventional genetic approaches with targeted alleles, the outlined experimental strategy could be used to assess the preclinical efficacy of a growing number of putative therapeutic hits in a rapid and cost-effective manner.


Subject(s)
Lentivirus/metabolism , Neoplasms/metabolism , Neoplasms/therapy , Animals , Cell Line, Tumor , DNA/metabolism , Disease Models, Animal , Humans , Lentivirus/genetics , Mice , Neoplasms/genetics , Plasmids/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Reproducibility of Results
11.
Biotechniques ; 53(1)2012 Jul 01.
Article in English | MEDLINE | ID: mdl-26307259

ABSTRACT

Huntington's disease (HD) is a fatal neurodegenerative disorder that is caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (htt) gene. None of the existing HD mouse models recapitulate the exact disease symptoms and course as it is seen in humans and the generation of further HD disease models is challenging because of the size and complexity of the htt gene locus. Starting from a single substrate plasmid harboring human htt cDNA comprising 98 glutamine (Q) residues, we applied Red/ET recombination to generate four BDNF-BAC transgenes harboring full-length or truncated (N171) htt cDNA comprising 98 or 15 Q residues. BDNF (brain-derived neurotrophic factor) is expressed in the cortical neurons projecting to the striatal medium spiny neurons, and was used to direct htt transgene expression to investigate the contribution of these cell types to HD.


Subject(s)
Chromosomes, Artificial, Bacterial/genetics , Genetic Engineering/methods , Nerve Tissue Proteins/genetics , Recombination, Genetic/genetics , Animals , DNA, Complementary/genetics , Humans , Huntingtin Protein , Huntington Disease , Mice , Nerve Tissue Proteins/metabolism , Oligonucleotides
12.
Biotechniques ; 46(7): 527-33, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19594452

ABSTRACT

Existing methods for site-directed plasmid mutagenesis are restrained by the small spectrum of modifications that can be introduced by mutagenic primers and the amplicon size limitations of in vitro DNA synthesis. As demonstrated here, the combined use of Red/ET recombination and unique restriction site elimination enables extensive manipulation regardless of plasmid size and DNA sequence elements. First, a selectable marker is PCR-amplified with synthetic primers attaching 50-bp homology target flanks for Red/ET recombination and an arbitrary restriction site absent in the substrate plasmid. The resulting cassette is co-electroporated with substrate plasmids in Red/ET-proficient Escherichia coli cells. Following isolation of recombinant plasmids, linear nonselectable DNA replaces the cassette and introduces the desired mutation(s) in a second Red/ET recombination step. Upon selective digestion of parental plasmids and retransformation, a 38% mutation efficiency was achieved using a synthetic 97-nucleotide oligonucleotide to cure a 17-bp deletion within lacZalpha of pUC19 (2,686 bp). A PCR fragment was used with similar efficiency to co-replace mouse Cdkn1b codons 9 and 76 in gene-targeting vector pGTC (13,083 bp).


Subject(s)
Gene Dosage , Mutagenesis, Site-Directed/methods , Plasmids/genetics , Recombination, Genetic/genetics , Restriction Mapping/methods , Animals , Base Sequence , Codon/genetics , Exons/genetics , Genetic Vectors/genetics , Mice , Molecular Sequence Data , Oligonucleotides/metabolism , Point Mutation/genetics , Transformation, Genetic
13.
Plant Physiol ; 138(2): 1083-96, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15923334

ABSTRACT

Comprehensive functional data on plant R2R3-MYB transcription factors is still scarce compared to the manifold of their occurrence. Here, we identified the Arabidopsis (Arabidopsis thaliana) R2R3-MYB transcription factor MYB12 as a flavonol-specific activator of flavonoid biosynthesis. Transient expression in Arabidopsis protoplasts revealed a high degree of functional similarity between MYB12 and the structurally closely related factor P from maize (Zea mays). Both displayed similar target gene specificity, and both activated target gene promoters only in the presence of a functional MYB recognition element. The genes encoding the flavonoid biosynthesis enzymes chalcone synthase, chalcone flavanone isomerase, flavanone 3-hydroxylase, and flavonol synthase were identified as target genes. Hence, our observations further add to the general notion of a close relationship between structure and function of R2R3-MYB factors. High-performance liquid chromatography analyses of myb12 mutant plants and MYB12 overexpression plants demonstrate a tight linkage between the expression level of functional MYB12 and the flavonol content of young seedlings. Quantitative real time reverse transcription-PCR using these mutant plants showed MYB12 to be a transcriptional regulator of CHALCONE SYNTHASE and FLAVONOL SYNTHASE in planta, the gene products of which are indispensable for the biosynthesis of flavonols.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/metabolism , Gene Expression Regulation, Plant , Phenylpropionates/metabolism , Transcription Factors/physiology , Acyltransferases/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Base Sequence , Cells, Cultured , Chromosome Mapping , Chromosomes, Plant , Flavonols/metabolism , Oxidoreductases/genetics , Phenotype , Plant Proteins/genetics , Seedlings/metabolism
14.
Mol Cell Proteomics ; 4(2): 205-13, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15604093

ABSTRACT

High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors.


Subject(s)
Computational Biology/methods , Protein Interaction Mapping/methods , Proteomics/methods , Receptors, Cytoplasmic and Nuclear/chemistry , Statistics as Topic/methods , Automation , Cell Nucleus/metabolism , DNA, Complementary/metabolism , Databases as Topic , Gene Library , Genome, Human , Humans , Protein Binding , Proteome , Receptors, Cytoplasmic and Nuclear/metabolism , Two-Hybrid System Techniques
15.
Genome ; 48(2): 207-16, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15838542

ABSTRACT

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 co-digested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P < or = 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 < or = 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 < or = 1 x 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/.


Subject(s)
Chromosomes, Artificial, Bacterial/genetics , Chromosomes, Fungal/genetics , Contig Mapping , Genome, Fungal , Ustilago/genetics , Genomic Library
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