Genome physical mapping with large-insert bacterial clones by fingerprint analysis: methodologies, source clone genome coverage, and contig map quality

Genome physical mapping with large-insert clones by fingerprint analysis is becoming an active area of genomics research. Here, we report two new capillary electrophoresis-based fingerprinting methods for genome physical mapping and the effects of different fingerprinting methods and source clone genome coverage on quality physical map construction revealed by computer simulations and laboratory experiments. It was shown that the manual sequencing gel-based two-enzyme fingerprinting method consistently generated larger and more accurate contigs, followed by the new capillary electrophoresis-based three-enzyme method, the new capillary electrophoresis-based five-enzyme (SNaPshot) method, the agarose gel-based one-enzyme method, and the automatic sequencing gel-based four-enzyme method, in descending order, when 1% or fewer questionable clones were allowed. Analysis of clones equivalent to 5x, 8x, 10x, and 15x genomes using the fingerprinting methods revealed that as the number of clones increased from 5x to 10x, the contig length rapidly increased for all methods. However, when the number of clones was increased from 10x to 15x coverage, the contig length at best increased at a lower rate or even decreased. The results will provide useful knowledge and strategies for effective construction of quality genome physical maps for advanced genomics research.     

HiTRACE: high-throughput robust analysis for capillary electrophoresis

MOTIVATION: Capillary electrophoresis (CE) of nucleic acids is a workhorse technology underlying high-throughput genome analysis and large-scale chemical mapping for nucleic acid structural inference. Despite the wide availability of CE-based instruments, there remain challenges in leveraging their full power for quantitative analysis of RNA and DNA structure, thermodynamics and kinetics. In particular, the slow rate and poor automation of available analysis tools have bottlenecked a new generation of studies involving hundreds of CE profiles per experiment. RESULTS: We propose a computational method called high-throughput robust analysis for capillary electrophoresis (HiTRACE) to automate the key tasks in large-scale nucleic acid CE analysis, including the profile alignment that has heretofore been a rate-limiting step in the highest throughput experiments. We illustrate the application of HiTRACE on 13 datasets representing 4 different RNAs, 3 chemical modification strategies and up to 480 single mutant variants; the largest datasets each include 87 360 bands. By applying a series of robust dynamic programming algorithms, HiTRACE outperforms prior tools in terms of alignment and fitting quality, as assessed by measures including the correlation between quantified band intensities between replicate datasets. Furthermore, while the smallest of these datasets required 7-10 h of manual intervention using prior approaches, HiTRACE quantitation of even the largest datasets herein was achieved in 3-12 min. The HiTRACE method, therefore, resolves a critical barrier to the efficient and accurate analysis of nucleic acid structure in experiments involving tens of thousands of electrophoretic bands.     

Genome mapping with anchored clones: Theoretical aspects

As part of our effort to construct a physical map of the genome of Arabidopsis thaliana we have made a mathematical analysis of our experimental approach of anchoring yeast artificial chromosome clones with genetically mapped RFLPs and RAPDs. The details of this analysis are presented and their implications for mapping the Arabidopsis genome are discussed.     

Genome physical mapping of polyploids: a BIBAC physical map of cultivated tetraploid cotton, Gossypium hirsutum L

Polyploids account for approximately 70% of flowering plants, including many field, horticulture and forage crops. Cottons are a world-leading fiber and important oilseed crop, and a model species for study of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. This study has addressed the concerns of physical mapping of polyploids with BACs and/or BIBACs by constructing a physical map of the tetraploid cotton, Gossypium hirsutum L. The physical map consists of 3,450 BIBAC contigs with an N50 contig size of 863 kb, collectively spanning 2,244 Mb. We sorted the map contigs according to their origin of subgenome, showing that we assembled physical maps for the A- and D-subgenomes of the tetraploid cotton, separately. We also identified the BIBACs in the map minimal tilling path, which consists of 15,277 clones. Moreover, we have marked the physical map with nearly 10,000 BIBAC ends (BESs), making one BES in approximately 250 kb. This physical map provides a line of evidence and a strategy for physical mapping of polyploids, and a platform for advanced research of the tetraploid cotton genome, particularly fine mapping and cloning the cotton agronomic genes and QTLs, and sequencing and assembling the cotton genome using the modern next-generation sequencing technology.     

Peptide mapping by capillary electrophoresis with Pluronic F127

Separation of peptides and proteins by capillary zone electrophoresis suffers from the interaction of these solutes with the capillary wall which results in the formation of broad peaks and low resolution. To minimize the protein/peptide-capillary wall interaction we tried to use Pluronic F127, a triblock copolymer of the general formula (polyethylene oxide)(x)(polypropylene oxide)(y)(polyethylene oxide)(z) when x=106, y=70 and z=106 which can be considered a surfactant capable of self-association both into isotropic and anisotropic gels. The analytes studied were enzymatic digests (obtained by trypsin or pepsin treatment) of insoluble matrix proteins from avian eggshell. The best separations were obtained by a system exploiting 10% Pluronic F127 in 20 mmol/l phosphate buffer, pH 2.5. Electrophoretic peptide profiles obtained were very complex owing to the complicated nature of the samples (the exact composition of the proteinous insoluble part of the eggshell is still unknown). The separation in phosphate buffer only offered complex maps of incompletely resolved peaks. The use of Pluronic F127 distinctly improved the separation with a considerably better resolution regarding both the number of peaks obtained and the quality of the separation.     

Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum

Enzymatic activation of PAA (phenylacetic acid) to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and POA (phenoxyacetic acid) act as acyl donors in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a PCL (phenylacetate-CoA ligase), was cloned in Escherichia coli as a maltose-binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA into phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with k(cat)/K(m) values of 0.23+/-0.06 and 7.8+/-1.2 mM(-1).s(-1) for PAA and POA respectively. Surprisingly, PCL was very efficient in catalysing the conversion of trans-cinnamic acids to the corresponding CoA thioesters [k(cat)/K(m)=(3.1+/-0.4)x10(2) mM(-1).s(-1) for trans-cinnamic acid]. Of all the substrates screened, medium-chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on the basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty-acid-activating ancestral enzyme that may have been involved in the beta-oxidation of fatty acids.     

GRAMA: genetic mapping analysis of temperature gradient capillary electrophoresis data

Temperature gradient capillary electrophoresis (TGCE) is a high-throughput method to detect segregating single nucleotide polymorphisms and InDel polymorphisms in genetic mapping populations. Existing software that analyzes TGCE data was, however, designed for mutation analysis rather than genetic mapping. Genetic recombinant analysis and mapping assistant (GRAMA) is a new tool that automates TGCE data analysis for the purpose of genetic mapping. Data from multiple TGCE runs are analyzed, integrated, and displayed in an intuitive visual format. GRAMA includes an algorithm to detect peaks in electropherograms and can automatically compare its peak calls with those produced by another software package. Consequently, GRAMA provides highly accurate results with a low false positive rate of 5.9% and an even lower false negative rate of 1.3%. Because of its accuracy and intuitive interface, GRAMA boosts user productivity more than twofold relative to previous manual methods of scoring TGCE data. GRAMA is written in Java and is freely available at .     

Aminoacyl-coenzyme A synthesis catalyzed by a CoA ligase from Penicillium chrysogenum

Coenzyme A ligases play an important role in metabolism by catalyzing the activation of carboxylic acids. In this study we describe the synthesis of aminoacyl-coenzyme As (CoAs) catalyzed by a CoA ligase from Penicillium chrysogenum. The enzyme accepted medium-chain length fatty acids as the best substrates, but the proteinogenic amino acids L-phenylalanine and L-tyrosine, as well as the non-proteinogenic amino acids D-phenylalanine, D-tyrosine and (R)- and (S)-β-phenylalanine were also accepted. Of these amino acids, the highest activity was found for (R)-β-phenylalanine, forming (R)-β-phenylalanyl-CoA. Homology modeling suggested that alanine 312 is part of the active site cavity, and mutagenesis (A312G) yielded a variant that has an enhanced catalytic efficiency with β-phenylalanines and D-α-phenylalanine.     

Quantification of autolysis in Penicillium chrysogenum by semiautomated image analysis

An image analysis method is described for the characterization of empty (autolyzed and inactive) regions within the mycelia of filamentous fungi. It extends a previous method that characterized only regions filled with cytoplasm or vacuoles (i.e., the active biomass). The method is semiautomatic, requiring some manual editing before automated measurements. When the method was used for samples from a batch fermentation of an industrial strain of Penicillium chrysogenum, the empty regions were observed to constitute up to 15% (by projected area) of the biomass during the growth phase. After nutrient exhaustion, however, the proportion of empty regions rose rapidly, eventually representing more than 50% of the biomass by the end of fermentation. The increase in the percentage of empty regions coincided with a decrease in biomass (as measured by dry cell weight) and a fall in penicillin titre. Further morphological analysis revealed that fragmentation of mycelia, particularly clumps, coincided with increases in the levels of empty regions. This new image analysis method gave additional information on hyphal differentiation and a measure of autolysis. It was also a useful indicator of the processes leading to autolysis.     

Genomic mapping by fingerprinting random clones: a mathematical analysis

Results from physical mapping projects have recently been reported for the genomes of Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans, and similar projects are currently being planned for other organisms. In such projects, the physical map is assembled by first "fingerprinting" a large number of clones chosen at random from a recombinant library and then inferring overlaps between clones with sufficiently similar fingerprints. Although the basic approach is the same, there are many possible choices for the fingerprint used to characterize the clones and the rules for declaring overlap. In this paper, we derive simple formulas showing how the progress of a physical mapping project is affected by the nature of the fingerprinting scheme. Using these formulas, we discuss the analytic considerations involved in selecting an appropriate fingerprinting scheme for a particular project.     

Bacterial artificial chromosome-based physical map of the rice genome constructed by restriction fingerprint analysis

Genome-wide physical mapping with bacteria-based large-insert clones (e.g., BACs, PACs, and PBCs) promises to revolutionize genomics of large, complex genomes. To accelerate rice and other grass species genome research, we developed a genome-wide BAC-based map of the rice genome. The map consists of 298 BAC contigs and covers 419 Mb of the 430-Mb rice genome. Subsequent analysis indicated that the contigs constituting the map are accurate and reliable. Particularly important to proficiency were (1) a high-resolution, high-throughput DNA sequencing gel-based electrophoretic method for BAC fingerprinting, (2) the use of several complementary large-insert BAC libraries, and (3) computer-aided contig assembly. It has been demonstrated that the fingerprinting method is not significantly influenced by repeated sequences, genome size, and genome complexity. Use of several complementary libraries developed with different restriction enzymes minimized the "gaps" in the physical map. In contrast to previous estimates, a clonal coverage of 6.0-8.0 genome equivalents seems to be sufficient for development of a genome-wide physical map of approximately 95% genome coverage. This study indicates that genome-wide BAC-based physical maps can be developed quickly and economically for a variety of plant and animal species by restriction fingerprint analysis via DNA sequencing gel-based electrophoresis.     

High-performance capillary electrophoresis: Protein charge estimations and peptide mapping by complexation electrophoresis

Two high-performance capillary electrophoretic (HPCE) methods are presented: The first methodology provides a procedure for estimating the isoelectric points of proteins in the absence of chaotropic agents with charge reversal Micro-Coat capillaries. The second method provides an optimized peptide mapping methodology for protein characterization that employs ion-pairing reagents to optimize the HPCE separation. Advantages and limitations of each methodology are discussed in terms of theory and practical experience. Both methodologies are applicable to a variety of proteins and both enhance our ability to characterize proteins on a molecular level.     

Towards a physical map of the Drosophila melanogaster genome: mapping of cosmid clones within defined genomic divisions.

A physical map of the D. melanogaster genome is being constructed, in the form of overlapping cosmid clones that are assigned to specific polytene chromosome sites. A master library of ca. 20,000 cosmids is screened with probes that correspond to numbered chromosomal divisions (ca. 1% of the genome); these probes are prepared by microdissection and PCR-amplification of individual chromosomes. The 120 to 250 cosmids selected by each probe are fingerprinted by Hinfl digestion and gel electrophoresis, and overlaps are detected by computer analysis of the fingerprints, permitting us to assemble sets of contiguous clones (contigs). Selected cosmids, both from contigs and unattached, are then localized by in situ hybridization to polytene chromosomes. Crosshybridization analysis using end probes links some contigs, and hybridization to previously cloned genes relates the physical to the genetic map. This approach has been used to construct a physical map of the 3.8 megabase DNA in the three distal divisions of the x chromosome. The map is represented by 181 canonical cosmids, of which 108 clones in contigs and 32 unattached clones have been mapped individually by in situ hybridization to chromosomes. Our current database of in situ hybridization results also includes the beginning of a physical map for the rest of the genome: 162 cosmids have been assigned by in situ hybridization to 129 chromosomal subdivisions elsewhere in the genome, representing 5 to 6 megabases of additional mapped DNA.     

Genomic mapping by anchoring random clones: a mathematical analysis.

A complete physical map of the DNA of an organism, consisting of overlapping clones spanning the genome, is an extremely useful tool for genomic analysis. Various methods for the construction of such physical maps are available. One approach is to assemble the physical map by "fingerprinting" a large number of random clones and inferring overlap between clones with sufficiently similar fingerprints. E.S. Lander and M.S. Waterman (1988, Genomics 2:231-239) have recently provided a mathematical analysis of such physical mapping schemes, useful for planning such a project. Another approach is to assemble the physical map by "anchoring" a large number of random clones--that is, by taking random short regions called anchors and identifying the clones containing each anchor. Here, we provide a mathematical analysis of such a physical mapping scheme.     

Degradation of phenol by a halotolerant strain of Penicillium chrysogenum

The lowest 50% lethal (effective) concentration, L(E)C₅₀, of phenol in a battery of seven microbiotests with species representing different trophic levels was 1–10 mg l⁻¹, classifying it as “toxic”. A phenol-degrading microorganism was isolated from soil samples of the salt mine of Clona in Portugal, after enrichment in the presence of phenol and high salt concentration. Based on cultural and morphological characteristics, the strain CLONA2 was identified as belonging to Penicillium chrysogenum. It was found to be a halotolerant fungus able to grow in a nutrient-rich medium with 5.8% NaCl. It degraded at least 300 mg l⁻¹ phenol as sole source of carbon and energy, without accumulation of intermediates. The samples were also tested for toxicity using the Microtox^® assay. Data showed that P. chrysogenum CLONA2 could be effectively utilized to reduce phenol toxicity. The results suggest also that phenol under saline conditions can be successfully mineralized by P. chrysogenum CLONA2.     

Libraries of large-insert genomic clones as a tool for molecular cytogenetic analysis of avian genome

Integration of molecular and cytegenetic levels of investigation results in complex understanding of structural and functional genome organization. Gridded libraries of large-insert genomic clones represent a powerful tool of the genome analysis. Their utilization provides coordination of data on molecular organization of nucleic acids with cytogenetic data on the chromosome structure. These libraries played an important role in sequencing of genomes of human, mouse, and other organisms as an instrument linking molecular biological and cytogenetic data via construction of contigs and their localization on the chromosomes. They also enabled analysis of orthology between the mammalian genomes. The existing avian libraries fit molecular cytogenetic analysis of the class Aves genome, and can be successfully used for the isolation and characterization of large genomic fragments. This provides utilization of these libraries not only for the chromosome mapping, but also for positional cloning and search for candidate genes for quantitative traits.     

Isolation and Characterization of a Double-Stranded Ribonucleic Acid from Penicillium chrysogenum

Double-stranded ribonucleic acid has been obtained from cells of the fungus Penicillium chrysogenum. This ribonucleic acid appears to be associated with mycophage and is an efficient inducer of interferon Its. extraction and partial purification are discussed, and evidence for its double-stranded and ribosidal nature is reviewed. The implications of viral nucleic acid in the life processes of fungi are considered.