Extensive Conserved Synteny of Genes Between the Karyotypes of \u3cem\u3eManduca sexta\u3c/em\u3e and \u3cem\u3eBombyx mori\u3c/em\u3e Revealed by BAC-FISH Mapping

Background: Genome sequencing projects have been completed for several species representing four highly diverged holometabolous insect orders, Diptera, Hymenoptera, Coleoptera, and Lepidoptera. The striking evolutionary diversity of insects argues a need for efficient methods to apply genome information from such models to genetically uncharacterized species. Constructing conserved synteny maps plays a crucial role in this task. Here, we demonstrate the use of fluorescence in situ hybridization with bacterial artificial chromosome probes as a powerful tool for physical mapping of genes and comparative genome analysis in Lepidoptera, which have numerous and morphologically uniform holokinetic chromosomes. Methodology/Principal Findings: We isolated 214 clones containing 159 orthologs of well conserved single-copy genes of a sequenced lepidopteran model, the silkworm, Bombyx mori, from a BAC library of a sphingid with an unexplored genome, the tobacco hornworm, Manduca sexta. We then constructed a BAC-FISH karyotype identifying all 28 chromosomes of M. sexta by mapping 124 loci using the corresponding BAC clones. BAC probes from three M. sexta chromosomes also generated clear signals on the corresponding chromosomes of the convolvulus hawk moth, Agrius convolvuli, which belongs to the same subfamily, Sphinginae, as M. sexta. Conclusions/Significance: Comparison of the M. sexta BAC physical map with the linkage map and genome sequence of B. mori pointed to extensive conserved synteny including conserved gene order in most chromosomes. Only a few rearrangements, including three inversions, three translocations, and two fission/fusion events were estimated to have occurred after the divergence of Bombycidae and Sphingidae. These results add to accumulating evidence for the stability of lepidopteran genomes. Generating signals on A. convolvuli chromosomes using heterologous M. sexta probes demonstrated that BAC-FISH with orthologous sequences can be used for karyotyping a wide range of related and genetically uncharacterized species, significantly extending the ability to develop synteny maps for comparative and functional genomics

Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species

<p>Abstract</p> <p>Background</p> <p><it>Manduca sexta, Heliothis virescens</it>, and <it>Heliconius erato </it>represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date.</p> <p>Results</p> <p>We report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152–175 kb. We estimated that the genome coverage of each library ranged from 6–9 ×, with the two combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6~8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of ~200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the <it>H. erato </it>genome is somewhat more abundant in repeat elements and simple repeats than those of <it>M. sexta </it>and <it>H. virescens</it>. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of <it>H. virescens </it>being the most conserved as a typical lepidopteran, whereas both genomes of <it>H. erato </it>and <it>M. sexta </it>appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences.</p> <p>Conclusion</p> <p>The high-quality and large-insert BAC libraries of the insects, together with the identified BACs containing genes of interest, provide valuable information, resources and tools for comprehensive understanding and studies of the insect genomes and for addressing many fundamental questions in Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and evolution of the insect genomes.</p

The Identification and Heterologous Expression of the Biosynthetic Gene Cluster Encoding the Antibiotic and Anticancer Agent Marinomycin

With the rise in antimicrobial resistance, there is an urgent need for new classes of antibiotic with which to treat infectious disease. Marinomycin, a polyene antibiotic from a marine microbe, has been shown capable of killing methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF), as well as having promising activity against melanoma. An attractive solution to the photoprotection of this antibiotic has been demonstrated. Here, we report the identification and analysis of the marinomycin biosynthetic gene cluster (BGC), and the biosynthetic assembly of the macrolide. The marinomycin BGC presents a challenge in heterologous expression due to its large size and high GC content, rendering the cluster prone to rearrangement. We demonstrate the transformation of Streptomyces lividans using a construct containing the cluster, and the heterologous expression of the encoded biosynthetic machinery and production of marinomycin B

The Identification and Heterologous Expression of the Biosynthetic Gene Cluster Encoding the Antibiotic and Anticancer Agent Marinomycin

With the rise in antimicrobial resistance, there is an urgent need for new classes of antibiotic with which to treat infectious disease. Marinomycin, a polyene antibiotic from a marine microbe, has been shown capable of killing methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF), as well as having promising activity against melanoma. An attractive solution to the photoprotection of this antibiotic has been demonstrated. Here, we report the identification and analysis of the marinomycin biosynthetic gene cluster (BGC), and the biosynthetic assembly of the macrolide. The marinomycin BGC presents a challenge in heterologous expression due to its large size and high GC content, rendering the cluster prone to rearrangement. We demonstrate the transformation of Streptomyces lividans using a construct containing the cluster, and the heterologous expression of the encoded biosynthetic machinery and production of marinomycin B

Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, Penicillium fuscum and P. camembertii/clavigerum, which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form “biosynthetic gene clusters” (BGCs), which might remain silent (inactive) under various fermentation conditions. Fungi may also harbor cryptic BGCs that are not associated with previously characterized molecules. We turned to the tools of Fungal Artificial Chromosomes (FAC)-Next-Gen-Sequencing (NGS) to understand how co-culture activated cryptic biosynthesis of BPLA and several related berkeleylactones and to further investigate the true biosynthetic potential of these two fungi. FAC-NGS enables the capture of BGCs as individual FACs for heterologous expression in a modified strain of Aspergillus nidulans (heterologous host, FAC-AnHH). With this methodology, we created ten BGC-FACs that yielded fourteen different SMs, including strobilurin, which was previously isolated exclusively from basidiomycetes. Eleven of these compounds were not detected in the extracts of the FAC-AnHH. Of this discrete set, only the novel compound citreohybriddional had been isolated from either Penicillium sp. before and only at very low yield. We propose that through heterologous expression, FACs activated these silent BGCs, resulting in the synthesis of new natural products (NPs) with yields as high as 50%–60% of the crude organic extracts

The Soybean Genome Database (SoyGD): a browser for display of duplicated, polyploid, regions and sequence tagged sites on the integrated physical and genetic maps of Glycine max

Genomes that have been highly conserved following increases in ploidy (by duplication or hybridization) like Glycine max (soybean) present challenges during genome analysis. At the Soybean Genome Database (SoyGD) genome browser has, since 2002, integrated and served the publicly available soybean physical map, bacterial artificial chromosome (BAC) fingerprint database and genetic map associated genomic data. The browser shows both build 3 and build 4 contiguous sets of clones (contigs) of the soybean physical map. Build 4 consisted of 2854 contigs that encompassed 1.05 Gb and 404 high-quality DNA markers that anchored 742 contigs. Many DNA markers anchored sets of 2–8 different contigs. Each contig in the set represented a homologous region of related sequences. GBrowse was adapted to show sets of homologous contigs at all potential anchor points, spread laterally and prevented from overlapping. About 8064 minimum tiling path (MTP2) clones provided 13 473 BAC end sequences (BES) to decorate the physical map. Analyses of BES placed 2111 gene models, 40 marker anchors and 1053 new microsatellite markers on the map. Estimated sequence tag probes from 201 low-copy gene families located 613 paralogs. The genome browser portal showed each data type as a separate track. Tetraploid, octoploid, diploid and homologous regions are shown clearly in relation to an integrated genetic and physical map

Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli

Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed ‘pJAZZ’ vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT—rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries

Extensive Conserved Synteny of Genes between the Karyotypes of Manduca sexta and Bombyx mori Revealed by BAC-FISH Mapping

BACKGROUND: Genome sequencing projects have been completed for several species representing four highly diverged holometabolous insect orders, Diptera, Hymenoptera, Coleoptera, and Lepidoptera. The striking evolutionary diversity of insects argues a need for efficient methods to apply genome information from such models to genetically uncharacterized species. Constructing conserved synteny maps plays a crucial role in this task. Here, we demonstrate the use of fluorescence in situ hybridization with bacterial artificial chromosome probes as a powerful tool for physical mapping of genes and comparative genome analysis in Lepidoptera, which have numerous and morphologically uniform holokinetic chromosomes. METHODOLOGY/PRINCIPAL FINDINGS: We isolated 214 clones containing 159 orthologs of well conserved single-copy genes of a sequenced lepidopteran model, the silkworm, Bombyx mori, from a BAC library of a sphingid with an unexplored genome, the tobacco hornworm, Manduca sexta. We then constructed a BAC-FISH karyotype identifying all 28 chromosomes of M. sexta by mapping 124 loci using the corresponding BAC clones. BAC probes from three M. sexta chromosomes also generated clear signals on the corresponding chromosomes of the convolvulus hawk moth, Agrius convolvuli, which belongs to the same subfamily, Sphinginae, as M. sexta. CONCLUSIONS/SIGNIFICANCE: Comparison of the M. sexta BAC physical map with the linkage map and genome sequence of B. mori pointed to extensive conserved synteny including conserved gene order in most chromosomes. Only a few rearrangements, including three inversions, three translocations, and two fission/fusion events were estimated to have occurred after the divergence of Bombycidae and Sphingidae. These results add to accumulating evidence for the stability of lepidopteran genomes. Generating signals on A. convolvuli chromosomes using heterologous M. sexta probes demonstrated that BAC-FISH with orthologous sequences can be used for karyotyping a wide range of related and genetically uncharacterized species, significantly extending the ability to develop synteny maps for comparative and functional genomics