Bacterial Artificial Chromosome Clones Research Articles

Complete Genome Sequence and Construction of an Infectious Bacterial Artificial Chromosome Clone of a Virulent Duck Enteritis Virus Strain XJ

In 2021, a highly virulent strain of duck enteritis virus (DEV), designated as DEV XJ, was isolated from Zhejiang, China, and its complete genome, spanning 162,234 bp with 78 predicted open reading frames (ORFs), was sequenced. While showing relative homology to the DEV CV strain, DEV XJ exhibited distinctions in 38 ORFs, including various immunogenic and virulence-related genes. Amino acid variation analysis, focusing on UL6 and LORF3, indicated a high degree of homology between DEV XJ and the 2085 strain from Europe, as well as the DEV DP-AS-Km-19 strain from India. Subsequently, a full-length infectious bacterial artificial chromosome clone (BAC) of DEV XJ was successfully constructed to delve into the pathogenic mechanisms of this virulent strain. XJ BAC demonstrated substantial similarity to the parental DEV XJ in both in vitro growth properties and the induction of typical pathogenic symptoms in sheldrakes. Furthermore, the US3, LORF3, UL21, and UL36 genes were individually deleted using a two-step RED recombination approach based on the infectious BAC clone. Our findings revealed that the UL21 and UL36 genes play crucial roles in viral proliferation. Although the US3 and LORF3 genes were dispensable for viral replication and cell-to-cell transmission in vitro, they attenuated the replication and transmission efficiency of DEV compared to the WT. In summary, this study accomplished the whole-genome sequencing of a clinically virulent DEV strain and the successful construction of an infectious DEV XJ clone. Moreover, the functional roles of the above-mentioned mutant genes were preliminarily explored through the analysis of their in vitro biological characteristics.

Isolation and Structure Elucidation of JBIR-157, a Skeletally Novel Aromatic Polyketide Produced by the Heterologous Expression of a Cryptic Gene Cluster.

Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.

Comparative karyotype analysis of the red brocket deer (M. americana sensu lato and M. rufa) complex: evidence of drastic chromosomal evolution and implications on speciation process.

Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

Capability of a large bacterial artificial chromosome clone harboring multiple biosynthetic gene clusters for the production of diverse compounds.

The biosynthetic gene clusters (BGCs) for the macrocyclic lactone-based polyketide compounds are extremely large-sized because the polyketide synthases that generate the polyketide chains of the basic backbone are of very high molecular weight. In developing a heterologous expression system for the large BGCs amenable to the production of such natural products, we selected concanamycin as an appropriate target. We obtained a bacterial artificial chromosome (BAC) clone with a 211-kb insert harboring the entire BGC responsible for the biosynthesis of concanamycin. Heterologous expression of this clone in a host strain, Streptomyces avermitilis SUKA32, permitted the production of concanamycin, as well as that of two additional aromatic polyketides. Structural elucidation identified these additional products as ent-gephyromycin and a novel compound that was designated JBIR-157. We describe herein sequencing and expression studies performed on these BGCs, demonstrating the utility of large BAC clones for the heterologous expression of cryptic or near-silent loci.

Local Genomic Instability of the SpTransformer Gene Family in the Purple Sea Urchin Inferred from BAC Insert Deletions.

The SpTransformer (SpTrf) gene family in the purple sea urchin, Strongylocentrotus purpuratus, encodes immune response proteins. The genes are clustered, surrounded by short tandem repeats, and some are present in genomic segmental duplications. The genes share regions of sequence and include repeats in the coding exon. This complex structure is consistent with putative local genomic instability. Instability of the SpTrf gene cluster was tested by 10 days of growth of Escherichia coli harboring bacterial artificial chromosome (BAC) clones of sea urchin genomic DNA with inserts containing SpTrf genes. After the growth period, the BAC DNA inserts were analyzed for size and SpTrf gene content. Clones with multiple SpTrf genes showed a variety of deletions, including loss of one, most, or all genes from the cluster. Alternatively, a BAC insert with a single SpTrf gene was stable. BAC insert instability is consistent with variations in the gene family composition among sea urchins, the types of SpTrf genes in the family, and a reduction in the gene copy number in single coelomocytes. Based on the sequence variability among SpTrf genes within and among sea urchins, local genomic instability of the family may be important for driving sequence diversity in this gene family that would be of benefit to sea urchins in their arms race with marine microbes.

Cytogenetic, molecular, and morphological characterization of Odocoileus pandora (Merriam, 1901) (Artiodactyla, Cervidae)

The Yucatan brown brocket deer was initially described as Mazama pandora Merriam, 1901. Phylogenetic analysis of mitochondrial DNA recovered the species as sister group of the genus Odocoileus and subsequently the species was repositioned to this genus naming Odocoileus pandora (Merriam, 1901). However, there is still a lack of taxonomic consensus that could assist in nomenclature definition of the species. We aim to clarify the taxonomy of O. pandora through an integrative assessment using morphological, cytogenetic, and molecular data from recently collected specimens. Morphological characterizations identified O. pandora as a medium-sized brocket deer. The skull shows an inflated auditory bulla and a narrow zygomatic arch. Males present long, broad, spike-like antlers converging inward, marked with deep longitudinal grooves. Odocoileus pandora shows a karyotype with 2 n = 60 FN = 74. Bacterial artificial chromosome clone hybridization showed that centric and tandem fusions, and inversions are involved in karyotypical divergences between O. pandora, S. gouazoubira, M. americana, and O. virginianus. Our phylogeny based on Bayesian Inference of mitogenome recovered O. pandora as monophyletic within the subtribe Odocoileina, with Odocoileus as sister taxa. Morphological and genetic characteristics of O. pandora agrees in differencing the Yucatan brown brocket from Mazama and Odocoileus genera. Therefore, a new generic name should be indicated for this taxon.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.

Transgenes with genomic DNA fragments that encompass genes of interest are the gold standard for complementing null alleles in rescue experiments in the fruit fly Drosophila melanogaster. Of particular interest are genomic DNA clones available as bacterial artificial chromosomes (BACs) or fosmids from publicly available genomic DNA libraries. Genes contained within BAC and fosmid clones can be easily modified by recombineering cloning to insert peptide or protein tags to localize, visualize, or manipulate gene products, and to create point mutations or deletions for structure-function analysis of the inserted genes. However, since transgenesis efficiency is inversely correlated with transgene size, obtaining transgenic animals for increasingly larger BAC and fosmid clones requires increasingly laborious screening efforts using the transgenesis marker commonly used for these transgenes, the dominant eye color marker white+ . We recently described a drug-based selectable genetic platform for Drosophila melanogaster, which included four resistance markers that allow direct selection of transgenic animals, eliminating the need to identify transgenic progeny by laborious phenotypic screening. By integrating these resistance markers into BAC transgenes, we were able to isolate animals containing large transgenes by direct selection, avoiding laborious screening. Here we present procedures on how to upgrade BAC clones by serial recombineering cloning to build both selectable and tagged BAC transgenes, for selection transgenesis and functional gene analysis, respectively. We illustrate these procedures using a BAC clone encompassing the gene encoding the synaptic vesicle protein, cysteine string protein. We demonstrate that the modified BAC clone, serially recombineered with a selectable marker for selection transgenesis and an N-terminal green fluorescent protein tag for gene expression analysis, is functional by showing the expression pattern obtained after successful selection transgenesis. The protocols cover: (1) cloning and preparation of the recombineering templates needed for serial recombineering cloning to incorporate selectable markers and protein tags; (2) preparing electrocompetent cells needed to perform serial recombineering cloning; and (3) the serial recombineering workflow to generate both selectable and tagged genomic BAC reporter transgenes for selection transgenesis and functional gene analysis in Drosophila melanogaster. The protocols we describe can be easily adapted to incorporate any of four selectable markers, protein tags, or any other modification for structure-function analysis of the genes present within any of the BAC or fosmid clones. A protocol for generating transgenic animals using serially recombineered BAC clones is presented in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023a). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning and preparation of recombineering templates used for serial recombineering cloning. Basic Protocol 2: Making electrocompetent cells of the bacterial strains used to perform serial recombineering cloning or induction of plasmid copy number. Basic Protocol 3: Serial recombineering cloning to generate both selectable and tagged genomic P[acman] BAC reporter transgenes for selection transgenesis and gene expression analysis in Drosophila melanogaster.

Evaluation of eGFP expression in the ChAT-eGFP transgenic mouse brain

BackgroundA historically definitive marker for cholinergic neurons is choline acetyltransferase (ChAT), a synthesizing enzyme for acetylcholine, (ACh), which can be found in high concentrations in cholinergic neurons, both in the central and peripheral nervous systems. ChAT, is produced in the body of the neuron, transported to the nerve terminal (where its concentration is highest), and catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline, yielding ACh. The creation of bacterial artificial chromosome (BAC) transgenic mice that express promoter-specific fluorescent reporter proteins (green fluorescent protein—[GFP]) provided an enormous advantage for neuroscience. Both in vivo and in vitro experimental methods benefited from the transgenic visualization of cholinergic neurons. Mice were created by adding a BAC clone into the ChAT locus, in which enhanced GFP (eGFP) is inserted into exon 3 at the ChAT initiation codon, robustly and supposedly selectively expressing eGFP in all cholinergic neurons and fibers in the central and peripheral nervous systems as well as in non-neuronal cells.MethodsThis project systematically compared the exact distribution of the ChAT-eGFP expressing neurons in the brain with the expression of ChAT by immunohistochemistry using mapping and also made comparisons with in situ hybridization (ISH).ResultsWe qualitatively described the distribution of ChAT-eGFP neurons in the mouse brain by comparing it with the distribution of immunoreactive neurons and ISH data, paying special attention to areas where the expression did not overlap, such as the cortex, striatum, thalamus and hypothalamus. We found a complete overlap between the transgenic expression of eGFP and the immunohistochemical staining in the areas of the cholinergic basal forebrain. However, in the cortex and hippocampus, we found small neurons that were only labeled with the antibody and not expressed eGFP or vice versa. Most importantly, we found no transgenic expression of eGFP in the lateral dorsal, ventral and dorsomedial tegmental nuclei cholinergic cells.ConclusionWhile the majority of the forebrain ChAT expression was aligned in the transgenic animals with immunohistochemistry, other areas of interest, such as the brainstem should be considered before choosing this particular transgenic mouse line.

Attenuation of equine herpesvirus 1 through deletion of gE gene and its pathological evaluation in murine model.

Equine herpesvirus 1 (EHV1) infection is aglobal health problem in equines and the virus is responsible for abortions, respiratory disease and myeloencephalitis in horses. Disease management requires proper biosecurity and immunoprophylactic measures. Vaccines strengthening both arms of immunity are essential for proper control and there has been acontinuous focus in this area for generation of better vaccines. Here we report construction of bacterial artificial chromosome (BAC) clone of EHV-1 strain Tohana for mutagenesis of the virus and generation of gE gene deletion mutant EHV1. The BAC clone was generated by inserting the mini-F plasmid replacing ORF71 of EHV1 and transforming into E. coli for generation of EHV1-BAC. The infectious virus was regenerated from EHV-1 BAC DNA in RK13 cells. To check utility of EHV1-BAC, we have generated mutant EHV1 by deleting the virulence-associated gE gene. The mutant virus (vToHΔgE) showed significantly reduced plaque size without affecting replication efficiency. Pathological evaluation of lesions in BALB/c mice infected with vToHΔgE revealed reduction in clinical signs and pathology in comparison to the wild-type virus. Generation of infectious BAC of EHV1 and its usage in construction of attenuated viruses shows potential of the technology for development of indigenous modified live vaccine for EHV1. Keywords: quine herpesvirus 1; bacterial artificial chromosome (BAC); mutation; glycoprotein E; vaccine.

Development and applications of chromosome-specific cytogenetic BAC-FISH probes in Larimichthys crocea

Large yellow croaker Larimichthys crocea (Richardson) is an important member in family Sciaenidae, and one of most productive mariculture fishes in China. Fluorescence in situ hybridization is a useful tool for cytogenetics and genomics research. Here, we demonstrated that bacterial artificial chromosome (BAC) clones could be used to identify individual chromosomes in large yellow croaker, and also to study chromosome evolution in the related species. By using BAC paired-end sequencing and sequence alignment, 435 BAC clones were anchored to 24 pseudochromosomes of large yellow croaker genome. Among them, 72 BAC clones with low repeat content were selected and passed PCR test, and then grouped by chromosome for FISH test. As a results, there were 67 BAC clones, 2 to 3 BAC clones per chromosome, generated specific and stable signal at expected position. Then, a dual-color FISH probe cocktail composed of 48 of these BAC clones was formulated and used to hybridize metaphase chromosome spreads, resulting in distinct signal patterns on each chromosome pair, which help to distinguish all chromosomes in the metaphase spreads of large yellow croaker. The chromosome-specific BAC-FISH probes were also applied to a close relative of large yellow croaker, Collichthys lucidus, demonstrating that its Y chromosome originated from the fusion of Chr.1 and Chr.7. Thus, our study provides the first set of chromosome-specific FISH probes in family Sciaenidae, which will play an important role in cytogenetics and genomics research in the family.

BAC-FISH Based Physical Map of Endangered Catfish Clarias magur for Chromosome Cataloguing and Gene Isolation through Positional Cloning.

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.

A New Autosomal Myh11-CreERT2 Smooth Muscle Cell Lineage Tracing and Gene Knockout Mouse Model-Brief Report.

The Myh11 promoter is extensively used as a smooth muscle cell (SMC) Cre-driver and is regarded as the most restrictive and specific promoter available to study SMCs. Unfortunately, in the existing Myh11-CreERT2 mouse, the transgene was inserted on the Y chromosome precluding the study of female mice. Given the importance of including sex as a biological variable and that numerous SMC-based diseases have a sex-dependent bias, the field has been tremendously limited by the lack of a model to study both sexes. Here, we describe a new autosomal Myh11-CreERT2 mouse (referred to as Myh11-CreERT2-RAD), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice. A Myh11-CreERT2-RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome clone RP23-151J22 modified to contain a Cre-ERT2 after the Myh11 start codon. Myh11-CreERT2-RAD mice were crossed with 2 different fluorescent reporter mice and tested for SMC-specific labeling by flow cytometric and immunofluorescence analyses. Myh11-CreERT2-RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreERT2-RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreERT2 mice. Labeling was equivalent in both male and female Cre+ mice and was limited to vascular and visceral SMCs and pericytes in various tissues as assessed by immunofluorescence. We generated and validated the function of an autosomal Myh11-CreERT2-RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.

Development of a Next-Generation Vaccine Platform for Porcine Epidemic Diarrhea Virus Using a Reverse Genetics System.

For the past three decades, the porcine epidemic diarrhea virus (PEDV) has remained an enormous threat to the South Korean swine industry. The scarcity of an effective method for manipulating viral genomes has impeded research progress in PEDV biology and vaccinology. Here, we report the development of reverse genetics systems using two novel infectious full-length cDNA clones of a Korean highly pathogenic-G2b strain, KNU-141112, and its live attenuated vaccine strain, S DEL5/ORF3, in a bacterial artificial chromosome (BAC) under the control of a eukaryotic promoter. Direct transfection of cells with each recombinant BAC clone induced cytopathic effects and produced infectious progeny. The reconstituted viruses, icKNU-141112 and icS DEL5/ORF3, harboring genetic markers, displayed phenotypic and genotypic properties identical to their respective parental viruses. Using the DNA-launched KNU-141112 infectious cDNA clone as a backbone, two types of recombinant viruses were generated. First, we edited the open reading frame 3 (ORF3) gene, as cell-adapted strains lose full-length ORF3, and replaced this region with an enhanced green fluorescent protein (EGFP) gene to generate icPEDV-EGFP. This mutant virus presented parental virus-like growth kinetics and stably retained robust EGFP expression, indicating that ORF3 is dispensable for PEDV replication in cell culture and is a tolerant location for exogeneous gene acceptance. However, the plaque size and syncytia phenotypes of ORF3-null icPEDV-EGFP were larger than those of icKNU-141112 but similar to ORF3-null icS DEL5/ORF3, suggesting a potential role of ORF3 in PEDV cytopathology. Second, we substituted the spike (S) gene with a heterologous S protein, designated S51, from a variant of interest (VOI), which was the most genetically and phylogenetically distant from KNU-141112. The infectious recombinant VOI, named icPEDV-S51, could be recovered, and the rescued virus showed indistinguishable growth characteristics compared to icKNU-141112. Virus cross-neutralization and structural analyses revealed antigenic differences in S between icKNU-141112 and icPEDV-S51, suggesting that genetic and conformational changes mapped within the neutralizing epitopes of S51 could impair the neutralization capacity and cause considerable immune evasion. Collectively, while the established molecular clones afford convenient, versatile platforms for PEDV genome manipulation, allowing for corroborating the molecular basis of viral replication and pathogenesis, they also provide key infrastructural frameworks for developing new vaccines and coronaviral vectors.

A transgenic bacterial artificial chromosome approach to identify regulatory regions that direct Amhr2 and Osterix expression in Müllerian duct mesenchyme.

A transgenic mouse approach using bacterial artificial chromosomes (BAC) was used to identify regulatory regions that direct Müllerian duct expression for Amhr2 and Osterix (Osx, also known as Sp7). Amhr2 encodes the receptor that mediates anti-Müllerian hormone (AMH) signaling for Müllerian duct regression in male embryos. Amhr2 is expressed in the Müllerian duct mesenchyme of both male and female embryos. A ∼147-kb BAC clone containing the Amhr2 locus was used to generate transgenic mice. The transgene was able to rescue the block in Müllerian duct regression of Amhr2-null males, suggesting that the BAC clone contains regulatory sequences active in male embryos. Osx is expressed in the developing skeleton of male and female embryos but is also an AMH-induced gene that is expressed in the Müllerian duct mesenchyme exclusively in male embryos. Osx-Cre transgenic mice were previously generated using a ∼204-kb BAC clone. Crosses of Osx-Cre mice to Cre-dependent lacZ reporter mice resulted in reporter expression in the developing skeleton and in the Müllerian duct mesenchyme of male but not female embryos. Osx-Cherry transgenic mice were previously generated using a 39-kb genomic region surrounding the Osx locus. Osx-Cherry embryos expressed red fluorescence in the developing skeleton and Müllerian duct mesenchyme of male but not female embryos. In addition, female Osx-Cherry embryos ectopically expressing human AMH from an Mt1-AMH transgene activated red fluorescence in the Müllerian duct mesenchyme. These results suggest that the 39-kb region used to generate Osx-Cherry contains male-specific Müllerian duct mesenchyme regulatory sequences that are responsive to AMH signaling. These BAC transgenic mouse approaches identify two distinct regions that direct Müllerian duct mesenchyme expression and contribute fundamental knowledge to define a gene regulatory network for sex differentiation.

Identification of additional fire blight resistance candidate genes following MinION Oxford Nanopore sequencing and assembly of BAC clone spanning the Malus fusca resistance locus

AbstractPyramiding different fire blight resistance genes and QTLs in future apple cultivars is the most eco-friendly way to combat this disease. Identification of strong fire blight resistance donors, and introgression of their resistance into apple breeding material are a continuing effort of breeding programs. Thus, enormous effort is been put into breeding research to understand host – pathogen interactions and mechanisms of resistance found in Malus. The crabapple Malus fusca (accession MAL0045) is highly resistant to fire blight, and although resistance is strain-dependent, resistance of MAL0045 is not overcome by any known strain of Erwinia amylovora to date. A strong fire blight resistance locus (FB_Mfu10) was fine mapped to an interval of 0.33 Centimorgan (cM) on linkage group (LG) 10 of MAL0045 using 1888 progenies. Subsequently, a single bacterial artificial chromosome (BAC) clone (46H22), which harbours FB_Mfu10-resistance alleles, was identified in a MAL0045 BAC library and sequenced using MiSeq illumina leading to the assembly of 45 contigs. Analyses of the sequence of 46H22 led to the identification of a receptor-like kinase candidate gene. Here, we report about resequencing 46H22 using MinION Oxford Nanopore and successfully assembled the sequences into a single contig, which allowed for identifying additional candidate genes.

Identification of sex-specific markers and ZW-chromosome DNA clones from the genomic BAC library of the Chinese mitten crab Eriocheir sinensis

A ZW-ZZ sex determination system was previously proposed based on sex linkage analysis in the Chinese mitten crab, Eriocheir sinensis. In this study, we successfully identified a sex-specific marker from sex-linked markers in the genetic map and verified in different wild populations of E. sinensis. To further identify ZW chromosome DNA, we constructed a highly redundant genomic bacterial artificial chromosome (BAC) library for the Chinese mitten crab. The library was generated from high-molecular weight genomic DNA isolated from hemocytes and muscle in a female crab, which was comprising of 153,984 clones arrayed in 401,384-well microtiter plates. The average inserts size is 72 kb, representing 8.7-fold haploid genome coverage. By using sex specific markers, PCR-based screening was performed to identify ZW-derived BAC clones. Three W and Z-derived BAC clones were isolated, respectively. One of W-derived (P282E18) BAC clones was sequenced and assembled into a 43 kb contig without any gap. The W-derived BAC inserts can be mapped to an assembled contig of E. sinensis genome with 95% similarity in the aligned region, demonstrating that the BAC library is a valuable resource for discriminating Z and W chromosome contigs in the assembled haploid genome. The sex-specific marker provides a powerful tool not only for distinguishing the gender of larval or early juvenile E. sinensis without sexual dimorphism and sex-reversed individuals in monosex crab breeding, but also for identification of ZW-derived BAC clones used in the assembly of Z and W chromosomes in E. sinensis.

Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor.

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

Intact ribosomal DNA arrays of Potentilla origin detected in Erythronium nucleus suggest recent eudicot-to-monocot horizontal transfer.

During our initial phylogenetic study of the monocot genus Erythronium (Liliaceae), we observed peculiar eudicot-type internal transcribed spacer (ITS) sequences in a dataset derived from genomic DNA of Erythronium dens-canis. This raised the possibility of horizontal transfer of a eudicot alien ribosomal DNA (rDNA) into the Erythronium genome. In this work we aimed to support this hypothesis by carrying out genomic, molecular, and cytogenetic analyses. Genome skimming coupled by PacBio HiFi sequencing of a bacterial artificial chromosome clone derived from flow-sorted nuclei was used to characterise the alien 45S rDNA. Integration of alien rDNA in the recipient genome was further proved by Southern blotting and fluorescence in situ hybridization using specific probes. Alien rDNA, nested among Potentilla species in phylogenetic analysis, likely entered the Erythronium lineage in the common ancestor of E. dens-canis and E. caucasicum. Transferred eudicot-type rDNA preserved its tandemly arrayed feature on a single chromosome and was found to be transcribed in the monocot host, albeit much less efficiently than the native counterpart. This study adds a new example to the rarely documented nuclear-to-nuclear jumps of DNA between eudicots and monocots while holding the scientific community continually in suspense about the mode of DNA transfer.

Mining and validation of novel simple sequence repeat (SSR) markers derived from coconut (Cocos nucifera L.) genome assembly

BackgroundIn the past, simple sequence repeat (SSR) marker development in coconut is achieved through microsatellite probing in bacterial artificial chromosome (BAC) clones or using previously developed SSR markers from closely related genomes. These coconut SSRs are publicly available in published literatures and online databases; however, the number is quite limited. Here, we used a locally established, coconut genome-wide SSR prediction bioinformatics pipeline to generate a vast amount of coconut SSR markers. ResultsA total of 7139 novel SSR markers were derived from the genome assembly of coconut ‘Catigan Green Dwarf’ (CATD). A subset of the markers, amounting to 131, were selected for synthesis based on motif filtering, contig distribution, product size exclusion, and success of in silico PCR in the CATD genome assembly. The OligoAnalyzer tool was also employed using the following desired parameters: %GC, 40–60%; minimum ΔG value for hairpin loop, −0.3 kcal/mol; minimum ΔG value for self-dimer, −0.9 kcal/mol; and minimum ΔG value for heterodimer, −0.9 kcal/mol. We have successfully synthesized, optimized, and amplified 131 novel SSR markers in coconut using ‘Catigan Green Dwarf’ (CATD), ‘Laguna Tall’ (LAGT), ‘West African Tall’ (WAT), and SYNVAR (LAGT × WAT) genotypes. Of the 131 SSR markers, 113 were polymorphic among the analyzed coconut genotypes. ConclusionThe development of novel SSR markers for coconut will serve as a valuable resource for mapping of quantitative trait loci (QTLs), assessment of genetic diversity and population structure, hybridity testing, and other marker-assisted plant breeding applications.