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We have previously developed a bacterial artificial chromosome (BAC)‐vectored SARS‐CoV‐2 replicon, namely BAC‐CoV2‐Rep, which, upon transfection into host cells, serves as a transcription template for SARS‐CoV‐2 replicon mRNA to initiate replicon replication and produce nanoluciferase (Nluc) reporter from the subgenomic viral mRNA. However, an inherent issue of such DNA‐launched replicon system is that the nascent full‐length replicon transcript undergoes process by host RNA splicing machinery, which reduces replicon replication and generates spliced mRNA species expressing NLuc reporter independent of replicon replication. To mitigate this problem, we employed Isoginkgetin, a universal eukaryotic host splicing inhibitor, to treat cells transfected with BAC‐CoV2‐Rep. Isoginkgetin effectively increased the level of full‐length replicon transcripts while concurrently reducing the level of Nluc signal derived from spliced replicon mRNA, making the Nluc reporter signal more correlated with replicon replication, as evidenced by treatment with known SARS‐CoV‐2 replication inhibitors including Remdesivir, GC376, and EIDD‐1931. Thus, our study emphasizes that host RNA splicing is a confounding factor for DNA‐launched SARS‐CoV‐2 replicon systems, which can be mitigated by Isoginkgetin treatment. [ABSTRACT FROM AUTHOR]

Background: Reverse genetics systems to rescue viruses from modified DNA are useful tools to investigate the molecular mechanisms of viruses. The COVID‐19 pandemic prompted the development of several reverse genetics systems for SARS‐CoV‐2. The circular polymerase extension reaction (CPER) method enables the rapid generation of recombinant SARS‐CoV‐2; however, such PCR‐based approaches could introduce unwanted mutations due to PCR errors. Methods: To compare the accuracy of CPER and a classic reverse genetics method using bacterial artificial chromosome (BAC), SARS‐CoV‐2 Wuhan/Hu‐1/2019 was generated five times using BAC and five times using CPER. These 10 independent virus stocks were then deep sequencing, and the number of substitutions for which the frequency was greater than 10% was counted. Results: No nucleotide substitutions with a frequency of greater than 10% were observed in all five independent virus stocks generated by the BAC method. In contrast, three to five unwanted nucleotide substitutions with a frequency of more than 10% were detected in four of the five virus stocks generated by the CPER. Furthermore, four substitutions with frequencies greater than 20% were generated in three virus stocks by using the CPER. Conclusions: We found that the accuracy of the CPER method is lower than that of the BAC method. Our findings suggest care should be used when employing the CPER method. [ABSTRACT FROM AUTHOR]

Objective: The mechanistic target of rapamycin (mTOR) kinase is one of the master coordinators of cellular stress responses, regulating metabolism, autophagy, and apoptosis. We recently reported that staufen1 (STAU1), a stress granule (SG) protein, was overabundant in fibroblast cell lines from patients with spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis, frontotemporal degeneration, Huntington's, Alzheimer's, and Parkinson's diseases as well as animal models, and patient tissues. STAU1 overabundance is associated with mTOR hyperactivation and links SG formation with autophagy. Our objective was to determine the mechanism of mTOR regulation by STAU1. Methods: We determined STAU1 abundance with disease‐ and chemical‐induced cellular stressors in patient cells and animal models. We also used RNA‐binding assays to contextualize STAU1 interaction with MTOR mRNA. Results: STAU1 and mTOR were overabundant in bacterial artificial chromosome (BAC)‐C9ORF72, ATXN2Q127, and Thy1‐TDP‐43 transgenic mouse models. Reducing STAU1 levels in these mice normalized mTOR levels and activity and autophagy‐related marker proteins. We also saw increased STAU1 levels in HEK293 cells transfected to express C9ORF72‐relevant dipeptide repeats (DPRs). Conversely, DPR accumulations were not observed in cells treated by STAU1 RNA interference (RNAi). Overexpression of STAU1 in HEK293 cells increased mTOR levels through direct MTOR mRNA interaction, activating downstream targets and impairing autophagic flux. Targeting mTOR by rapamycin or RNAi normalized STAU1 abundance in an SCA2 cellular model. Interpretation: STAU1 interaction with mTOR drives its hyperactivation and inhibits autophagic flux in multiple models of neurodegeneration. Staufen, therefore, constitutes a novel target to modulate mTOR activity and autophagy, and for the treatment of neurodegenerative diseases. ANN NEUROL 2023;93:398–416 [ABSTRACT FROM AUTHOR]

Endometrial cancer (EC) is the most common gynaecological malignancy with increasing incidence in developed countries. As gold standard, hysteroscopy confirms only 30% of suspected ECs. The detection of EC cells in the vagina by fluorescence in situ hybridization (FISH) after a smear test could reduce invasive procedures in the future. Using array‐based comparative genome hybridization (aCGH) on 65 endometrial carcinomas, most frequently imbalanced regions of the tumour genome were identified. Bacterial artificial chromosomes were used to generate FISH‐probes homologue to these human regions. The FISH test was hybridized on swabs specimens collected from the vaginal cavity. Samples from six patients without EC were selected as a negative control and on 13 patients with known EC as a positive control. To distinguish between benign and EC cases, the cut‐off value has been defined. A first validation of this EC‐FISH Test was performed with swabs from 41 patients with suspected EC. The most common genomic imbalances in EC are around the CTNNB1, FBXW7 and APC genes. The cut‐off is defined at 32% of analysed cells without diploid signal pattern. This differs significantly between the positive and negative controls (p < 0.001). In a first validation cohort of 41 patients with suspected EC, the EC‐FISH Test distinguishes patients with and without EC with a sensitivity of 91% and a specificity of 83%. The negative predictive value is 96%. This is the first report of a non‐invasive EC‐FISH Test to predict EC in women with suspected EC. [ABSTRACT FROM AUTHOR]

Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock‐in to the Dopachrome tautomerase locus or knock‐in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte‐specific expression of CreERT2, nuclear localised H2B‐Cerulean and membrane localised marcks‐mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R‐EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4‐OHT. The fluorescent proteins H2B‐Cerulean and marcks‐mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: Wheat stem (or black) rust, caused by Puccinia graminis f. sp. tritici (Pgt), has been historically among the most devastating global fungal diseases of wheat. The recent occurrence and spread of new virulent races such as Ug99 have prompted global efforts to identify and isolate more effective stem rust resistance (Sr) genes. Here, we report the map‐based cloning of the Ug99‐effective SrTm5 gene from diploid wheat Triticum monococcum accession PI 306540 that encodes a typical coiled‐coil nucleotide‐binding leucine‐rich repeat protein. This gene, designated as Sr22b, is a new allele of Sr22 with a rare insertion of a large (13.8‐kb) retrotransposon into its second intron. Biolistic transformation of an ~112‐kb circular bacterial artificial chromosome plasmid carrying Sr22b into the susceptible wheat variety Fielder was sufficient to confer resistance to stem rust. In a survey of 168 wheat genotypes, Sr22b was present only in cultivated T. monococcum subsp. monococcum accessions but absent in all tested tetraploid and hexaploid wheat lines. We developed a diagnostic molecular marker for Sr22b and successfully introgressed a T. monococcum chromosome segment containing this gene into hexaploid wheat to accelerate its deployment and pyramiding with other Sr genes in wheat breeding programmes. Sr22b can be a valuable component of gene pyramids or transgenic cassettes combining different resistance genes to control this devastating disease. [ABSTRACT FROM AUTHOR]

Background: This study aimed to delineate the clinical phenotype of patients with 9p deletions, pinpoint the chromosomal breakpoints, and identify the critical region for trigonocephaly, which is a frequent finding in 9p terminal deletion. Methods: We investigated a cohort of nine patients with chromosome 9p terminal deletions who all displayed developmental delay, intellectual disability, hypotonia, and dysmorphic features. Of them, eight had trigonocephaly, seven had brain anomalies, seven had autistic manifestations, seven had fair hair, and six had a congenital heart defect (CHD). Results: Karyotyping revealed 9p terminal deletion in all patients, and patients 8 and 9 had additional duplication of other chromosomal segments. We used six bacterial artificial chromosome (BAC) clones that could identify the breakpoints at 17–20 Mb from the 9p terminus. Array CGH identified the precise extent of the deletion in six patients; the deleted regions ranged from 16 to 18.8 Mb in four patients, patient 8 had an 11.58 Mb deletion and patient 9 had a 2.3 Mb deletion. Conclusion: The gene deletion in the 9p24 region was insufficient to cause ambiguous genitalia because six of the nine patients had normal genitalia. We suggest that the critical region for trigonocephaly lies between 11,575 and 11,587 Mb from the chromosome 9p terminus. To the best of our knowledge, this is the minimal critical region reported for trigonocephaly in 9p deletion syndrome, and it warrants further delineation. [ABSTRACT FROM AUTHOR]

With the advent of chromatin‐interaction maps, chromosome‐level genome assemblies have become a reality for a wide range of organisms. Scaffolding quality is, however, difficult to judge. To explore this gap, we generated multiple chromosome‐scale genome assemblies of an emerging wild animal model for carcinogenesis, the California sea lion (Zalophus californianus). Short‐read assemblies were scaffolded with two independent chromatin interaction mapping data sets (Hi‐C and Chicago), and long‐read assemblies with three data types (Hi‐C, optical maps and 10X linked reads) following the "Vertebrate Genomes Project (VGP)" pipeline. In both approaches, 18 major scaffolds recovered the karyotype (2n = 36), with scaffold N50s of 138 and 147 Mb, respectively. Synteny relationships at the chromosome level with other pinniped genomes (2n = 32–36), ferret (2n = 34), red panda (2n = 36) and domestic dog (2n = 78) were consistent across approaches and recovered known fissions and fusions. Comparative chromosome painting and multicolour chromosome tiling with a panel of 264 genome‐integrated single‐locus canine bacterial artificial chromosome probes provided independent evaluation of genome organization. Broad‐scale discrepancies between the approaches were observed within chromosomes, most commonly in translocations centred around centromeres and telomeres, which were better resolved in the VGP assembly. Genomic and cytological approaches agreed on near‐perfect synteny of the X chromosome, and in combination allowed detailed investigation of autosomal rearrangements between dog and sea lion. This study presents high‐quality genomes of an emerging cancer model and highlights that even highly fragmented short‐read assemblies scaffolded with Hi‐C can yield reliable chromosome‐level scaffolds suitable for comparative genomic analyses. [ABSTRACT FROM AUTHOR]

Summary: Intra‐specific variability is a cornerstone of evolutionary success of species. Acquiring genetic material from distant sources is an important adaptive mechanism in bacteria, but it can also play a role in eukaryotes. In this paper, we investigate the nature and evolution of a chromosomal segment of panicoid (Poaceae, Panicoideae) origin occurring in the nuclear genomes of species of the barley genus Hordeum (Pooideae). The segment, spanning over 440 kb in the Asian Hordeumbogdanii and 219 kb in the South American Hordeumpubiflorum, resides on a pair of nucleolar organizer region (NOR)‐bearing chromosomes. Conserved synteny and micro‐collinearity of the segment in both species indicate a common origin of the segment, which was acquired before the split of the respective barley lineages 5–1.7 million years ago. A major part of the foreign DNA consists of several approximately 68 kb long repeated blocks containing five stress‐related protein‐coding genes and transposable elements (TEs). Whereas outside these repeats, the locus was invaded by multiple TEs from the host genome, the repeated blocks are rather intact and appear to be preserved. The protein‐coding genes remained partly functional, as indicated by conserved reading frames, a low amount of non‐synonymous mutations, and expression of mRNA. A screen across Hordeum species targeting the panicoid protein‐coding genes revealed the presence of the genes in all species of the section Stenostachys. In summary, our study shows that grass genomes can contain large genomic segments obtained from distantly related species. These segments usually remain undetected, but they may play an important role in the evolution and adaptation of species. [ABSTRACT FROM AUTHOR]

SUMMARY: Self‐incompatibility in Petunia is controlled by the polymorphic S‐locus, which contains S‐RNase encoding the pistil determinant and 16–20 S‐locus F‐box (SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S2‐haplotype of the S‐locus in Petunia inflata using bacterial artificial chromosome clones collectively containing all 17 SLF genes, SLFLike1, and S‐RNase. Two SLF pseudogenes and 28 potential protein‐coding genes were identified, 20 of which were also found at the S‐loci of both the S6a‐haplotype of P. inflata and the SN‐haplotype of self‐compatible Petunia axillaris, but not in the S‐locus remnants of self‐compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses of S‐locus sequences of these three S‐haplotypes revealed potential genetic exchange in the flanking regions of SLF genes, resulting in highly similar flanking regions between different types of SLF and between alleles of the same type of SLF of different S‐haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at the S‐loci of all three S‐haplotypes and in the flanking regions of all S‐locus genes examined. We also found evidence of the association of transposable elements with SLF pseudogenes. Based on the hypothesis that SLF genes were derived by retrotransposition, we identified 10 F‐box genes as putative SLF parent genes. Our results shed light on the importance of non‐coding sequences in the evolution of the S‐locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLF genes. Significance Statement: Sequencing the Petunia S‐locus region of the S2‐haplotype containing 17 S‐locus F‐box (SLF) genes, SLFLike1, and the S‐RNase gene allows comparative analyses of the approximately 3.1 Mb assembled sequence with the S‐locus sequences of other S‐haplotypes of Petunia and S‐locus remnants of several self‐compatible solanaceous species. The results from comparison in both the coding and non‐coding flanking sequences of SLF genes shed light on the genomic organization and evolution of the S‐locus in Petunia. [ABSTRACT FROM AUTHOR]

Human herpesvirus 6A (HHV‐6A) is a member of the genus Roseolovirus and the subfamily Betaherpesvirinae. It is similar to and human cytomegalovirus (HCMV). HHV‐6A encodes a 41 kDa nuclear phosphoprotein, U27, which acts as a processivity factor in the replication of the viral DNA. HHV‐6A U27 has 43% amino acid sequence homology with HCMV UL44, which is important for DNA replication. A previous study on HHV‐6A U27 revealed that it greatly increases the in vitro DNA synthesis activity of HHV‐6A DNA polymerase. However, the role of U27 during the HHV‐6A virus replication process remains unclear. In this study, we constructed a U27‐deficient HHV‐6A mutant (HHV‐6ABACU27mut) with a frameshift insertion at the U27 gene using an HHV‐6A bacterial artificial chromosome (BAC) system. Viral reconstitution from the mutant BAC DNA was not detected, in contrast to the wild type and the revertant from the U27 mutant. This suggests that U27 plays a critical role in the life cycle of HHV‐6A. [ABSTRACT FROM AUTHOR]

Keywords: familial thrombocytosis; thrombopoietin; splicing EN familial thrombocytosis thrombopoietin splicing e104 e107 4 07/20/20 20200715 NES 200715 Familial thrombocytosis can be caused by germline mutations in I THPO i (encoding thrombopoietin), I MPL i (encoding the thrombopoietin receptor) or I JAK2 i (Janus kinase 2) genes.1-3 Several different mutations described in the I THPO i gene cause thrombocytosis through increased production of thrombopoietin (TPO), the primary megakaryocyte growth factor.4-7 Typically, these variants affect the 5'-untranslated region (5'-UTR) of the I THPO i mRNA and result in increased I THPO i mRNA translation efficiency. However, mutations changing the last guanine of the exon for adenine have been shown to induce exon splicing in several genes.12,13 GLO:1XW/15jul20:bjh16742-fig-0002.jpg PHOTO (COLOR): 2 Splicing reporter minigene assay. [Extracted from the article]

Histamine is a bioactive monoamine that is synthesized by the enzymatic activity of histidine decarboxylase (HDC) in basophils, mast cells, gastric enterochromaffin‐like (ECL) cells and histaminergic neuronal cells. Upon a series of cellular stimuli, these cells release stored histamine, which elicits allergies, inflammation, and gastric acid secretion and regulates neuronal activity. Recent studies have shown that certain other types of myeloid lineage cells also produce histamine with HDC induction under various pathogenic stimuli. Histamine has been shown to play a series of pathophysiological roles by modulating immune and inflammatory responses in a number of disease conditions, whereas the mechanistic aspects underlying induced HDC expression remain elusive. In the present review, we summarize the current understanding of the regulatory mechanism of Hdc gene expression and the roles played by histamine in physiological contexts as well as pathogenic processes. We also introduce a newly developed histaminergic cell‐monitoring transgenic mouse line (Hdc‐BAC‐GFP) that serves as a valuable experimental tool to identify the source of histamine and dissect upstream regulatory signals. [ABSTRACT FROM AUTHOR]

Summary: The papaya diminutive mutant exhibits miniature stature, retarded growth and reduced fertility. This undesirable mutation appeared in the variety 'Sunset', the progenitor of the transgenic line 'SunUp', and was accidentally carried forward into breeding populations.The diminutive mutation was mapped to chromosome 2 and fine mapped to scaffold 25. Sequencing of a bacterial artificial chromosome in the fine mapped region led to the identification of the target gene responsible for the diminutive mutant, a gene orthologous to MMS19 with a 36.8 kb deletion co‐segregating with the diminutive mutant.The genomic sequence of CpMMS19 is 62 kb, consisting of 20 exons and 19 introns. It encodes a protein of 1143 amino acids while the diminutive allele encodes a truncated protein of 287 amino acids. Expression of the full‐length CpMMS19 was able to complement the thermosensitive growth of the yeast mms19 deletion mutant while expression of the diminutive allele resulted in increased thermosensitivity. Over‐expression of the diminutive allele in Arabidopsis met18 mutant results in a high frequency of seed abortion.The papaya diminutive phenotype is caused by an alteration in gene function rather than a loss‐of‐function mutation. SCAR (sequence characterized amplified region) markers were developed for rapid detection of the diminutive allele in breeding populations. [ABSTRACT FROM AUTHOR]

Summary: Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication‐related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement. [ABSTRACT FROM AUTHOR]

Summary: Traditional approaches for sequencing insertion ends of bacterial artificial chromosome (BAC) libraries are laborious and expensive, which are currently some of the bottlenecks limiting a better understanding of the genomic features of auto‐ or allopolyploid species. Here, we developed a highly efficient and low‐cost BAC end analysis protocol, named BAC‐anchor, to identify paired‐end reads containing large internal gaps. Our approach mainly focused on the identification of high‐throughput sequencing reads carrying restriction enzyme cutting sites and searching for large internal gaps based on the mapping locations of both ends of the reads. We sequenced and analysed eight libraries containing over 3 200 000 BAC end clones derived from the BAC library of the tetraploid potato cultivar C88 digested with two restriction enzymes, Cla I and Mlu I. About 25% of the BAC end reads carrying cutting sites generated a 60–100 kb internal gap in the potato DM reference genome, which was consistent with the mapping results of Sanger sequencing of the BAC end clones and indicated large differences between autotetraploid and haploid genotypes in potato. A total of 5341 Cla I‐ and 165 Mlu I‐derived unique reads were distributed on different chromosomes of the DM reference genome and could be used to establish a physical map of target regions and assemble the C88 genome. The reads that matched different chromosomes are especially significant for the further assembly of complex polyploid genomes. Our study provides an example of analysing high‐coverage BAC end libraries with low sequencing cost and is a resource for further genome sequencing studies. [ABSTRACT FROM AUTHOR]

Summary: To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site‐directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR‐amplified DNA fragments. Application of fast‐track recombineering is illustrated by the simultaneous labelling of CYCLIN‐DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD‐activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18‐StrepII‐HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T‐DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co‐localization and co‐immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA‐replication‐coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast‐track recombineering is well suited to promote a wide range of protein interaction and proteomics studies. Significance Statement: A PCR‐based BAC‐recombineering protocol accelerating site‐specific modification of plant genes with suitable tags is described and exploited for purification, cellular localization, co‐immunoprecipitation, and mass spectrometry analysis of CDKD;2 and CYCLIN H associated components of TFIIH general transcription factor. [ABSTRACT FROM AUTHOR]

Neurotoxicology is hampered by the inability to predict regional and cellular targets of toxicant‐induced damage. Evaluating astrogliosis overcomes this problem because reactive astrocytes highlight the location of toxicant‐induced damage. While enhanced expression of glial fibrillary acidic protein is a hallmark of astrogliosis, few other biomarkers have been identified. However, bacterial artificial chromosome ‐ translating ribosome affinity purification (bacTRAP) technology allows for characterization of the actively translating transcriptome of a particular cell type; use of this technology in aldehyde dehydrogenase 1 family member L1 (ALDH1L1) bacTRAP mice can identify genes selectively expressed in astrocytes. The aim of this study was to characterize additional biomarkers of neurotoxicity‐induced astrogliosis using ALDH1L1 bacTRAP mice. The known dopaminergic neurotoxicant 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP; 12.5 mg/kg s.c.) was used to induce astrogliosis. Striatal tissue was obtained 12, 24, and 48 h following exposure for the isolation of actively translating RNA. Subsequently, MPTP‐induced changes in this RNA pool were analyzed by microarray and 184 statistically significant, differentially expressed genes were identified. The dataset was interrogated by gene ontology, pathway, and co‐expression network analyses, which identified novel genes, as well as those with known immune and inflammatory functions. Using these analyses, we were directed to several genes associated with reactive astrocytes. Of these, TIMP1 and miR‐147 were identified as candidate biomarkers because of their robust increased expression following both MPTP and trimethyl tin exposures. Thus, we have demonstrated that bacTRAP can be used to identify new biomarkers of astrogliosis and aid in the characterization of astrocyte phenotypes induced by toxicant exposures. Open Science Badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14518. [ABSTRACT FROM AUTHOR]

Early Huntington disease is characterized by involuntary motor symptoms, especially chorea, due to dominance of direct pathway Striatal Projection Neurons (SPN) which facilitate movement. AMPA receptors are inserted into the membrane during increases in synaptic strength mediated by PKM by preventing endocytosis, but a substrate of PKM involved in this has not been identified. Amyloid beta 25-35 impairs docosahexaenoic acid efflux by down-regulating fatty acid transpor. [Extracted from the article]

Nicotine dependence and schizophrenia are two mental health disorders with remarkably high comorbidity. Cigarette smoking is particularly prevalent amongst schizophrenic patients and it is hypothesised to comprise a form of self‐medication for relieving cognitive deficits in these patients. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin in the modulation of drug addiction, as well as schizophrenia symptomology; however, the underlying mechanism remains unclear. Therefore, we sought to investigate the effects of chronic nicotine administration on oxytocin receptor (OTR) binding in the brain of a transgenic mouse model of schizophrenia that carries a bacterial artificial chromosome of the human G72/G30 locus (G72Tg). Female wild‐type (WT) and heterozygous G72 transgenic CD‐1 mice were treated with a chronic nicotine regimen (24 mg/kg/day, osmotic minipumps for 14 days) and quantitative autoradiographic mapping of oxytocin receptors was carried out in brains of these animals. OTR binding levels were higher in the cingulate cortex (CgCx), nucleus accumbens (Acb), and central amygdala (CeA) of saline treated G72Tg mice compared to WT control mice. Chronic nicotine administration reversed this upregulation in the CgCx and CeA. Interestingly, chronic nicotine administration induced an increase in OTR binding in the CeA of solely WT mice. These results indicate that nicotine administration normalises the dysregulated central oxytocinergic system of this mouse model of schizophrenia and may contribute towards nicotine's ability to modulate cognitive deficits which are common symptoms of schizophrenia. [ABSTRACT FROM AUTHOR]

Karyotype evolution in one of the most diverse and species-rich group of insects, moths and butterflies (Lepidoptera), has interesting features that remain to be resolved. Recent studies showed that fluorescence in situ hybridization using bacterial artificial chromosome clones (BAC-FISH) is an efficient cytogenetic method for identification and gene mapping of lepidopteran chromosomes. Using comparative mapping by BAC-FISH, extensive synteny of genes was revealed between chromosomes of different lepidopteran species based on Bombyx mori genomic information. However, this comparative mapping has been done only in representatives of advanced groups of Lepidoptera. Here we constructed a BAC library of Endoclita excrescens, which belongs to the primitive lepidopteran family Hepialidae. High molecular weight DNA for the library construction was prepared from the pupae by using a rapid nuclear isolation method known in plants. The BAC clones of E. excrescens contain 66.6 kb inserts on average. The successful application of BAC-FISH showed that the BAC library of E. excrescens is a useful tool for comparative gene mapping on chromosomes of this species. [ABSTRACT FROM AUTHOR]

Elf5 is a transcription factor known to regulate critical developmental processes and has been shown to act as a tumour suppressor in multiple cancers. Elf5 knockout mice are embryonically lethal, limiting in vivo studies pertaining to its function. Moreover, haploinsufficiency of Elf5 limits the use of current mouse models to investigate adult tissue distribution of Elf5. Here, we successfully generated Elf5CreERT2‐GFP bacterial artificial chromosome (BAC) transgenic mice and show that Elf5+ cells are present in several adult tissues, where its expression was previously not known. Our study demonstrates the unique distribution of Elf5+ cells in multiple adult organs, which will facilitate future studies investigating the function of Elf5 in these tissues during homeostasis, repair and cancer. [ABSTRACT FROM AUTHOR]

Tumor predisposition in an individual with chromosomal rearrangements of 1q31.2-q41 encompassing cell division cycle protein 73 Hyperparathyroidism-jaw tumor (HPT-JT) is caused by a loss-of-function mutation or haploinsufficiency of cell division cycle protein 73 ( I CDC73 i ), a tumor-suppressor gene in chromosome 1q31.2. I CDC73 i mutation leads to parathyroid tumor development, parathyroid carcinoma, and mandible/maxilla ossifying fibroma in 90%, 15%, and 25% cases, respectively; approximately 75% female patients develop benign or malignant uterine tumors. [Extracted from the article]

ABSTRACT Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar ( Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts. [ABSTRACT FROM AUTHOR]

Furanocoumarins are specialized metabolites that are involved in the defense of plants against phytophagous insects. The molecular and functional characterization of the genes involved in their biosynthetic pathway is only partially complete. Many recent reports have described gene clusters responsible for the biosynthesis of specialized metabolites in plants. To investigate possible co-localization of the genes involved in the furanocoumarin pathway, we sequenced parsnip BAC clones spanning two different gene loci. We found that two genes previously identified in this pathway, CYP71 AJ3 and CYP71 AJ4, were located on the same BAC, whereas a third gene, Ps PT1, belonged to a different BAC clone. Chromosome mapping using fluorescence in situ hybridization ( FISH) indicated that Ps PT1 and the CYP71 AJ3- CYP71 AJ4 clusters are located on two different chromosomes. Sequencing the BAC clone harboring Ps PT1 led to the identification of a gene encoding an Fe( II) α-ketoglutarate-dependent dioxygenase (Ps DIOX) situated in the neighborhood of Ps PT1 and confirmed the occurrence of a second gene cluster involved in the furanocoumarin pathway. This enzyme metabolizes p-coumaroyl CoA, leading exclusively to the synthesis of umbelliferone, an important intermediate compound in furanocoumarin synthesis. This work provides an insight into the genomic organization of genes from the furanocoumarin biosynthesis pathway organized in more than one gene cluster. It also confirms that the screening of a genomic library and the sequencing of BAC clones represent a valuable tool to identify genes involved in biosynthetic pathways dedicated to specialized metabolite synthesis. [ABSTRACT FROM AUTHOR]

Cowpea ( Vigna unguiculata L. Walp.) is a legume crop that is resilient to hot and drought-prone climates, and a primary source of protein in sub-Saharan Africa and other parts of the developing world. However, genome resources for cowpea have lagged behind most other major crops. Here we describe foundational genome resources and their application to the analysis of germplasm currently in use in West African breeding programs. Resources developed from the African cultivar IT97K-499-35 include a whole-genome shotgun ( WGS) assembly, a bacterial artificial chromosome ( BAC) physical map, and assembled sequences from 4355 BACs. These resources and WGS sequences of an additional 36 diverse cowpea accessions supported the development of a genotyping assay for 51 128 SNPs, which was then applied to five bi-parental RIL populations to produce a consensus genetic map containing 37 372 SNPs. This genetic map enabled the anchoring of 100 Mb of WGS and 420 Mb of BAC sequences, an exploration of genetic diversity along each linkage group, and clarification of macrosynteny between cowpea and common bean. The SNP assay enabled a diversity analysis of materials from West African breeding programs. Two major subpopulations exist within those materials, one of which has significant parentage from South and East Africa and more diversity. There are genomic regions of high differentiation between subpopulations, one of which coincides with a cluster of nodulin genes. The new resources and knowledge help to define goals and accelerate the breeding of improved varieties to address food security issues related to limited-input small-holder farming and climate stress. [ABSTRACT FROM AUTHOR]

Background: Hyperimmunoglobulins are frequently applied for prophylaxis and treatment of human cytomegalovirus (HCMV) infections but were only marginally effective in meta-analyses of clinical studies. This might be partially due to selection of donors rather for total anti-HCMV titers than for neutralizing capacities. To improve efficacy against HCMV infection, we aimed at developing a high-throughput screening method for identification of blood donors with highly and broadly neutralizing capacities.Study Design and Methods: Using a Gaussia luciferase-expressing reporter virus, 1000 HCMV immunoglobulin (Ig)G-positive plasma samples with known anti-HCMV immunoglobulin titers were analyzed regarding their neutralization titers against fibroblast and endothelial cell infection. Based on these results, a high-throughput screening was designed. Highly neutralizing plasma samples were further tested 1) by an enzyme-linked immunosorbent assay-based neutralization assay regarding efficiency against different HCMV strains and 2) for their efficiency compared to commercially available hyperimmunoglobulins.Results: Total anti-HCMV immunoglobulin titers did not correlate with neutralization. Mean neutralization capacities were 15-fold higher in endothelial cells compared to fibroblasts. All plasma samples neutralizing fibroblast infection were at least equally effective against infection of endothelial cells, providing the possibility to simplify our screening method by testing only fibroblasts as target cells with a plasma dilution of 1 in 400. Of the nine tested top HCMV neutralizers, four were broadly effective against different HCMV strains. All nine were significantly superior to hyperimmunoglobulins.Conclusion: Donors with highly and broadly neutralizing capacities can be identified by a two-step high-throughput screening approach. This may provide a basis for improved antibody-based treatment or prophylaxis of HCMV infections. [ABSTRACT FROM AUTHOR]

Contents771I.771II.772III.780IV.781V.786786References786 Summary: Plant natural products are of great value for agriculture, medicine and a wide range of other industrial applications. The discovery of new plant natural product pathways is currently being revolutionized by two key developments. First, breakthroughs in sequencing technology and reduced cost of sequencing are accelerating the ability to find enzymes and pathways for the biosynthesis of new natural products by identifying the underlying genes. Second, there are now multiple examples in which the genes encoding certain natural product pathways have been found to be grouped together in biosynthetic gene clusters within plant genomes. These advances are now making it possible to develop strategies for systematically mining multiple plant genomes for the discovery of new enzymes, pathways and chemistries. Increased knowledge of the features of plant metabolic gene clusters – architecture, regulation and assembly – will be instrumental in expediting natural product discovery. This review summarizes progress in this area. [ABSTRACT FROM AUTHOR]

Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes ( BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs ( Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path. [ABSTRACT FROM AUTHOR]

• Duckweeds are aquatic monocotyledonous plants of potential economic interest with fast vegetative propagation, comprising 37 species with variable genome sizes (0.158-1.88 Gbp). The genomic sequence of Spirodela polyrhiza, the smallest and the most ancient duckweed genome, needs to be aligned to its chromosomes as a reference and prerequisite to study the genome and karyotype evolution of other duckweed species. • We selected physically mapped bacterial artificial chromosomes (BACs) containing Spirodela DNA inserts with little or no repetitive elements as probes for multicolor fluorescence in situ hybridization (mcFISH), using an optimized BAC pooling strategy, to validate its physical map and correlate it with its chromosome complement. • By consecutive mcFISH analyses, we assigned the originally assembled 32 pseudomolecules (supercontigs) of the genomic sequences to the 20 chromosomes of S. polyrhiza. A Spirodela cytogenetic map containing 96 BAC markers with an average distance of 0.89 Mbp was constructed. Using a cocktail of 41 BACs in three colors, all chromosome pairs could be individualized simultaneously. • Seven ancestral blocks emerged from duplicated chromosome segments of 19 Spirodela chromosomes. The chromosomally integrated genome of S. polyrhiza and the established prerequisites for comparative chromosome painting enable future studies on the chromosome homoeology and karyotype evolution of duckweed species. [ABSTRACT FROM AUTHOR]

Barley ( Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome ( BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software Harv EST:Barley provides facile access to BAC sequences and their annotations, along with the barley- Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant. [ABSTRACT FROM AUTHOR]

ABSTRACT Human herpesvirus 6 (HHV-6) is a T-lymphotrophic virus belongs to the genus Roseolovirus within the beta herpesvirus subfamily. The U20-U24 gene cluster is unique to Roseoloviruses; however, both their function and whether they are essential for virus growth is unknown. Recently, bacterial artificial chromosome (BAC) techniques have been used to investigate HHV-6A. This study describes generation of a virus genome lacking U21-U24 (HHV-6ABACΔU21-24) and shows that infectious virus particles can be reconstituted from this BAC DNA. Our data indicate that the HHV-6 U21-U24 gene cluster is dispensable for virus propagation. [ABSTRACT FROM AUTHOR]

Background Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. Methods The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. Results The porcine B4 GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4 GALNT2 genes, respectively. The B4 GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4 GALNT2 in human HEK cells ( HEK-B4T) results in increased binding of antibody to the B4 GALNT2 enzyme, and increased reactivity with anti-Sda and DBA. HEK-B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO: CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells. Conclusion The functional isolation of the porcine B4 GALNT2 gene from a PAEC expression library, the pattern of B4 GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4 GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response detected after pig-to-baboon cardiac xenotransplantation. [ABSTRACT FROM AUTHOR]

Bread wheat ( Triticum aestivum L.) is the most important staple food crop for 35% of the world's population. International efforts are underway to facilitate an increase in wheat production, of which the International Wheat Genome Sequencing Consortium ( IWGSC) plays an important role. As part of this effort, we have developed a sequence-based physical map of wheat chromosome 6A using whole-genome profiling ( WGP™). The bacterial artificial chromosome ( BAC) contig assembly tools fingerprinted contig ( fpc) and linear topological contig ( ltc) were used and their contig assemblies were compared. A detailed investigation of the contigs structure revealed that ltc created a highly robust assembly compared with those formed by fpc. The ltc assemblies contained 1217 contigs for the short arm and 1113 contigs for the long arm, with an L50 of 1 Mb. To facilitate in silico anchoring, WGP™ tags underlying BAC contigs were extended by wheat and wheat progenitor genome sequence information. Sequence data were used for in silico anchoring against genetic markers with known sequences, of which almost 79% of the physical map could be anchored. Moreover, the assigned sequence information led to the 'decoration' of the respective physical map with 3359 anchored genes. Thus, this robust and genetically anchored physical map will serve as a framework for the sequencing of wheat chromosome 6A, and is of immediate use for map-based isolation of agronomically important genes/quantitative trait loci located on this chromosome. [ABSTRACT FROM AUTHOR]

A complete and high-quality genome reference sequence of an organism provides a solid foundation for a wide research community and determines the outcomes of relevant genomic, genetic, molecular and evolutionary research. Rice is an important food crop and a model plant for grasses, and therefore was the first chosen crop plant for whole genome sequencing. The genome of the japonica representative rice variety, Nipponbare, was sequenced using a gold standard, map-based clone-by-clone strategy. However, although the Nipponbare reference sequence ( RefSeq) has the best quality for existing crop genome sequences, it still contains many assembly errors and gaps. To improve the Nipponbare RefSeq, first a robust method is required to detect the hidden assembly errors. Through alignments between BAC-end sequences ( BESs) embedded in the Nipponbare bacterial artificial chromosome ( BAC) physical map and the Nipponbare RefSeq, we detected locations on the Nipponbare RefSeq that were inversely matched with BESs and could therefore be candidates for spurious inversions of assembly. We performed further analysis of five potential locations and confirmed assembly errors at those locations; four of them, two on chr4 and two on chr11 of the Nipponbare RefSeq ( IRGSP build 5), were found to be caused by reverse repetitive sequences flanking the locations. Our approach is effective in detecting spurious inversions in the Nipponbare RefSeq and can be applied for improving the sequence qualities of other genomes as well. [ABSTRACT FROM AUTHOR]

Some transposable elements ( TEs) show extraordinary variance in abundance along sex chromosomes but the mechanisms responsible for this variance are unknown. Here, we studied Ogre long terminal repeat ( LTR) retrotransposons in Silene latifolia, a dioecious plant with evolutionarily young heteromorphic sex chromosomes. Ogre elements are ubiquitous in the S. latifolia genome but surprisingly absent on the Y chromosome., Bacterial artificial chromosome (BAC) library analysis and fluorescence in situ hybridization (FISH) were used to determine Ogre structure and chromosomal localization. Next generation sequencing ( NGS) data were analysed to assess the transcription level and abundance of small RNAs. Methylation of Ogres was determined by bisulphite sequencing. Phylogenetic analysis was used to determine mobilization time and selection forces acting on Ogre elements., We characterized three Ogre families ubiquitous in the S. latifolia genome. One family is nearly absent on the Y chromosome despite all the families having similar structures and spreading mechanisms. We showed that Ogre retrotransposons evolved before sex chromosomes appeared but were mobilized after formation of the Y chromosome. Our data suggest that the absence of one Ogre family on the Y chromosome may be caused by 24-nucleotide (24-nt) small RNA-mediated silencing leading to female-specific spreading., Our findings highlight epigenetic silencing mechanisms as potentially crucial factors in sex-specific spreading of some TEs, but other possible mechanisms are also discussed. [ABSTRACT FROM AUTHOR]

The fragment contains protein-coding genes, ribosomal DNA genes (rDNA) and transposable elements (TEs). Whereas the lineages of pooid and panicoid grasses split about 50 million years ago, the Panicum-like DNA fragment in Hordeum was obtained by horizontal gene transfer between 2.9 and 1.7 million years ago. Recently, they established that the foreign DNA is indeed present in wild barley species, and was transferred by horizontal gene transfer (Mahelka I et al i ., 2017). [Extracted from the article]

Comparative analysis using available genomic resources within closely related species is an effective way to investigate genomic sequence and structural diversity. Rice ( Oryza sativa L.) has undergone significant physiological and morphological changes during its domestication and local adaptation. We present a complete bacterial artificial chromosome ( BAC) physical map for the aus rice cultivar ' Kasalath', which covers 90% of the sequence of temperate japonica rice cultivar ' Nipponbare'. Examination of physical distances between computational and experimental measurements of ' Kasalath' BAC insert size revealed the presence of more than 500 genomic regions that appear to have significant chromosome structural changes between the two cultivars. In particular, a genomic region on the long arm of ' Kasalath' chromosome 11 carrying a disease-resistance gene cluster was greatly expanded relative to the 'Nipponbare' genome. We also decoded 41.37 Mb of high-quality genomic sequence from 'Kasalath' chromosome 1. Extensive comparisons of chromosome 1 between 'Kasalath' and 'Nipponbare' led to the discovery of 317 843 single-nucleotide polymorphisms ( SNPs) and 66 331 insertion/deletion (indel) sites. Nearly two-thirds of the expressed genes on rice chromosome 1 carried natural variations involving SNPs and/or indels that resulted in substitutions, insertions or deletions of amino acids in one cultivar relative to the other. We also observed gain and loss of genes caused by large indels. This study provides an important framework and an invaluable dataset for further understanding of the molecular mechanisms underlying the evolution and functions of the rice genome. [ABSTRACT FROM AUTHOR]

The physical map of the hexaploid wheat chromosome 3 B was screened using centromeric DNA probes. A 1.1-Mb region showing the highest number of positive bacterial artificial chromosome ( BAC) clones was fully sequenced and annotated, revealing that 96% of the DNA consisted of transposable elements, mainly long terminal repeat ( LTR) retrotransposons (88%). Estimation of the insertion time of the transposable elements revealed that CRW (also called Cereba) and Quinta are the youngest elements at the centromeres of common wheat ( Triticum spp.) and its diploid ancestors, with Quinta being younger than CRW in both diploid and hexaploid wheats. Chromatin immunoprecipitation experiments revealed that both CRW and Quinta families are targeted by the centromere-specific histone H3 variant CENH3. Immuno colocalization of retroelements and CENH3 antibody indicated that a higher proportion of Quinta than CRWs was associated with CENH3, although CRWs were more abundant. Long arrays of satellite repeats were also identified in the wheat centromere regions, but they lost the ability to bind with CENH3. In addition to transposons, two functional genes and one pseudogene were identified. The gene density in the centromere appeared to be between three and four times lower than the average gene density of chromosome 3 B. Comparisons with related grasses also indicated a loss of microcollinearity in this region. Finally, comparison of centromeric sequences of Aegilops tauschii ( DD), Triticum boeoticum ( AA) and hexaploid wheat revealed that the centromeres in both the polyploids and diploids are still undergoing dynamic changes, and that the new CRWs and Quintas may have undertaken the core role in kinetochore formation. [ABSTRACT FROM AUTHOR]

Contents IZUMO1, belonging to the family of mammalian immunoglobulin proteins, has been well characterized in the mouse. Here, we describe the molecular cloning and expression analysis of porcine IZUMO1 (pIZUMO1). Partial sequence information published in the National Center for Biotechnology Information (NCBI) database was used to generate the full-length sequence for IZUMO1 using rapid amplification of cDNA ends (RACE). A search of the porcine genomic sequence in the NCBI database identified a bacterial artificial chromosome (BAC) encoding the pIZUMO1 gene. This BAC is derived from porcine chromosome 6 and is syntenic with the corresponding regions of mouse, bovine, and human genomes encoding the IZUMO gene family. This BAC was found to encode an IZUMO1 protein with a predicted amino acid sequence having high similarity with mouse and human IZUMO1. Western blot analysis of proteins from porcine tissues indicated that pIZUMO1 was specifically expressed in the sperm. Furthermore, to confirm whether pIZUMO1 forms complexes, we overexpressed pIZUMO1 in HEK293 cells. The recombinant pIZUMO1 from cell extracts was found to form complexes. Our finding suggests that pIZUMO1 forms homodimeric complex on the sperm membrane. Furthermore, an IVF inhibition assay with an antibody for the porcine IZUMO1 Ig-like domain showed that Ig-like domain effectively prevented pig sperm-egg interactions. [ABSTRACT FROM AUTHOR]

We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH ( fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North-South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome-specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S-5.8S-25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber-FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA. [ABSTRACT FROM AUTHOR]

Genome structure exhibits remarkable plasticity within Zea mays. To examine how haplotype structure has evolved within the Andropogoneae tribe, we have analyzed the bz gene-rich region of maize ( Zea mays), the Zea teosintes mays ssp. mexicana, luxurians and diploperennis, Tripsacum dactyloides, Coix lacryma-jobi and Sorghum propinquum. We sequenced and annotated BAC clones from these species and re-annotated the orthologous Sorghum bicolor region. Gene colinearity in the region is well conserved within the genus Zea. However, the orthologous regions of Coix and Sorghum exhibited several micro-rearrangements relative to Zea, including addition, truncation and deletion of genes. The stc1 gene, involved in the production of a terpenoid insect defense signal, is evolving particularly fast, and its progressive disappearance from some species is occurring by microhomology-mediated recombination. LTR retrotransposons are the main contributors to the dynamic evolution of the bz region. Common transposon insertion sites occur among haplotypes from different Zea mays sub-species, but not outside the species. As in Zea, different patterns of interspersion between genes and retrotransposons are observed in Sorghum. We estimate that the mean divergence times between maize and Tripsacum, Coix and Sorghum are 8.5, 12.1 and 12.4 million years ago, respectively, and that between Coix and Sorghum is 9.3 million years ago. A comparison of the bz orthologous regions of Zea, Sorghum and Coix with those of Brachypodium, Setaria and Oryza allows us to infer how the region has evolved by addition and deletion of genes in the approximately 50 million years since these genera diverged from a common progenitor. [ABSTRACT FROM AUTHOR]

Aside from polyploidy, transposable elements are the major drivers of genome size increases in plants. Thus, understanding the diversity and evolutionary dynamics of transposable elements in sunflower ( Helianthus annuus L.), especially given its large genome size (∼3.5 Gb) and the well-documented cases of amplification of certain transposons within the genus, is of considerable importance for understanding the evolutionary history of this emerging model species. By analyzing approximately 25% of the sunflower genome from random sequence reads and assembled bacterial artificial chromosome (BAC) clones, we show that it is composed of over 81% transposable elements, 77% of which are long terminal repeat (LTR) retrotransposons. Moreover, the LTR retrotransposon fraction in BAC clones harboring genes is disproportionately composed of chromodomain-containing Gypsy LTR retrotransposons ('chromoviruses'), and the majority of the intact chromoviruses contain tandem chromodomain duplications. We show that there is a bias in the efficacy of homologous recombination in removing LTR retrotransposon DNA, thereby providing insight into the mechanisms associated with transposable element (TE) composition in the sunflower genome. We also show that the vast majority of observed LTR retrotransposon insertions have likely occurred since the origin of this species, providing further evidence that biased LTR retrotransposon activity has played a major role in shaping the chromatin and DNA landscape of the sunflower genome. Although our findings on LTR retrotransposon age and structure could be influenced by the selection of the BAC clones analyzed, a global analysis of random sequence reads indicates that the evolutionary patterns described herein apply to the sunflower genome as a whole. [ABSTRACT FROM AUTHOR]

An 8-year-old male neutered Labrador Retriever was referred to the University of Wisconsin Veterinary Medical Teaching Hospital with a presumptive diagnosis of leukemia. Hematologic abnormalities included normal neutrophil count with a left shift, monocytosis, eosinophilia, thrombocytopenia, and circulating immature mononuclear cells. Bone marrow was effaced by immature hematopoietic cells of various morphologic appearances. In addition, large multinucleated cells were observed frequently. Flow cytometric analysis of nucleated cells in blood revealed 34% CD34+ cells, consistent with acute leukemia. By immunocytochemical analysis of cells in blood and bone marrow, some mononuclear cells expressed CD18, myeloperoxidase, and CD11b, indicating myeloid origin; some, but not all, large multinucleated cells expressed CD117 and CD42b, the latter supporting megakaryocytic lineage. The diagnosis was acute myeloblastic leukemia without maturation ( AML-M1). To identify genetic aberrations associated with this malignancy, cells from formalin-fixed paraffin-embedded bone marrow were analyzed cytogenetically by multicolor fluorescence in situ hybridization ( FISH). Co-localization of bacterial artificial chromosome ( BAC) containing BCR and ABL was evident in 32% of cells. This confirmed the presence of the canine BCR- ABL translocation or Raleigh chromosome. In people, the analogous translocation or Philadelphia chromosome is characteristic of chronic myelogenous leukemia ( CML) and is rarely reported in AML. BCR- ABL translocation also has been identified in dogs with CML; however, to our knowledge this is the first report of AML with a BCR- ABL translocation in a domestic animal. [ABSTRACT FROM AUTHOR]

Chromosomal rearrangements are relatively rare evolutionary events and can be used as markers to study karyotype evolution. This research aims to use such rearrangements to study chromosome evolution in Solanum., Chromosomal rearrangements between Solanum crops and several related wild species were investigated using tomato and potato bacterial artificial chromosomes (BACs) in a multicolour fluorescent in situ hybridization (FISH). The BACs selected are evenly distributed over seven chromosomal arms containing inversions described in previous studies. The presence/absence of these inversions among the studied Solanum species were determined and the order of the BAC-FISH signals was used to construct phylogenetic trees., Compared with earlier studies, data from this study provide support for the current grouping of species into different sections within Solanum; however, there are a few notable exceptions, such as the tree positions of S. etuberosum (closer to the tomato group than to the potato group) and S. lycopersicoides (sister to S. pennellii). These apparent contradictions might be explained by interspecific hybridization events and/or incomplete lineage sorting., This cross-species BAC painting technique provides unique information on genome organization, evolution and phylogenetic relationships in a wide variety of species. Such information is very helpful for introgressive breeding. [ABSTRACT FROM AUTHOR]

To test the feasibility of using bacterial artificial chromosomes (BAC) containing kinases for pathological diagnosis using fluorescence in situ hybridization (FISH), 10 BAC probes containing a gene amplified in 5% or more of a pilot cohort were selected from a previous survey using arbitrarily selected BAC clones harboring 100 kinases. In this report, we describe the prevalence and association with the clinico-pathological profile of these selected 10 BAC probes in 365 gastric cancer tissues. FISH analyses using these 10 BAC probes containing loci encoding EGFR, ERBB2(HER2), EPHB3, PIK3CA, MET, PTK7, ACK1, STK15, SRC, and HCK showed detectable amplifications in paraffin-embedded tissue in 2.83% to 13.6% of the gastric cancer tissues. Considerable numbers of the cases showed the co-amplification of two or more of the probes that were tested. BAC probes located within a genome neighborhood, such as PIK3CA, EPHB3, and ACK1 at 3q26-29 or HCK, SRC, and STK15 at 20q11-13.1, were often co-amplified in the same cases, but non-random co-amplifications of genes at distant genomic loci were also observed. These findings provide basic information regarding the creation of a strategy for personalizing gastric cancer therapy, especially when using multiple kinase inhibitors. [ABSTRACT FROM AUTHOR]

Summary The chicken major histocompatibility complex (MHC-B locus) has a strong association with resistance and susceptibility to numerous diseases. We have found a B haplotype designated WLA that associated with the regression of tumours caused by Rous sarcoma virus J strain (RSV-J). Haplotype WLA was identical to the regressive B6 haplotype when partial genotyping was performed (Poultry Science, 89, 2010, 651). We then constructed a bacterial artificial chromosome (BAC) library from a WLA homozygote chicken to evaluate the structure of this regression haplotype and compared it to those of the B6 haplotype. Comparison between WLA and B6 above 59 kb within the 167 kb, including 14 genes from BG1 to BF2, revealed 75 SNPs and 14 indels. However, several genes were identical between WLA and B6, including the BF1 and BF2 genes, which encode a class I molecule previously suggested to be related to the regression phenotype. The BLB2 gene encoding the MHC class II beta chain showed the greatest diversity, with 19 non-synonymous SNPs. A comparison of WLA and B6 haplotpyes that are associated with tumour regression and RIRa and B24 haplotypes associated with tumour progression suggests that DMA1, DMA2, BRD2, TAPBP and BLB2 genes are not involved in the intensity of RSV J tumour regression. [ABSTRACT FROM AUTHOR]

Acyl-coenzyme A (Acyl-CoA) desaturases play a key role in the biosynthesis of female moth sex pheromones. Desaturase genes are encoded by a large multigene family, and they have been divided into five subgroups on the basis of biochemical functionality and phylogenetic affinity. In this study both copy numbers and transcriptional levels of desaturase genes in the European corn borer (ECB), Ostrinia nubilalis, were investigated. The results from genome-wide screening of ECB bacterial artificial chromosome (BAC) library indicated there are many copies of some desaturase genes in the genome. An open reading frame (ORF) has been isolated for the novel desaturase gene ECB ezi-Δ11β from ECB gland complementary DNA and its functionality has been analyzed by two yeast expression systems. No functional activities have been detected for it. The expression levels of the four desaturase genes both in the pheromone gland and fat body of ECB and Asian corn borer (ACB), O. furnacalis, were determined by real-time polymerase chain reaction. In the ECB gland, Δ11 is the most abundant, although the amount of Δ14 is also considerable. In the ACB gland, Δ14 is the most abundant and is 100 times more abundant than all the other three combined. The results from the analysis of evolution of desaturase gene transcription in the ECB, ACB and other moths indicate that the pattern of Δ11 gene transcription is significantly different from the transcriptional patterns of other desaturase genes and this difference is tied to the underlying nucleotide composition bias of the genome. [ABSTRACT FROM AUTHOR]