. In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(؉)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding ␤-lactamase. pSKO(؉)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(؉)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing ␤-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (␤-lactamase) gene.Coxiella burnetii is the etiological agent of human Q fever (9). It is an obligate intracellular bacterium replicating within the acidic phagolysosomes of eukaryotic cells (1,4,15,24). Although C. burnetii becomes metabolically active and synthesizes protein and DNA during in vitro acid activation assays, axenic growth has not been observed (7,15,43). This obligate intracellular parasitism poses difficulties in studying virulence factors associated with this organism. Classical genetic studies involve the mutation of genes to eliminate a virulent phenotype and then complementation using DNA from wild-type organisms to restore virulence; this approach ascertains the contribution of specific genes to virulence. However, these types of studies have been unavailable for C. burnetii, since genetic transformation of this organism has not yet been demonstrated.Information on enzymes produced by C. burnetii and on gene structure within the chromosome and the endogenous plasmid QpHI has been obtained by the cloning and expression of C. burnetii genes in Escherichia coli (19). The use of E. coli as a host for C. burnetii genes was also implemented for the cloning of the C. burnetii chromosomal origin of DNA replication. These origin search techniques resulted in the isolation of a 5.8-kb C. burnetii chromosomal DNA fragment which initiates plasmid DNA replication within E. coli (36). The minimal ars sequence required for plasmid replication in an E. coli polA strain is 403 bp (6, 36); it demonstrates limited similarity to origins of other bacteria (Fig. 1). The ars sequence contains two consensus sequences for the binding of DnaA, an AϩT-rich region, and a potential binding site for integration host factor and for factor of inversion stimulation; these sequences are characteristic of bacterial chromosomal origins (3,12,28). The open reading frames flanking one side of the C. burnetii ars sequence demonstrate similarity to genes located in bacterial origin regions; these open reading frames include rnpA and rpmH genes and genes encoding 9K and 60K proteins (Fig. 1). ...

BackgroundAs one of the most popular and valuable commercial marine fishes in China and East Asian countries, the Chinese black porgy (Acanthopagrus schlegelii), also known as the blackhead seabream, has some attractive characteristics such as fast growth rate, good meat quality, resistance to diseases, and excellent adaptability to various environments. Furthermore, the black porgy is a good model for investigating sex changes in fish due to its protandrous hermaphroditism. Here, we obtained a high-quality genome assembly of this interesting teleost species and performed a genomic survey on potential genes associated with the sex-change phenomenon.FindingsWe generated 175.4 gigabases (Gb) of clean sequence reads using a whole-genome shotgun sequencing strategy. The final genome assembly is approximately 688.1 megabases (Mb), accounting for 93% of the estimated genome size (739.6 Mb). The achieved scaffold N50 is 7.6 Mb, reaching a relatively high level among sequenced fish species. We identified 19 465 protein-coding genes, which had an average transcript length of 17.3 kb. By performing a comparative genomic analysis, we found 3 types of genes potentially associated with sex change, which are useful for studying the genetic basis of the protandrous hermaphroditism.ConclusionsWe provide a draft genome assembly of the Chinese black porgy and discuss the potential genetic mechanisms of sex change. These data are also an important resource for studying the biology and for facilitating breeding of this economically important fish.

To understand the molecular mechanism of tumorigenesis of pulmonary lymphoepithelioma-like carcinoma and explore potential therapeutic strategies, we investigated the genomic profiles and PD-L1 expression of 29 Chinese pulmonary lymphoepithelioma-like carcinoma patients at various stages. We performed capture-based targeted sequencing on tissue samples collected from 27 patients with sufficient samples using a panel consisting of 520 cancer-related genes, spanning 1.64 Mb of the human genome. We identified 184 somatic mutations in 109 genes from 26 patients. One patient had no mutations detected by this panel. Copy number variations were detected in 52% (14/27) of the patients, with a majority having advancedstage disease (10/14). Except for the detection of ERBB2 amplification and KRAS mutation in two patients, no other classic lung cancer driver mutations were detected. Interestingly, 78% (21/27) of the patients had mutations in epigenetic regulators. Of the 184 mutations identified, 51 occurred in 29 epigenetics-related genes. Furthermore, we performed PD-L1 immunohistochemistry staining using the Dako 22C3 assay and demonstrated that 69% (20/29) of the cohort had positive PD-L1 expression, of which three patients received and benefited from a PD-1 inhibitor. In conclusion, we elucidated a distinct genomic landscape associated with pulmonary lymphoepithelioma-like carcinoma with no classic lung cancer driver mutation but an enrichment of mutations in epigenetic regulators. The detection of high PD-L1 expression and lack of any canonical druggable driver mutations raises the potential of checkpoint immunotherapy for pulmonary lymphoepithelioma-like carcinoma.

A Coxiella burnetii chromosomal fragment capable of functioning as an origin for the replication of a kanamycin resistance (Kanr) plasmid was isolated by use of origin search methods utilizing an Escherichia coli host. The 5.8-kb fragment was subcloned into phagemid vectors and was deleted progressively by an exonuclease III-S1 technique. Plasmids containing progressively shorter DNA fragments were then tested for their capability to support replication by transformation of an E. coli polA strain. A minimal autonomous replication sequence (ARS) was delimited to 403 bp. Sequencing of the entire 5.8-kb region revealed that the minimal ARS contained two consensus DnaA boxes, three A + T-rich 21-mers, a transcriptional promoter leading rightwards, and potential integration host factor and factor of inversion stimulation binding sites. Database comparisons of deduced amino acid sequences revealed that open reading frames located around the ARS were homologous to genes often, but not always, found near bacterial chromosomal origins; these included identities with rpmH and rnpA in E. coli and identities with the 9K protein and 60K membrane protein in E. coli and Pseudomonas species. These and direct hybridization data suggested that the ARS was chromosomal and not associated with the resident plasmid QpH1. Two-dimensional agarose gel electrophoresis did not reveal the presence of initiating intermediates, indicating that the ARS did not initiate chromosome replication during laboratory growth of C. burnetii.

Two populations of CoxieIlu burnetii were isolated from fibroblast tissue cultures and examined for their ability to synthesize DNA when incubated in a defined medium. Both the populations released by mechanical lysis of heavily infected host cells, as well as those recovered from the tissue culture medium, incorporated H332P04 into DNA. Incorporation occurred at pH 4.5 but not at pH 7-0, and proceeded for 12-15 h. When incorporation of [ 3H]thymidine was studied, only the organisms obtained by mechanical lysis of host cells were active. Those which had been released by natural means into the tissue culture medium, and then recovered for study, did not incorporate precursor thymidine but were extremely active in protein biosynthesis. In mechanically released organisms, thymidine incorporation was inhibited immediately by rifamycin (40 p~) and hydroxyurea (10 mM), but it was not affected by chloramphenicol(310 p~) until 4 h after addition of the drug. Incorporation of H332P04 by both populations of organisms was also inhibited by rifamycin, chloramphenicol and hydroxyurea, but the time sequence of inhibition differed. Southern hybridization utilizing 32P-labelled DNA suggested that both populations synthesized authentic chromosomal DNA sequences, as well as QpHl plasmid DNA, during acid activation of metabolism .