Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species.

BACKGROUND: Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. RESULTS: We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. CONCLUSIONS: This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins.

Characterization of Streptomyces plasmid-phage pFP4 and its evolutionary implications.

Autonomous-replicating plasmid pFP4 of Streptomyces sp. FR1 isolated from a heavy metal-contaminated land was cloned and sequenced. Surprisingly, the 40,949-bp pFP4 contains a cluster of 20 genes, resembling these chromosome-integrated prophages of Streptomyces sp. SPB78 and Streptomyces scabiei 87.22. Plasmid pFP4 could transfer by conjugation and a replication locus, iteron/repA/repB, was identified. The filtered FR1 culture could infect both FR1 and FR1 cured of pFP4 to form plaques, and also six out of 13 strains from the same land, but failed to form plaques on other seven strains from same source and all ten Streptomyces species from different sources. pFP4 phage particles were observed by transmission electron microscopy. Major structural proteins (capsid, portal and tail, etc.) of pFP4 virions were encoded by twelve pFP4 genes. pFP4 phage DNA contained 3' protruding cohesive ends of 9-nt. Streptomyces pFP4 represents a novel plasmid-phage.

Development of a vector and host system and characterization of replication of plasmid pSQ10 in moderately halophilic Nocardiopsis.

The genus of Nocardiopsis is a new source of antibiotics, chemicals, and enzymes. Here we reported the development of a vector and host system in moderately halophilic Nocardiopsis via an oriT-mediated conjugation. By screening about 80 Nocardiopsis strains, 6 of them harbored 8 plasmids (18-80 kb). The complete nucleotide sequence of pSQ10 consisted of 18,219 bp, with 71.9% G + C content, encoding 17 open reading frames, 5 of them resembled those of Streptomyces plasmids. A rep locus (iteron within the gene) was identified for replication in Nocardiopsis sp. YIM 90083, and rep protein bound to its iteron sequence. This system may be useful for gene cloning and expression in Nocardiopsis.

[Cloning, sequencing and identification of replication origin of Rhodococcus linear plasmid pNSL1].

UNLABELLED: Two linear plasmids, pNSL1 and pNSL1, were detected from Rhodocuccus sp. NS1. OBJECTIVE: Cloning, sequencing and identification of replication origin of the Rhodococcus linear plasmid pNSL1. METHODS: Large amount of linear plasmid DNA was recovered from pulsed-field gels for shotgun-cloning and sequencing, and identification of its replication locus. RESULTS: The complete nucleotide sequence of pNSL1 consisted of 117252 bp, including the conserved 1282-bp telomere sequences among Rhodococcus linear plasmids. pNSL1 encoded 103 open reading frames, including functions of replication, maintenance and transfer etc. A locus, pNSL1. 038 and upstream 767-bp non-coding sequence, was identified for autonomous replication by cloning in an E. coli vector and introduced by electroporation into Nocardia coralline 4. 1037. CONCLUSION: Cloning and sequencing of Rhodococcus linear plasmid pNSL1, and identification of its replication origin.

Does baseline renal function always decrease after unilateral ureteral severe obstruction? -experimental validation and novel findings by Tc-99m diethylene triamine pentaacetate acid (DTPA) dynamic renal scintigraphy.

BACKGROUND: There is a lack of consensus concerning changes in renal function after unilateral ureteral obstruction. The aim of this study was to assess the influence of ureteral obstruction on renal morphology and function and to explore the effectiveness of dynamic renal scintigraphy in evaluating obstructive renal function. METHODS: We established a model of right ureteral obstruction using New Zealand white rabbits. We measured the glomerular filtration rate (GFR) before the operation and from days 1 to 82 after obstruction, observed the changes in bilateral kidney sizes and the GFR, and then compared the differences between the left and right kidneys. RESULTS: The difference between left and right kidney sizes was not significant before obstruction (t=-0.430, P=0.674); the right kidneys increased in size after obstruction and were larger than the left kidneys (P≤0.001). Obstructed kidneys demonstrated a morphological process of decelerated expansion and retraction. The difference in GFR between the left and right kidneys was not significant before obstruction (t=1.77, P=0.098); during days 1-21 and 42-82 after obstruction, the GFR of the right kidneys decreased and was lower than that of the left kidneys (P<0.001); on day 28, the GFR difference between the left and right kidneys (t=1.62, P=0.130) and the difference in the right kidney GFR before and after obstruction (t=1.03, P=0.323) were not significant. The GFR of obstructed kidneys rapidly declined initially, experienced a tortuous process of repeated dormancies and multiple self-recoveries, and then gradually declined. CONCLUSIONS: The GFR in hydronephrotic kidneys is fluctuating. Thus, evaluating the true function of hydronephrotic kidneys using only baseline GFR is difficult; however, combining baseline GFR with renal morphology to assess obstructive renal function and its recoverability can provide more meaningful results.

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus.

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.

Mixed Epithelial and Stromal Tumor of the Kidney Mimicking Malignancy on 18F-FDG PET/CT in a Patient With Breast Cancer.

Mixed epithelial and stromal tumor of the kidney (MESTK) is a rare renal tumor composed of solid and cystic components. Mixed epithelial and stromal tumor of the kidney has no pathognomonic imaging feature. It is difficult to distinguish MESTK from other cystic renal neoplasms using current radiological methods. We report a rare case of MESTK that was diagnosed in a 46-year-old woman with breast cancer. F-FDG PET/CT and contrast-enhanced CT features of MESTK are presented, which could potentially provide useful information in the differential diagnoses of cystic renal neoplasms.

Replication and inheritance of Nocardia plasmid pC1.

Nocardia sp. C-14-1, isolated from acrylic fiber wastewater, can degrade long-chain alkanes and succinonitrile efficiently. Here we report the characterization of an indigenous plasmid pC1. The overall nucleotide sequence of pC1 consisted of 5841 bp. The five ORFs, encoding a DNA recombinase, replication protein (Rep(pC1)) and three proteins of unknown function, were predicted on pC1. The Rep(pC1) displayed its homology with the Rep of Rhodococcus large plasmid p33701, suggesting a theta type of replication. An Escherichia coli plasmid (containing the single rep(pC1) gene) propagated autonomously in low copy number in Nocardia or Rhodococcus, suggesting that rep(pC1) was an essential gene for plasmid replication. The plasmid (containing the single rep(pC1) gene) presented as inheritance unstable hints that other pC1 loci were required for the stable inheritance of plasmids. By comparison of the plasmid-borne Rep proteins, we classify Rhodococcus or Nocardia plasmids into four groups.

99mTcO4- scintigraphic detection of follicular thyroid cancer and multiple metastatic lesions: A case report.

99mTcO4- thyroid imaging is often used to detect thyroid diseases that are confined to the neck. However, this examination is not frequently used to detect metastatic lesions of thyroid cancer in the whole body, while 131I imaging is often used to detect the metastases of differentiated thyroid cancers. The present study performed 99mTcO4- thyroid imaging for a 69-year-old patient with a thyroid nodule and incidentally identified a lesion with abnormally increased 99mTcO4- uptake in the chest of the patient. Furthermore, a whole-body scan was performed for this patient and multiple lesions with increased 99mTcO4- uptake were identified. The results confirmed that these lesions were follicular thyroid cancer and the metastatic lesions were distributed in numerous locations. The results revealed that analysis of the whole body is significant when regional lesions with abnormally increased 99mTcO4- uptake outside of thyroid tissues are identified by routine 99mTcO4- thyroid imaging.

Comparison of distribution characteristics of metastatic bone lesions between breast and prostate carcinomas.

The purpose of the present study was to investigate the distribution characteristics of bone metastases in breast and prostate carcinomas. Bone scintigraphies were performed in 504 cancer patients. We studied the correlation between the distribution and total number of metastatic bone lesions, and compared the distribution of metastatic bone lesions between breast and prostate carcinomas. In the early stage, the distribution in the thoracic vertebrae, lumbar vertebrae and pelvis of the metastatic lesions of the prostate carcinoma (81.0%, 47/58) was significantly higher than that of the breast carcinoma (41.7%, 63/151; χ(2)=27.6, P=0.000). The distribution of the lesions in the thoracic skeleton in the cases of the breast carcinoma (65.6%, 99/151) was significantly higher than that of the prostate carcinoma (27.6%, 16/58; χ(2)=24.8, P=0.000); however, the distributions in the advanced cases were not markedly different. The differences in the proportions of the metastatic lesions in the lumbar vertebrae (χ(2)=56.1, P=0.000) and ribs (χ(2)=39.1, P=0.000) in the cases of the prostate carcinoma, and in the sternum (χ(2)=31.2, P=0.000), skull (χ(2)=26.5, P=0.000) and femur (χ(2)=13.6, P=0.001) in the cases of the breast carcinoma were significant. Between the breast and prostate carcinomas, the differences in the proportions of the metastatic lesions of certain bones were also significant. In cases with few bone metastases, the proportion of sternum metastases of patients with breast carcinoma (17.9%) was significantly higher than that of patients with prostate carcinoma (1.7%; χ(2)=12.7, P=0.000); the proportion of metastases in the lumbar vertebrae of prostate carcinoma (39.7%) was significantly higher than that of breast carcinoma (13.9%; χ(2)=15.4, P=0.000); the proportion of rib metastases of breast carcinoma (27.2%) was significantly higher than that of prostate carcinoma (8.6%; χ(2)=9.6, P=0.002). In cases with extensive bone metastases, the proportions of metastatic lesions in the sternum and lumbar vertebrae in breast and prostate carcinomas were not significantly different (P>0.05). In conclusion, the distribution of bone metastases is correlated with the total number of metastatic bone lesions in breast and prostate carcinoma patients, and has different characteristics in different lesions.

Treating oligohydramnios with extract of Salvia miltiorrhiza: A randomized control trial.

OBJECTIVE: To determine whether purified herbal extract of Salvia miltiorrhiza can improve the amniotic fluid volume in pre-term oligohydramnios by improving uteroplacental circulation. METHODS: Forty-three pregnant women with oligohydramnios received a daily intravenous dose of 30 mL of salvia extract mixed with 5% glucose 500 mL. A control group of 41 women received daily 5% glucose 500 mL only. The amniotic fluid index (AFI) was assessed at least twice a week by ultrasonographists who were blinded to the treatment. Both women and fetuses were monitored closely. The change in AFIwas calculated and compared by paired t test within and between groups. The revised recommendations for improving the quality of reports of parallel group randomized trials were used. RESULTS: After a mean of 7.2 +/- 2.7 days' therapy, ranging from 3 to 18 days, the AFIincreased significantly from a mean of 4.9 +/- 2.3 cm to a mean of 7.12 +/- 2.36 cm, by a mean of AFI0.18 +/- 0.06 cm/day (paired t = 3.62, p < 0.005). In the control group, the AFIincreased from a mean of 5.1 +/- 2.4 cm to a mean of 5.5 +/- 3.1 cm after a mean of 6.1 +/- 3.3 days' treatment, ranging from 4 to 15 days. The effect of salvia treatment on AFIin the salvia group was significantly greater than in the control group (p < 0.001). No side effects were observed in treated patients. CONCLUSION: Salvia miltiorrhiza is an effective Chinese medicine for the treatment of oligohydramnios.

Characterization of the genetic components of Streptomyces lividans linear plasmid SLP2 for replication in circular and linear modes.

The nucleotide sequence of Streptomyces lividans linear plasmid SLP2 consists of 50,410 bp (C. H. Huang, C. Y. Chen, H. H. Tsai, C. Chen, Y. S. Lin, and C. W. Chen, Mol. Microbiol. 47:1563-1576, 2003). Here we report that the basic SLP2 locus for plasmid replication in circular mode resembles that of Streptomyces linear plasmids pSLA2 and SCP1 and comprises iterons(SLP2) and the adjacent rep(SLP2) gene. More efficient replication additionally required the 47-bp sequence between bp 581 and 628 upstream of the iterons. Replacement of either the iterons or the rep gene of SLP2 by the corresponding genes of pSLA2 or SCP1 still allows propagation in Streptomyces, although the transformation frequencies were 3 orders of magnitude lower than the original plasmids, suggesting that these plasmids share similar replication mechanisms. To replicate SLP2 in linear mode, additional SLP2 loci--either mtap(SLP2)/tpg(SLP2) or mtap(SLP2)/ilrA(SLP2)--were required. IlrA(SLP2) protein binds specifically to the iterons(SLP2) in vitro. Interactions were detected between these SLP2-borne replication proteins (Mtap(SLP2), Tpg(SLP2), and IlrA(SLP2)) and the telomeric replication proteins (TpgL, TapL, and TpgL) of the S. lividans chromosome, respectively, but the SLP2 proteins failed to interact. These results suggest that SLP2 recruits chromosomally encoded replication proteins for its telomere replication.

Diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids.

Streptomyces sp. linear plasmids and linear chromosomes usually contain conserved terminal palindromic sequences bound by the conserved telomeric proteins Tap and Tp, encoded by the tap and tpg genes, respectively, as well as plasmid loci required for DNA replication in circular mode when the telomeres are deleted. These consist of iterons and an adjacent rep gene. By using PCR, we found that 8 of 17 newly detected linear plasmids in Streptomyces strains lack typical telomeric tap and tpg sequences. Instead, two novel telomeres in plasmids pRL1 and pRL2 from the eight strains and one conserved telomere in pFRL1 from the other strains were identified, while multiple short palindromes were also found in the plasmids. The complete nucleotide sequence of pRL2 revealed a gene encoding a protein containing two domains, resembling Tap of Streptomyces and a helicase of Thiobacillus, and an adjacent gene encoding a protein similar to Tpg of Streptomyces and a portion of the telomere terminal protein pTP of adenoviruses. No typical iterons-rep loci were found in the three plasmids. These results indicate an unexpected diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids.

Identification and characterization of a pSLA2 plasmid locus required for linear DNA replication and circular plasmid stable inheritance in Streptomyces lividans.

Streptomyces linear plasmids and linear chromosomes can replicate also in a circular form when their telomeres are deleted. The 17-kb linear plasmid pSLA2 has been a useful model in studies of such replicons. Here we report that the minimal origin initiating replication of pSLA2-derived plasmids as circular molecules cannot propagate these plasmids in a linear mode unless they also contain a novel plasmid-encoded locus, here named rlrA (required for linear replication). In contrast with the need for rlrA to accomplish replication of telomere-containing linear plasmids, expression of rlrA, which encodes two LuxR family regulatory domains, interferes with the establishment of pSLA2 in circular form in Streptomyces lividans transformants. The additional presence of an adjacent divergently transcribed locus, rorA (rlrA override), which strongly resembles the kor (kil override) transcription control genes identified previously on Streptomyces plasmids, reversed the detrimental effects of rlrA on plasmid establishment and additionally stabilized circular plasmid inheritance by spores during the S. lividans life cycle. While the effects of the rlrA/rorA locus of pSLA2 were seen also on linear plasmids derived from the unrelated SLP2 replicon, they did not extend to plasmids whose replication was initiated at a cloned chromosomal origin. Our results establish the existence of, and provide the initial description of, a novel plasmid-borne regulatory system that differentially affects the propagation of linear and circular plasmids in Streptomyces.