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1.  Gene structure and expression of the MboI restriction--modification system. 
Nucleic Acids Research  1993;21(10):2309-2313.
The genes from Moraxella bovis encoding the MboI restriction--modification system were cloned and expressed in Escherichia coli. Three open reading frames were found in the sequence containing the genes. These genes, which we named mboA, mboB, and mboC, had the same orientation in the genome. Genes mboA and mboC encoded MboI methyltransferases (named M.MboA and M.MboC) with 294 and 273 amino acid residues, respectively. The mboB gene coded for MboI restriction endonuclease (R.MboI) with 280 amino acid residues. Recombinant E.coli-MBOI, which contained the whole MboI system, overproduced R.MboI. R.MboI activity from E.coli-MBOI was 480-fold that of M.bovis. The amino acid sequences deduced from these genes were compared with those of other restriction--modification systems. The protein sequences of the MboI system had 38-49% homology with those of the DpnII system.
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PMCID: PMC309525  PMID: 8506128
2.  Cloning and characterization of the MboII restriction-modification system. 
Nucleic Acids Research  1991;19(5):1007-1013.
The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.
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PMCID: PMC333773  PMID: 2020540
3.  Results-oriented management though MBO. 
Management by Objectives (MBO) as it has been implemented in the Houston Academy of Medicine--Texas Medical Center Library is described. That MBO must be a total management system and not just another library program is emphasized throughout the discussion and definitions of the MBO system parts: (1) mission statement; (2) role functions; (3) role relationships; (4) effectiveness areas; (5) objective; (6) action plans; and (7) performance review and evaluation. Examples from the library's implementation are given within the discussion of each part to give the reader a clearer picture of the library's actual experiences with the MBO process. Tables are included for further clarification. In conclusion some points are made which the author feels are particularly crucial to any library MBO implementation.
PMCID: PMC226932  PMID: 476316
4.  Malignant bowel obstruction in advanced cancer patients: epidemiology, management, and factors influencing spontaneous resolution 
Malignant bowel obstruction (MBO) is a frequent complication in advanced cancer patients, especially in those with abdominal tumors. Clinical management of MBO requires a specific and individualized approach that is based on disease prognosis and the objectives of care. The global prevalence of MBO is estimated to be 3% to 15% of cancer patients. Surgery should always be considered for patients in the initial stages of the disease with a preserved general status and a single level of occlusion. Less invasive approaches such as duodenal or colonic stenting should be considered when surgery is contraindicated in obstructions at the single level. The priority of care for inoperable and consolidated MBO is to control symptoms and promote the maximum level of comfort possible. The spontaneous resolution of an inoperable obstructive process is observed in more than one third of patients. The mean survival is of no longer than 4–5 weeks in patients with consolidated MBO. Polymodal medical treatment based on a combination of glucocorticoids, strong opioids, antiemetics, and antisecretory drugs achieves very high symptomatic control. This review focuses on the epidemiological aspects, diagnosis, surgical criteria, medical management, and factors influencing the spontaneous resolution of MBO in advanced cancer patients.
doi:10.2147/CMAR.S29297
PMCID: PMC3421464  PMID: 22904637
malignant bowel obstruction; cancer; intestinal obstruction; bowel occlusion
5.  Malignant bowel obstruction: A retrospective clinical analysis 
Malignant bowel obstruction (MBO) is a disease with a poor prognosis, particularly in patients with advanced bowel or gynecological cancers. Multimodality teatments may be used to relieve the symptoms in patients with MBO; however, there is currently no consensus regarding the optimal treatment and no strong evidence supporting the efficacy of any treatment in improving the quality of life (QOL) and prolonging survival. We conducted a search through our medical center database of cancer registries for MBO cases between January, 1995 and December, 2008 and analyzed the clinicopathological characteristics and association between treatments and prognosis or QOL. The primary type of cancer causing MBO was found to be adenocarcinoma of colon. The overall survival time was found to be significantly higher among patients presenting with MBO as the initial symptom and improved QOL was achieved in patients who received surgical treatment. The mean survival time and the functional status of colorectal cancer patients receiving targeted therapy and chemotherapy were more satisfactory compared with those receiving surgery alone or conservative treatment. Furthermore, for end-stage cancer patients with MBO, hospice care was effective in reducing pain scores and relieving the symptoms of the disease.
doi:10.3892/mco.2013.216
PMCID: PMC3915666  PMID: 24649301
bowel obstruction; cancer
6.  Mutant strains of Chlamydomonas reinhardtii that move backwards only 
The Journal of Cell Biology  1984;98(6):2026-2034.
Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility. Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern. The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants. This defect in motility was found to be a property of the mutant axonemes themselves: the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition. Axonemes obtained from mbo1 , mbo2 , and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S- or 32P-labeled axonemes. One of the six polypeptides has previously been identified; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes. Detailed ultrastructural analysis of the mbo1 flagella and the mbo1 , mbo2A , and mbo3 axonemes revealed that the mutants specifically lack the beak- like projections found within the B-tubules of outer doublets 5 and 6.
PMCID: PMC2113042  PMID: 6725408
7.  Organosulfates as Tracers for Secondary Organic Aerosol (SOA) Formation from 2-Methyl-3-Buten-2-ol (MBO) in the Atmosphere 
Environmental Science & Technology  2012;46(17):9437-9446.
2-Methyl-3-buten-2-ol (MBO) is an important biogenic volatile organic compound (BVOC) emitted by pine trees and a potential precursor of atmospheric secondary organic aerosol (SOA) in forested regions. In the present study, hydroxyl radical (OH)-initiated oxidation of MBO was examined in smog chambers under varied initial nitric oxide (NO) and aerosol acidity levels. Results indicate measurable SOA from MBO under low-NO conditions. Moreover, increasing aerosol acidity was found to enhance MBO SOA. Chemical characterization of laboratory-generated MBO SOA reveals that an organosulfate species (C5H12O6S, MW 200) formed and was substantially enhanced with elevated aerosol acidity. Ambient fine aerosol (PM2.5) samples collected from the BEARPEX campaign during 2007 and 2009, as well as from the BEACHON-RoMBAS campaign during 2011, were also analyzed. The MBO-derived organosulfate characterized from laboratory-generated aerosol was observed in PM2.5 collected from these campaigns, demonstrating that it is a molecular tracer for MBO-initiated SOA in the atmosphere. Furthermore, mass concentrations of the MBO-derived organosulfate are well correlated with MBO mixing ratio, temperature, and acidity in the field campaigns. Importantly, this compound accounted for an average of 0.25% and as high as 1% of the total organic aerosol mass during BEARPEX 2009. An epoxide intermediate generated under low-NO conditions is tentatively proposed to produce MBO SOA.
doi:10.1021/es301648z
PMCID: PMC3557936  PMID: 22849588
8.  Identification and Characterization of Two Novel Proteins Affecting Fission Yeast γ-tubulin Complex FunctionV⃞ 
Molecular Biology of the Cell  2004;15(5):2287-2301.
The γ-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe γ-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to γ-tubulin–associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1+ or gfh1+ are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Δ and gfh1Δ cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of γ-tubulin complex components in organizing different types of microtubule structures in S. pombe.
doi:10.1091/mbc.E03-10-0728
PMCID: PMC404023  PMID: 15004232
9.  Postpyloric decompression tube placement through a gastrostomy for malignant bowel obstruction 
BMC Research Notes  2013;6:217.
Background
Malignant bowel obstruction affect a patient’s quality of life, but, management of MBO is controversial.
Case presentation
A 51-year-old woman who had been diagnosed as uterine cervix cancer 2 years ago and had undergone surgery, chemotherapy and radiotherapy, was admitted to our hospital. She was diagnosed as having a recurrence of peritoneal metastasis and bowel obstruction. For her nasal pain, we considered insertion of a postpyloric decompression tube through the gastrostomy instead of via the nasal cavity. After insertion of a percutaneous gastrostomy tube was performed endoscopically, we introduced a postpyloric decompression tube through her gastrostomy. She could be discharged home, and 91 days later, she died in her home under hospice care, as she had wished.
Conclusions
Insertion of a postpyloric decompression tube through a gastrostomy might be useful in the management of advanced cancer patients with bowel obstruction.
doi:10.1186/1756-0500-6-217
PMCID: PMC3680198  PMID: 23731859
Malignant bowel obstruction; Gastrostomy; Palliative care; Quality of life
10.  Do transcriptional enhancers also augment DNA replication? 
Nucleic Acids Research  1988;16(23):11207-11222.
Enhancers are DNA elements that augment transcription in cis, independent of distance and orientation. Evidence such as hormone dependent neoplastic cell growth and the stimulation of viral replication by sequences present in enhancers suggests that enhancers may also directly affect DNA replication. We tested this hypothesis in recombinant plasmids by asking whether sequences that stimulated DNA replication shared the properties of transcriptional enhancers. The homologous simian virus 40 (SV40) core enhancer was ligated either adjacent to or 2.6 kb distant from the SV40 minimal origin of replication (ori) in both orientations. Plasmids were transfected into T antigen producing COS cells, and episomal DNA was harvested for quantitation of replication. Replication could be assessed either as accumulation of fmol of MboI sensitive progeny DNA, or as a transition in % DNA in replicated (MboI sensitive) versus unreplicated (DpnI sensitive) form. The two measures were related exponentially (r = 0.86). The SV40 enhancer augmented replication 1.5-10 fold. The effect was time dependent, distance dependent (only the adjacent enhancer locus stimulated replication), partially orientation dependent, and enhancer copy number independent. Phorbol ester did not affect replication. The heterologous glucocorticoid enhancer had no effect on replication. We conclude that the SV40 enhancer's cis-effect on replication seems to be dependent on the close proximity to the replication origin of specific homologous sequences within the enhancer, rather than a typical enhancer-like effect.
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PMCID: PMC339005  PMID: 2849759
11.  Mechanism and Kinetics of Inducible Nitric Oxide Synthase Auto-S-Nitrosation and Inactivation† 
Biochemistry  2012;51(5):1028-1040.
Nitric oxide (NO), the product of the nitric oxide synthase (NOS) reaction, was previously shown to result in S-nitrosation of the NOS Zn2+-tetrathiolate and inactivation of the enzyme. To probe the potential physiological significance of NOS S-nitrosation, the inactivation timescale of the inducible NOS isoform (iNOS) was determined and found to directly correlate with an increase in iNOS S-nitrosation. A kinetic model of NOS inactivation in which arginine is treated as a suicide substrate was developed. In this model, NO synthesized at the heme cofactor is partitioned between release into solution (NO release pathway) and NOS S-nitrosation followed by NOS inactivation (inactivation pathway). Experimentally determined progress curves of NO formation were fit to the model. The NO release pathway was perturbed through addition of the NO traps oxymyoglobin (MbO2) and β2 H-NOX, which yielded partition ratios between NO release and inactivation of ~100 at 4 μM MbO2 and ~22,000 at saturating trap concentrations. The results suggest that a portion of the NO synthesized at the heme cofactor reacts with the Zn2+-tetrathiolate without being released into solution. Perturbation of the inactivation pathway through addition of the reducing agents GSH or TCEP resulted in a concentration-dependent decrease in iNOS S-nitrosation that directly correlated with protection from iNOS inactivation. iNOS inactivation was most responsive to physiological concentrations of GSH with an apparent Km value of 13 mM. NOS turnover that leads to NOS S-nitrosation might be a mechanism to control NOS activity, and NOS S-nitrosation could play a role in the physiological generation of nitrosothiols.
doi:10.1021/bi201818c
PMCID: PMC3277664  PMID: 22242685
12.  The International Conference on Malignant Bowel Obstruction: A Meeting of the Minds to Advance Palliative Care Research 
Journal of pain and symptom management  2007;34(1 Suppl):S1-S6.
There is a dearth of well-designed clinical research focusing on palliative care in cancer patients, especially those who are near the end of life. Reasons for this include ethical dilemmas in conducting such trials, communication barriers between specialties, and unclear standards for best care practices. To ensure that patients with incurable illnesses are offered the best available care, it is essential to develop and disseminate research methodologies well suited to this population. Given the multidimensional and culture-dependent nature of the end-of-life experience, it is necessary to adopt an interdisciplinary approach to developing research methods. As a means of initiating the process of palliative clinical research methodology development, malignant bowel obstruction (MBO) was used as a model to develop a research protocol. Although many treatment options for MBO have been proposed, existing literature offers little guidance with regard to algorithms for optimal management. To this end, an interdisciplinary summit of international leaders in quality-of-life research, ethno-cultural variability, palliative medicine, surgical oncology, gastroenterology, major consortium research, medical ethics, and patient advocacy/cancer survivors was convened in Pasadena, California, on November 12-13, 2004. Participants also represented the broad ethnic and racial perspectives required to develop culturally sensitive research methods. Consensus on methodological approaches was attained through vigorous debate. Using the conference-developed MBO model to implement trials will advance palliative care research.
doi:10.1016/j.jpainsymman.2007.04.005
PMCID: PMC2834265  PMID: 17544251
Palliative care research; malignant bowel obstruction; quality of life; end of life care
13.  Novel F141L Pre-S2 Mutation in Hepatitis B Virus Increases the Risk of Hepatocellular Carcinoma in Patients with Chronic Genotype C Infections ▿  
Journal of Virology  2010;85(1):123-132.
Several lines of evidence have suggested that some naturally occurring mutations of hepatitis B virus (HBV) play a critical role in hepatocellular carcinoma (HCC). Here, we describe a novel HCC-related pre-S2 mutation, F141L. To prove the relationship between the F141L mutation and HCC, molecular epidemiology studies using MboII PCR restriction analysis (PRA) were performed, and the molecular mechanism was investigated through construction of a stable hepatocyte cell line expressing the large surface HB protein (LHB) with the F141L mutation (F141L-LHB). Application of MboII PRA to samples from 241 Korean patients with chronic liver diseases of different clinical stages confirmed that F141L mutants were significantly related to HCC, even in comparison to liver cirrhosis (HCC, 26.3% of patients, or 26/99; liver cirrhosis, 3.8% of patients, or 2/52; P = 0.001). By studying stable cell lines, we found that F141L-LHBs could induce cell cycle progression by downregulating the p53 and p21 pathways and upregulating CDK4 and cyclin A. Furthermore, we found that in a colony-forming assay, the colony-forming rates in cell lines expressing F141L-LHBs were about twice as high as those of the wild type. In conclusion, our results suggest that F141L-LHBs may contribute importantly to the pathogenesis of HCC by inducing cell proliferation and transformation. So, the F141L mutation examined in this study could serve as a diagnostic marker for the prognosis of HCC.
doi:10.1128/JVI.01524-10
PMCID: PMC3014212  PMID: 20962085
14.  Mangotoxin production of Pseudomonas syringae pv. syringae is regulated by MgoA 
BMC Microbiology  2014;14:46.
Background
The antimetabolite mangotoxin is a key factor in virulence of Pseudomonas syringae pv. syringae strains which cause apical necrosis of mango trees. Previous studies showed that mangotoxin biosynthesis is governed by the mbo operon. Random mutagenesis led to the identification of two other gene clusters that affect mangotoxin biosynthesis. These are the gacS/gacA genes and mgo operon which harbors the four genes mgoBCAD.
Results
The current study shows that disruption of the nonribosomal peptide synthetase (NRPS) gene mgoA resulted in loss of mangotoxin production and reduced virulence on tomato leaves. Transcriptional analyses by qPCR and promoter reporter fusions revealed that mbo expression is regulated by both gacS/gacA and mgo genes. Also, expression of the mgo operon was shown to be regulated by gacS/gacA. Heterologous expression under the native promoter of the mbo operon resulted in mangotoxin production in non-producing P. syringae strains, but not in other Pseudomonas species. Also introduction of the mbo and mgo operons in nonproducing P. protegens Pf-5 did not confer mangotoxin production but did enhance transcription of the mbo promoter.
Conclusions
From the data obtained in this study, we conclude that both mbo and mgo operons are under the control of the gacS/gacA two-component system and that the MgoA product acts as a positive regulator of mangotoxin biosynthesis.
doi:10.1186/1471-2180-14-46
PMCID: PMC3945005  PMID: 24555804
Antimetabolite toxin; mgo operon; GacS/GacA; Plant-microbe interaction
15.  A possible structure for calf satellite DNA I. 
Nucleic Acids Research  1976;3(10):2677-2696.
Calf satellite DNA I (p = 1.715) has been hydrolysed by a number or restriction endonucleases. It consists of a repeating unit of 1460 nucleotide pairs within which the sites of Eco R II Mbo I, Sac I, Alu I, Ava II and Hha I were localised in comparison with those of Eco R I and Hind II. The distribution of the Hpa II, Sac I, Hha I, Hinf I and Mbo II sites within calf satellite DNA I, as well as that of several restriction endonuclease sites within calf satellite DNA III (p = 1.705) allowed me to define subsatellite fractions. Furthermore, some of the sites of the CpG containing restriction enzymes Hpa II and Hha I are lacking. The possible implications of these results are discussed.
PMCID: PMC343121  PMID: 995648
16.  A novel strategy for the identification of protein–DNA contacts by photocrosslinking and mass spectrometry 
Nucleic Acids Research  2004;32(16):e132.
Photochemical crosslinking is a method for studying the molecular details of protein–nucleic acid interactions. In this study, we describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocrosslinking, proteolytic digestion, Fe3+-IMAC (immobilized metal affinity chromatography) purification of peptide–oligodeoxynucleotide heteroconjugates and hydrolysis of oligodeoxynucleotides by hydrogen fluoride (HF), with efficient matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The new method is illustrated by the identification of the DNA-binding site of the restriction endonuclease MboI. Photoactivatable 5-iododeoxyuridine was incorporated into a single site within the DNA recognition sequence (GATC) of MboI. Ultraviolet irradiation of the protein–DNA complex with a helium/cadmium laser at 325 nm resulted in 15% crosslinking yield. Proteolytic digestion with different proteases produced various peptide–oligodeoxynucleotide adducts that were purified together with free oligodeoxynucleotide by Fe3+-IMAC. A combination of MS analysis of the peptide–nucleosides obtained after hydrolysis by HF and their fragmentation by MS/MS revealed that Lys209 of MboI was crosslinked to the MboI recognition site at the position of the adenine, demonstrating that the region around Lys209 is involved in specific binding of MboI to its DNA substrate. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of crosslinked complexes.
doi:10.1093/nar/gnh131
PMCID: PMC519130  PMID: 15383647
17.  Control of partial digestion combining the enzymes dam methylase and MboI. 
Nucleic Acids Research  1989;17(23):9571-9582.
A method is described which allows the preparation of reproducible partial digests without previous establishment of the incubation conditions. It is based on a combined application of dam methylase and the restriction endonuclease MboI, both recognizing the sequence 5'-GATC-3' but MboI unable to cut the methylated site. Due to their competition for the same substrate the DNA is partially digested, with the size of the resulting fragments strongly dependent on the ratio of enzymes. The Km of the dam methylase was determined to be 115 ng DNA/microliters indicating a variance in fragment sizes generated at low DNA-concentrations. This effect is minimized above 150 ng/microliters. Any influence of digestion time is avoided, because the reaction runs until complete modification of all sites. The dependence on enzyme concentration and presence of agarose was checked. Knowledge of these parameters allows an accurate prediction of fragment sizes generated at different conditions. The technique was successfully used to construct libraries from different sources, in particular chromosome-specific libraries from small amounts of flow-sorted material.
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PMCID: PMC335198  PMID: 2602137
18.  It's the Data! 
Three articles from the early years of Molecular Biology of the Cell (MBoC) have had remarkably many citations in the literature since their publication ∼10 years ago. As a coauthor of these articles and the former editor of MBoC, I was asked for possible explanations. I believe the answer lies in the unusual nature of these articles: each presents and summarizes gene expression data for nearly every gene in the yeast or human genomes. Continuing interest in the data themselves by cell biologists, rather than results or conclusions drawn by the authors, best accounts for the citation history. The flatness of the numbers of citations over time, the continuing high rate of accesses to individual Web sites set up to allow searching and display of the underlying data, and the large fraction of citations in journals focused on mathematics and computation all support the same conclusion: it's the data.
doi:10.1091/mbc.E09-07-0575
PMCID: PMC2801718  PMID: 20048255
19.  Magnetic Structures of LiMBO3 (M = Mn, Fe, Co) Lithiated Transition Metal Borates 
Inorganic Chemistry  2013;52(20):11966-11974.
The magnetic ordering within LiMBO3 compounds (M = Mn, Fe, and Co) has been explored by magnetization measurements and neutron powder diffraction. For all M, an incommensurately ordered magnetic phase is established on cooling, followed by a change to a commensurate long-range antiferromagnetic state below TN2 = 12(1) K for LiMnBO3, TN2 = 25(1) K for LiFeBO3, and TN2 = 12(1) K for LiCoBO3. For LiMnBO3, the magnetic ordering at T = 2 K exhibits a propagation vector k = (1, 0, 0) and consists of antiferromagnetic chains that are coupled antiferromagnetically to each other, the magnetic moments being oriented along the [001] direction. In contrast, the magnetic order at T = 2 K in LiFeBO3 and LiCoBO3 exhibits a propagation vector of k = (1/2, 1/2, 1/2) and consists of ferromagnetic chains that are antiferromagnetically coupled. The magnetic moments lie roughly along the [023̅] direction within the bc plane for LiFeBO3, and along the [301̅] direction within the ac plane for LiCoBO3. The moment orientations in both LiMnBO3 and LiFeBO3 suggest an Ising character arising from unquenched orbital momentum due to unusual trigonal bipyrimidal coordination environments. No evidence of Ising behavior is found in the case of LiCoBO3.
Magnetization measurements and neutron powder diffraction reveal incommensurate and then commensurate long-range antiferromagnetic ordering below 12 K for LiCoBO3 and LiMnBO3, and below 25 K for LiFeBO3. Uncommon trigonal bipyramid environments can give rise to novel electronic and magnetic phenomena.
doi:10.1021/ic401671m
PMCID: PMC3805329  PMID: 24087888
20.  Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus. 
Streptomyces cinnamomeus Tü89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.
PMCID: PMC168661  PMID: 9293006
21.  Mouse mammary tumor proviruses from a T-cell lymphoma are associated with the retroposon L1Md. 
Journal of Virology  1988;62(2):472-478.
Four Charon 4A clones containing mouse mammary tumor virus (MMTV) proviruses and their cellular flanking sequences were obtained from partial EcoRI libraries of a C57BL/6 T-cell lymphoma with both endogenous and newly acquired MMTV proviruses. The cellular flanking sequences of three of four MMTV proviruses contained DNA homologous to the 3' end of the long interspersed retroposon L1Md. Two of the three proviruses were newly acquired in the lymphoma DNA, and these MMTV proviruses appeared to be 5 kilobases downstream and in the same transcriptional orientation as the L1 sequence. The third provirus was endogenous Mtv-9 and was located less than 500 base pairs from the 3' end of L1. Seven additional clones containing MMTV proviruses were isolated from partial MboI libraries of a B6 T-cell lymphoma. Five of the seven clones contained L1 elements in the cellular DNA flanking MMTV DNA. At least two clones (including one with the Mtv-8 provirus) had multiple L1 copies flanking the MMTV provirus, and one clone contained a single MMTV long terminal repeat directly integrated into a truncated L1 sequence. Although the frequencies of B1 and L1 in random library clones were similar, only one MMTV-containing clone hybridized to the abundant repetitive element B1. These data suggest a nonrandom association between MMTV and L1Md.
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PMCID: PMC250557  PMID: 2826809
22.  Purification and properties of the MboII, a class-IIS restriction endonuclease. 
Nucleic Acids Research  1992;20(3):433-438.
After five purification steps a homogeneous preparation of endonuclease MboII was obtained, and several properties of the enzyme were determined. MboII is a monomer, with Mr under native and denaturing conditions being 47-49 x 10(3) Da. Endonuclease MboII is a basic protein (pI 8.3) which remains active when Mg2+ is replaced by Mn2+, Co2+, Ca2+, or Fe2+. MboII exhibits a star activity in the presence of some of the following reagents or ions: DMSO, glycerol, ethanol (and Co2+ or Mn2+ at pH 6). MboII does not bend DNA and is heat sensitive, losing activity after 15 min at 50 degrees C.
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PMCID: PMC310404  PMID: 1741276
23.  Cleavage map of BK virus DNA with restriction endonucleases MboI and HaeIII. 
Journal of Virology  1978;27(3):700-712.
Specific cleavage of BK virus (MM) DNA with restriction endonuclease MboI gives rise to 10 fragments. Two techniques were used to determine the location of these fragments on the viral genome with respect to the three known sites for HindIII cleavage. In the first method, reciprocal digestion, individual MboI fragments were digested with HindIII and individual HindIII fragments were digested with MboI. In the second method, single-end 32P-labeled HindIII subfragments were partially digested with MboI, and then the sizes of the radioactive partial products were used to deduce the nearest neighboring fragment. Information from these two methods is more than adequate to map all the MboI enzyme sites. Cleavage of BK virus (MM) DNA with restriction enzyme HaeIII produces 21 fragments. With the aid of the same two methods, these fragments have also been ordered with respect to the known map locations of the HindIII and MboI sites.
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PMCID: PMC525858  PMID: 212590
24.  The Mangotoxin Biosynthetic Operon (mbo) Is Specifically Distributed within Pseudomonas syringae Genomospecies 1 and Was Acquired Only Once during Evolution 
Mangotoxin production was first described in Pseudomonas syringae pv. syringae strains. A phenotypic characterization of 94 P. syringae strains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair specific for the mbo operon to examine its distribution within the P. syringae complex. These primers amplified a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor the mbo operon, whereas 35 non-mangotoxin-producing strains did not yield any amplification. This, together with the analysis of draft genomes, allowed the identification of the mbo operon in five pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of the mbo genes in the P. syringae complex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing the mbo operon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. The mbo operon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that the mbo operon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within the P. syringae complex.
doi:10.1128/AEM.03007-12
PMCID: PMC3568555  PMID: 23144138
25.  Isolation and partial characterization of plasmid DNA from Streptococcus thermophilus. 
Of 23 strains of Streptococcus thermophilus examined, 5 were found to contain a single small cryptic plasmid, designated pHM1 through pHM5. Through analysis by restriction endonuclease mapping and DNA-DNA hybridization, the five plasmids were found to be closely related. They were present in 4 to 18 copies per cell and ranged in size from 1.4 to 2.2 megadaltons. Plasmids pHM1 and pHM5, as well as pHM2 and pHM4, were found to be identical. Single restriction endonuclease sites were observed on each plasmid for PvuII and MboI. Plasmids pHM2 and pHM3 each had an additional single site for HhaI, and pHM1 had an additional single site for HindIII. The characteristics of these plasmids may make them useful for the development of cloning vectors for use in S. thermophilus.
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PMCID: PMC291801  PMID: 3002269

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