Dan S. Ray

Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

A type II DNA topoisomerase (topollmt), purified to near homogeneity from the trypanosomatid C. fasciculata has been shown to be localized to the single mitochondrion of these kinetoplastid protozoa. Immunoblots show at least a 10-fold higher level of topollmt (per milligram of protein) in preparations of partially purified mitochondria as compared with those from whole cells. Analyses of type I and type II topoisomerase activities in both mitochondrial and whole cell extracts show a 4- to 5-fold higher specific activity of topollmt in mitochondrial extracts while a nuclear type I topoisomerase has a 4- to 5-fold lower specific activity in the same extract. Immunolocalizations using anti-topollmt antibodies show the enzyme to be present in close association with the mitochondrial DNA networks (kinetoplast DNA or kDNA). This association appears at two distinct locations on opposite sides of the kDNA network.

High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA

Bacteriophage M13 gene II has been cloned in the plasmid expression vector pING1 and thereby placed under the control of the inducible araB promoter of Salmonella typhimurium. Upon induction with arabinose, gene II is transcribed as part of a polycistronic messenger RNA which initiates at the araB promoter. Subsequent translation of this message results in the coordinate, high-level expression of several proteins, including the gene II protein. Using this expression system, we have been able to overproduce gene II protein to a level of almost 15% of the total protein in Escherichia coli cells, thus providing an abundant source for its purification.

Nucleus-encoded histone H1-like proteins are associated with kinetoplast DNA in the trypanosomatid Crithidia fasciculata.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His6-tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

Mitochondrial DNA Ligases of Trypanosoma brucei

ABSTRACT The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG kα and LIG kβ) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG kα and LIG kβ are mitochondrial DNA ligases. Epitope-tagged LIG kα localizes throughout the kDNA, whereas LIG kβ shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG kα mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.

Replication of bacteriophage M13: I. Sedimentation analysis of crude lysates of M13-infected bacteria

Velocity sedimentation at high ionic strength, of DNA released from M13-infected bacteria lysed under gentle conditions, provides a simple method for separating phage-specific DNA components from bacterial DNA. 1. 1. Components sedimenting at rates of 0.71 and 0.57 times that of viral single strands are identified as component I replicative form (RFI-DNA) and component II replicative form (RFII-DNA), respectively. 2. 2. The major phage DNA component early ( 45 min) the major phage DNA component is intracellular single strands. 3. 3. The source of intracellular single strands is a pool of free DNA molecules rather than progeny phage particles.

Palliative care in the ICU: relief of pain, dyspnea, and thirst—A report from the IPAL-ICU Advisory Board

AbstractPurposePain, dyspnea, and thirst are three of the most prevalent, intense, and distressing symptoms of intensive care unit (ICU) patients. In this report, the interdisciplinary Advisory Board of the Improving Palliative Care in the ICU (IPAL-ICU) Project brings together expertise in both critical care and palliative care along with current information to address challenges in assessment and management.MethodsWe conducted a comprehensive review of literature focusing on intensive care and palliative care research related to palliation of pain, dyspnea, and thirst.ResultsEvidence-based methods to assess pain are the enlarged 0–10 Numeric Rating Scale (NRS) for ICU patients able to self-report and the Critical Care Pain Observation Tool or Behavior Pain Scale for patients who cannot report symptoms verbally or non-verbally. The Respiratory Distress Observation Scale is the only known behavioral scale for assessment of dyspnea, and thirst is evaluated by patient self-report using an 0–10 NRS. Opioids remain the mainstay for pain management, and all available intravenous opioids, when titrated to similar pain intensity end points, are equally effective. Dyspnea is treated (with or without invasive or noninvasive mechanical ventilation) by optimizing the underlying etiological condition, patient positioning and, sometimes, supplemental oxygen. Several oral interventions are recommended to alleviate thirst. Systematized improvement efforts addressing symptom management and assessment can be implemented in ICUs.ConclusionsRelief of symptom distress is a key component of critical care for all ICU patients, regardless of condition or prognosis. Evidence-based approaches for assessment and treatment together with well-designed work systems can help ensure comfort and related favorable outcomes for the critically ill.

Replication of bacteriophage M13: II. The role of replicative forms in single-strand synthesis

Abstract Sucrose-gradient sedimentation of DNA from cells pulse-labeled with tritiated thymidine at 130 minutes after M13 infection shows a flow of material from RF ‡ to progeny single strands. For pulses less than 40 seconds, most of the phagospecific DNA sediments at the rates of RFI and RFII. Pulses for 15 seconds or less show a third component sedimenting even more rapidly than RFI. All of the pulse label in this component is contained in linear viral strands, some of which sediment even more rapidly than circular viral DNA in alkaline sucrose gradients. The label in RFII is contained almost entirely in linear viral strands of unit length. The relative amount of label in RFII decreases with increasing pulse lengths up to about 180 seconds. After a lag of at least 40 seconds, label rapidly accumulates in progeny single strands; by five minutes the distribution of label is similar to that observed in long-term labeling with 70 to 80% of the label in single-stranded DNA. When pulse-labeling is followed by growth in unlabeled medium, most of the incorporated label is chased into progeny single strands. Label is found in both viral and complementary strands of RF only after long-term labeling from the time of infection. Labeled complementary strands of RF molecules are conserved during further growth in unlabeled medium while labeled viral strands are displaced from the RF as new viral strands are synthesized.

Formation of the parental replicative form DNA of bacteriophage φX174 and initial events in its replication

Intracellular φX174 DNA was studied under a variety of conditions that prevent the replication of the parental replicative form DNA. These conditions included treatment with 150 μg of chloramphenicol per ml., the use of the rep3− mutation of the host cell, amber mutation (am 8) in the viral gene responsible for RF replication (gene A)† and combinations thereof. In all cases the majority of the parental RF was in the covalently closed form (RFI). The relative amount of RF with a discontinuity in one strand (RFII) in these cases was between 2 and 10% of the total RF and independent of the multiplicity of infection. The only exception was seen in infections of rep3 cells with φX am 3 (a mutant in the lysis gene, gene E, used as a wild-type representative). In this case a fairly constant absolute amount of RFII (1 to 4 per cell), independent of the multiplicity of infection, was formed, consisting almost exclusively of a closed complementary and an open parental viral strand. Since the formation of this type of RFII was dependent on protein synthesis and the presence of the product of φX gene A, it is concluded that the discontinuity in the parental viral strand represents the result of the action of the gene A product on the DNA. Possible mechanisms for the mode of action of the gene A product are discussed. Intermediates during the synthesis of the first complementary strand were isolated from cells infected with ultraviolet light-irradiated phages. Such intermediates contained incomplete linear complementary strands and circular parental viral strands. It is therefore concluded that the virus-specified discontinuity in RFII is introduced after the first complementary strand is completed and closed. Some residual virus-specific DNA synthesis beyond the formation of the parental RF was seen in rep3− cells in infections where the viral gene for RF replication was functioning. This residual synthesis resulted in replicative intermediate structures containing circular complementary strands and elongated linear viral strands. The significance of this type of RI for the existing models of φX174 RF replication is discussed.

Cloning of a functional replication origin of phage G4 into the genome of phage M13.

Abstract A chimeric single-stranded DNA phage, M13G ori 1, has been formed as a result of the in vitro insertion of a 2216 base-pair Hae II fragment of bacteriophage G4 replicative form DNA into the replicative form DNA of bacteriophage M13. The inserted G4 DNA carries the dna G-dependent origin for G4 complementary strand synthesis. The cloned G4 origin functions both in vivo and in vitro in the conversion of M13G ori 1 single-stranded viral DNA to the duplex replicative form by a rifampicin-resistant mechanism. Labelling of the 3′ terminus of the single discontinuity in M13G ori 1 replicative form II molecules synthesized in crude extracts and subsequent restriction analysis indicate that M13G ori 1 complementary strand synthesis can be initiated at either the RNA polymeraseprimed M13 origin or at the dna G-primed G4 origin. The M13G ori 1 complementary strand initiated at the G4 origin terminates in the vicinity of the G4 origin after progressing around the circular template and traversing the M13 origin region, indicating the absence of a specific nucleotide sequence in the M13 origin for termination of the newly formed complementary strand. The ability of this chimeric phage to utilize the cloned G4 origin in vivo even in the presence of the presumed M13 pilot protein (gene 3 protein) indicate that the nucleotide sequence of the replication origin is sufficient for recognizing the appropriate initiation enzymes. Since decapsidation of M13 is tightly coupled to replicative form formation, initiation at the G4 origin, located over 1000 nucleotides from the M13 complementary strand origin, indicates that widely separated nucleotide sequences contained in the filamentous virion can be exposed to the cell cytoplasm during eclipse.