Search

Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.

Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

An efficient procedure for the generation of sequence-specific alterations of the vaccinia virus genome was demonstrated. Homologous DNA recombination within cells infected with vaccinia virus was used for the deletion or replacement of promoter sequences of the viral growth factor gene by a procedure comparable to transplacement in Saccharomyces cerevisiae. This DNA replacement procedure can potentially be used to generate any sequence alteration within the vaccinia virus genome. Deletion of growth factor promoter sequences resulted in a dramatic reduction in growth factor gene transcription and protein synthesis. Replacement of growth factor promoter sequences with promoter sequences of the strong constitutive 40-kDa gene resulted in an increase in gene transcription and protein synthesis and an altered temporal pattern of expression. Virus containing mutations in the growth factor gene demonstrated different plaque morphologies on cell culture monolayers.

Vaccination with irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting gene-transduced cancer vaccines induces tumoricidal immune responses. In a Phase I human gene therapy trial, eight immunocompetent prostate cancer (PCA) patients were treated with autologous, GM-CSF-secreting, irradiated tumor vaccines prepared from ex vivo retroviral transduction of surgically harvested cells. Expansion of primary cultures of autologous vaccine cells was successful to meet trial specifications in 8 of 11 cases (73%); the yields of the primary culture cell limited the number of courses of vaccination. Side effects were pruritus, erythema, and swelling at vaccination sites. Vaccine site biopsies manifested infiltrates of dendritic cells and macrophages among prostate tumor vaccine cells. Vaccination activated new T-cell and B-cell immune responses against PCA antigens. T-cell responses, evaluated by assessing delayed-type hypersensitivity (DTH) reactions against untransduced autologous tumor cells, were evident in two of eight patients before vaccination and in seven of eight patients after treatment. Reactive DTH site biopsies manifested infiltrates of effector cells consisting of CD45RO+ T-cells, and degranulating eosinophils consistent with activation of both Th1 and Th2 T-cell responses. A distinctive eosinophilic vasculitis was evident near autologous tumor cells at vaccine sites, and at DTH sites. B-cell responses were also induced. Sera from three of eight vaccinated men contained new antibodies recognizing polypeptides of 26, 31, and 150 kDa in protein extracts from prostate cells. The 150-kDa polypeptide was expressed by LNCaP and PC-3 PCA cells, as well as by normal prostate epithelial cells, but not by prostate stromal cells. No antibodies against prostate-specific antigen were detected. These data suggest that both T-cell and B-cell immune responses to human PCA can be generated by treatment with irradiated, GM-CSF gene-transduced PCA vaccines.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced, irradiated tumor vaccines induce potent, T-cell-mediated antitumor immune responses in preclinical models. We report the initial results of a Phase I trial evaluating this strategy for safety and the induction of immune responses in patients with metastatic renal cell carcinoma (RCC). Patients were treated in a randomized, double-blind dose-escalation study with equivalent doses of autologous, irradiated RCC vaccine cells with or without ex vivo human GM-CSF gene transfer. The replication-defective retroviral vector MFG was used for GM-CSF gene transfer. No dose-limiting toxicities were encountered in 16 fully evaluable patients. GM-CSF gene-transduced vaccines were equivalent in toxicity to nontransduced vaccines up to the feasible limits of autologous tumor vaccine yield. No evidence of autoimmune disease was observed. Biopsies of intradermal sites of injection with GM-CSF gene-transduced vaccines contained distinctive macrophage, dendritic cell, eosinophil, neutrophil, and T-cell infiltrates similar to those observed in preclinical models of efficacy. Histological analysis of delayed-type hypersensitivity responses in patients vaccinated with GM-CSF-transduced vaccines demonstrated an intense eosinophil infiltrate that was not observed in patients who received nontransduced vaccines. An objective partial response was observed in a patient treated with GM-CSF gene-transduced vaccine who displayed the largest delayed-type hypersensitivity conversion. No replication-competent retrovirus was detected in vaccinated patients. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous GM-CSF gene-transduced tumor vaccine for RCC patients.

The role of women as leaders has undergone considerable review in recent years, both outside and within the dental profession. Growing awareness of gender as an issue in leadership has focused attention on leadership styles and qualities with particular emphasis on the differences between male and female approaches. Further research is proposed, specifically on leadership within the dental profession. Trends are identified which lead to optimistic conclusions as to the future involvement of women in leadership in dentistry.

Preclinical studies with murine tumor models have demonstrated that autologous tumor cell vaccines engineered to secrete certain cytokines in a paracrine fashion elicit systemic immune responses capable of eliminating small amounts of established tumor. These results have engendered much interest in developing this strategy for gene therapy of human cancer. The major limitation to creating genetically modified autologous human tumor vaccines is efficient gene transfer into primary tumor explants, since the majority of human tumors fail to proliferate in long-term culture. Using the retroviral vector MFG in conjunction with short-term culture techniques, we have achieved, in the absence of selection, a mean transduction efficiency of 60% in primary renal, ovarian, and pancreatic tumor explants, and we have developed an autologous granulocyte-macrophage colony-stimulating factor secreting tumor vaccine for clinical trials.

Dental associations throughout the world face many new challenges. For example, many industrial countries confront the potential of excess manpower to meet current demands, while developing countries try to cope with scarce resources to control existing and increasing disease levels. This guide is intended to facilitate the development of oral promotion programmes in both situations. In industrialized countries, an oral health promotion programme should increase awareness and interest among consumers, thus facilitating the conversion of unmet need to demand. In developing countries, the oral health promotion guidelines demonstrate ways to extend resources to meet a broader base of need. In all countries, oral health promotion can demonstrate the benefit of self-care and emphasize the consumer's responsibility for personal health. Dental associations enjoy a long history of promoting oral health, yet developing a coherent programme or realizing the results of these efforts can sometimes be disappointing or frustrating. The guide is organized around a recommended sequence of activities: policy formation and dissemination; planning group; information gathering; goal setting; strategic planning; implementation; and evaluation. This document presents guidelines, not recipes. Given the range of economic, social and health needs of the Fédération Dentaire Internationale member countries, a recipe would be inappropriate. These guidelines can be tailored to the needs of your association based on the oral health needs of your population. They can be used to determine new or modified policies, to assess long- and short-term goals, to monitor progress and to educate the membership. Case studies derived from actual experiences of dental associations as they planned and analysed their efforts to promote oral health are presented. This information is included to highlight different approaches as well as problems encountered. The case studies illustrate various parts of the planning implementation and evaluation process, while the text should serve as a comprehensive review of all these components.

'Promoting Oral Health: Guidelines for Dental Associations' is the product of Working Group 3 on Oral Health Promotion of the Commission on Oral Health, Research and Epidemiology of the FDI. This paper describes the guidelines document, its rationale and its potential utility. An organized planned sequence of activities, including policy formation and dissemination, planning group structure and function, information gathering, goal setting, strategic planning of objectives and interventions, implementation as well as monitoring and evaluation, are reviewed. Relevant to both industrialized and developing countries, these oral health promotion guidelines can be used to develop programmes to demonstrate the benefit of self-care and appropriate demand for dental services.

The characterization of the three major membrane polypeptides indicated to be subunits of the light-harvesting bacteriochlorophyll-protein complexes of Rhodopseudomonas sphaeroides is described. Molecular weight determinations of the three polypeptides have been performed by sedimentation analysis and electrophoretic mobility and the values obtained were in agreement with each other as well as with those derived from compositional studies. NH2- and COOH-terminal amino acid determinations were performed for each of the species; two of the polypeptides produces no reaction with dansyl chloride and were concluded to have blocked amino termini. In each instance, the data were consistent with each of the polypeptides being a single, pure species. Further characterization of the proteins was accomplished by amino acid compositional analysis and peptide mapping of tryptic and chymotryptic digestions of the polypeptides. The number of peptides obtained from the digestions was in agreement with the amino acid compositional data. Two of the polypeptides, 15B and 15C, which possess very similar molecular weights, identical carboxyl termini and apparently blocked amino termini were shown by amino acid analysis and peptide mapping to be structurally discrete species, although related. The relationship between these purified proteins and the known activities and characteristics of the light-harvesting bacteriochlorophyll-protein complexes of R. sphaeroides is discussed.

A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.

Gas-phase dissociation of fluorine (1Ʃ+g) molecules in an agron bath at 3000 K was studied by using the 3D Monte Carlo classical trajectory (3DMCCT) method. To assess the importance of the potential energy surface (PES) in such calculations, three surfaces, with a fixed, experimentally determined F2dissociation energy, were constructed. These surfaces span the existing experimental uncertainties in the shape of the F2potential. The first potential was the widest and softest; in the second potential the anharmonicity was minimized. The intermediate potential was constructed to ‘localize’ anharmonicity in the energy range in which the collisions are most reactive. The remaining parameters for each PES were estimated from the best available data on interatomic potentials. By using the single uniform ensemble (SUE) method (Kutz, H. D. & Burns, G. J.chem. Phys. 72, 3652-3657 (1980)), large ensembles of trajectories (LET) were generated for the PES. Two such ensembles consisted of 30000 trajectories each and the third of 26200. It was found that the computed one-way-flux equilibrium rate coefficients (Widom, B.Science148, 1555-1560 (1965)) depend in a systematic way upon the anharmonicity of the potential, with the most anharmonic potential yielding the largest rate coefficient. Steady-state reaction-rate constants, which correspond to experimentally observable rate constants, were calculated by the SUE method. It was determined that this method yields (for a given trajectory ensemble, PES and translational temperature) a unique steady-state rate constant, independent of the initial, arbitrarily chosen, state (Tolman, R. C.The principles of statistical mechanics, p. 17. Oxford University Press (1938)) of the LET, and consequently independent of the corresponding initial value of the reaction rate coefficient. For each initial state of the LET, the development of the steady-state rate constant from the equilibrium rate coefficient was smooth, monotonic, and consistent with the detailed properties of the PES. It was found that, although the increased anharmonicity of the F2potential enhanced the equilibrium rate coefficients, it also enhanced the non-equilibrium effects. As a result, the steady-state rate constants were found to be insensitive to the variation of the PES. Thus, the differences among the steady-state rate constants for the three potentials were of the order of their standard errors, which was about 15% or less. On the other hand, the calculated rate constants exceeded the experimental rate constant by a factor of five to six. Because within the limitations of classical mechanics the calculations wereab initio, it was tentatively concluded that the discrepancy of five to six is due to the use of classical mechanics rather than details of the PES structure.

A nonspecific solubilization of the intracytoplasmic membrane proteins from Rhodopseudomonas sphaeroides has been achieved in the absence of detergents. Following removal of the photopigments and phospholipids by acetone/methanol extractions, the protein is quantitatively solubilized in 6 M guanidine thiocyanate which is subsequently exchanged by dialysis for 7 M urea. A urea-soluble protein fraction is obtained which represents 80% of the initial protein and qualitatively contains all the polypeptides of the membrane resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the exception of the reaction center polypeptides M and L, and the 35.2-kilodalton species. Fractionation of the urea-soluble polypeptides on a Sepharose CL-6B column resolves into separate fractions the two major low molecular weight polypeptides identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further examination of these fractions by isoelectric focusing and electrophoresis at pH 4.5 in the presence of 8 M urea reveals the presence of three major species each of which has been purified to homogeneity by a combination of ion exchange chromatography and preparative electrophoretic techniques. It is believed that these three major polypeptides are components of the light-harvesting pigment-protein complexes.

The transfer of phospholipids between two membrane substrates catalyzed by a soluble protein fraction from Rhodopseudomonas sphaeroides has been demonstrated. The assay employs purified intracytoplasmic membrane (ICM) vesicles derived from cells of R. sphaeroides grown on [3H]acetate as the phospholipid donor substrate and phosphatidylcholine (70%)/phosphatidylethanolamine (30%) unilamellar liposomes containing [14C]triolein, a nontransferable marker, as the acceptor substrate for transferred phospholipids. Incubation of these two membrane substrates with a 40 to 80% (NH4)2SO4 protein fraction from R. sphaeroides results in the transfer of tritium-labeled ICM phospholipids to the acceptor membrane substrate. Upon completion of the incubation period, the donor ICM vesicles are quantitatively separated from the acceptor liposomes by precipitation with antibody prepared against whole, purified ICM vesicles. Phospholipid transfer is linear with respect to time and protein concentration, is inhibited by trypsin and heat, and shows an absolute dependence upon the presence of acceptor liposomes and the 40 to 80% (NH4)2SO4 protein fraction. Control experiments indicate that no fusion of the donor and acceptor membrane occurs during the incubation period and that, following prolonged incubation there is no detectable degradation of the labeled lipid components. Preliminary data on the phospholipid specificity of the transfer reaction is also presented.

Sera from a patient with a chronic Epstein Barr virus (EBV) infection contained unusually high anti-EBV antibody titers (1:2560 to 1:10,240 for EA(D) and 1:5,120 to 1:40,960 for VCA). One of these serum samples was shown by immunoprecipitation to recognize at least 11 EBV-specific proteins from virus producer cells labeled in vivo and 10 EBV-specific proteins from in vitro translations of producer cell mRNA. Six of the in vivo labeled proteins (135,000, 89,000, 50,000 to 55,000 doublet, 46,000, and 34,000 daltons) are "early" by their resistance to phosphonoacetic acid, and five (350,000, 220,000, 160,000, 140,000, and 85,000 daltons) are "late" membrane and capsid proteins. The EBV-specific proteins immunoprecipitated from in vitro translations had molecular masses of 150,000, 140,000, 115,000, 52,000, 50,000, 45,000, 34,000, 29,000, 17,000, and 15,000. Subcellular fractionation studies of cells labeled in vivo revealed that the 135,000-dalton protein and part of the 50,000 to 55,000 dalton protein doublet were found in both the nuclear and the cytoplasmic fractions, and thus are good candidates to be components of the EA(D) diffuse-type immunofluorescence observed with many EA-positive sera.

It is shown that there will be strong orienting effects in the collision of two radicals which will invalidate attempts to calculate disproportionation–recombination ratios by simple geometric arguments.

A new type II sequence-specific endonuclease, RsaI, has been identified from Rhodopseudomonas sphaeroides strain 28/5. An RsaI purification scheme that yields enzyme which is free of contaminating exonuclease and phosphatase activities after a single column fractionation has been developed. The enzyme recognized the tetranucleotide sequence 5'-GTAC-3' and cleaved between the T and A, thereby generating flush ends. RsaI should be extremely useful in deoxyribonucleic acid sequencing experiments.

Principal progress was achieved in the field classical studies of diatom dissociation. 3-D trajectory calculations of dissociating bromine and fluorine were conducted over a range of temperatures, and using several significantly different potential energy surfaces. A library consisting over a two million trajectories was accummulated. These trajectories were used to test the strong collision assumption, important in several theories of unimolecular reactions and in some theories of thermal diatom dissociation. The assumption was shown to be not valid for the case of dissociating bromine and fluorine. Properties of the steady state of dissociating diatoms were investigated. Papers, now in preparation, involve study of nonequilibrium energy and angular momentum distribution functions; accumulation, interpretation and classification of data on inelastic and reactive collisions; and study of scaling factors and vibrational-rotational coupling in dissociating diatoms. Experiments, still in progress, aimed at measuring recombination rates of fluorine atoms. Time delayed photoelectric effect was explained in terms of a release of trapped electrons at the surface of photocathode.

Sera from a patient with a chronic Epstein Barr virus (EBV) infection contained unusually high anti-EBV antibody titers (1:2560 to 1:10,240 for EA(D) and 1:5,120 to 1:40,960 for VCA). One of these serum samples was shown by immunoprecipitation to recognize at least 11 EBV-specific proteins from virus producer cells labeled in vivo and 10 EBV-specific proteins from in vitro translations of producer cell mRNA. Six of the in vivo labeled proteins (135,000, 89,000, 50,000 to 55,000 doublet, 46,000, and 34,000 daltons) are "early" by their resistance to phosphonoacetic acid, and five (350,000, 220,000, 160,000, 140,000, and 85,000 daltons) are "late" membrane and capsid proteins. The EBV-specific proteins immunoprecipitated from in vitro translations had molecular masses of 150,000, 140,000, 115,000, 52,000, 50,000, 45,000, 34,000, 29,000, 17,000, and 15,000. Subcellular fractionation studies of cells labeled in vivo revealed that the 135,000-dalton protein and part of the 50,000 to 55,000 dalton protein doublet were found in both the nuclear and the cytoplasmic fractions, and thus are good candidates to be components of the EA(D) diffuse-type immunofluorescence observed with many EA-positive sera.

The study originated from the common need felt by the United States Public Health Service and the World Health Organization for objective information about various national dental care delivery systems and their effectiveness in the societies in which they were used. The relationships between consumers, providers and administrators within each system were emphasized rather than the comparison between systems since each operated within a specific biological and ecological environment. Six systems all of which had been in existence for at least twenty years were originally selected for study to encompass those dependent on a majority of government or private enterprise, use or non-use of auxiliaries, differeing systems of financing and of definition of target groups for receipt of services. The countries originally taking part were Australia, the Federal Republic of Germany, Japan, New Zealand, Norway and Bulgaria, though Bulgaria withdrew early in 1975.

The psychosocial sequelae of craniofacial disfigurement may have as great an impact on the patient as the strictly physical aspects of the problem. Very little systematic work has been focused directly on these effects. The following broad recommendations would constitute initial research steps in this field: Development of satisfactory measures of physical attractiveness and their use in studies to explore the role of craniofacial features in over-all physical attractiveness. The establishment of valid metrics for assessing the severity of craniofacial anomalies through the use of both physiologic and behavioral measures, thus constructing a broader definition of what constitutes a craniofacial handicap. Studies of the relationships among physiologic and behavioral variables using recently developed statistical techniques and computer methods to determine the psychosocial consequences of craniofacial disfigurement. Studies of the process through which persons with various types of malocclusion decide to seek and complete treatment. The studies would include the patients' demographic characteristics, self-perceptions, perceptions of them by others, and the complex patient-clinician interactions during the treatment programs.

Strategies to lower the barriers to both the receipt of personal dental services and to self-care are discussed in the light of the supporting body of socio-dental research. Planning to reach populations in need of care but who have not obtained such care involves both external marketing strategies as well as subsequent internal marketing strategies. Modifications of elements in the dental care delivery system, and approaches which impact on personal lifestyles of populations in need of care as well as on their environments, constitute a comprehensive marketing plan.

The characterization of the three major membrane polypeptides indicated to be subunits of the light-harvesting bacteriochlorophyll-protein complexes of Rhodopseudomonas sphaeroides is described. Molecular weight determinations of the three polypeptides have been performed by sedimentation analysis and electrophoretic mobility and the values obtained were in agreement with each other as well as with those derived from compositional studies. NH2- and COOH-terminal amino acid determinations were performed for each of the species; two of the polypeptides produces no reaction with dansyl chloride and were concluded to have blocked amino termini. In each instance, the data were consistent with each of the polypeptides being a single, pure species. Further characterization of the proteins was accomplished by amino acid compositional analysis and peptide mapping of tryptic and chymotryptic digestions of the polypeptides. The number of peptides obtained from the digestions was in agreement with the amino acid compositional data. Two of the polypeptides, 15B and 15C, which possess very similar molecular weights, identical carboxyl termini and apparently blocked amino termini were shown by amino acid analysis and peptide mapping to be structurally discrete species, although related. The relationship between these purified proteins and the known activities and characteristics of the light-harvesting bacteriochlorophyll-protein complexes of R. sphaeroides is discussed.

A nonspecific solubilization of the intracytoplasmic membrane proteins from Rhodopseudomonas sphaeroides has been achieved in the absence of detergents. Following removal of the photopigments and phospholipids by acetone/methanol extractions, the protein is quantitatively solubilized in 6 M guanidine thiocyanate which is subsequently exchanged by dialysis for 7 M urea. A urea-soluble protein fraction is obtained which represents 80% of the initial protein and qualitatively contains all the polypeptides of the membrane resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the exception of the reaction center polypeptides M and L, and the 35.2-kilodalton species. Fractionation of the urea-soluble polypeptides on a Sepharose CL-6B column resolves into separate fractions the two major low molecular weight polypeptides identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further examination of these fractions by isoelectric focusing and electrophoresis at pH 4.5 in the presence of 8 M urea reveals the presence of three major species each of which has been purified to homogeneity by a combination of ion exchange chromatography and preparative electrophoretic techniques. It is believed that these three major polypeptides are components of the light-harvesting pigment-protein complexes.

Plasmids containing the vaccinia virus thymidine kinase gene, its flanking DNA sequences, and the Escherichia coli beta-galactosidase gene were used in conjunction with a thymidine kinase-deficient virus to examine the viral products of recombination. Progeny derived from single-crossover events could be distinguished from those generated by gene conversion or double-crossover events when the beta-galactosidase gene was separated from the thymidine kinase gene by the flanking sequences. Using methotrexate to select for recombinant virus and a chromogenic indicator to detect beta-galactosidase, the generation of viral recombinants was measured over a 48-h period. Recombinant progeny were first observed at 12 h and increased to a maximum of 2.5% at 48 h. Single-crossover products, as determined by beta-galactosidase expression, reached a maximum of 57% of the recombinant population at 24 h and thereafter declined. DNA hybridization analysis was used to examine genomic structures of the progeny of the initial viral plaques, plaques purified three times, and those subject to a 10(4)-fold amplification. These analyses confirmed that single-crossover events within either the 5'- or 3'-homologous flanking sequences generated unstable recombinant structures. These structures were shown to contain a single copy of the intact thymidine kinase gene within the corresponding copy of the duplicated thymidine kinase flanking sequences, separated by the beta-galactosidase gene and plasmid DNA. Significantly, these duplicated structures could undergo further recombination to produce repeats of either the intact or the deleted thymidine kinase sequences. These intermediate structures ultimately degenerated to produce either the parental thymidine kinase-deleted or the wild-type genome. The wild-type genome was also shown to be generated directly by gene conversion or double-crossover events.

The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5' and 3' termini were precisely located by S1 nuclease analysis.

The transfer of phospholipids between two membrane substrates catalyzed by a soluble protein fraction from Rhodopseudomonas sphaeroides has been demonstrated. The assay employs purified intracytoplasmic membrane (ICM) vesicles derived from cells of R. sphaeroides grown on [3H]acetate as the phospholipid donor substrate and phosphatidylcholine (70%)/phosphatidylethanolamine (30%) unilamellar liposomes containing [14C]triolein, a nontransferable marker, as the acceptor substrate for transferred phospholipids. Incubation of these two membrane substrates with a 40 to 80% (NH4)2SO4 protein fraction from R. sphaeroides results in the transfer of tritium-labeled ICM phospholipids to the acceptor membrane substrate. Upon completion of the incubation period, the donor ICM vesicles are quantitatively separated from the acceptor liposomes by precipitation with antibody prepared against whole, purified ICM vesicles. Phospholipid transfer is linear with respect to time and protein concentration, is inhibited by trypsin and heat, and shows an absolute dependence upon the presence of acceptor liposomes and the 40 to 80% (NH4)2SO4 protein fraction. Control experiments indicate that no fusion of the donor and acceptor membrane occurs during the incubation period and that, following prolonged incubation there is no detectable degradation of the labeled lipid components. Preliminary data on the phospholipid specificity of the transfer reaction is also presented.