The National Institutes of Health system for enhancing the science,safety, and ethics of recombinant DNA research

Oversight of recombinant DNA research by the National Institutes of Health (NIH) is predicated on ethical and scientific responsibilities that are akin, in many ways, to those that pertain to the oversight of animal research. The NIH system of oversight, which originated more than 25 years ago, is managed by the NIH Office of Biotechnology Activities (OBA), which uses various tools to fulfill its oversight responsibilities. These tools include the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) and the Recombinant DNA Advisory Committee. The OBA also undertakes special initiatives to promote the analysis and dissemination of information key to our understanding of recombinant DNA, and in particular, human gene transfer research. These initiatives include a new query-capable database, an analytical board of scientific and medical experts, and conferences and symposia on timely scientific, safety, and policy issues. Veterinary scientists can play an important role in the oversight of recombinant DNA research and in enhancing our understanding of the many safety and scientific dimensions of the field. These roles include developing appropriate animal models, reporting key safety data, enhancing institutional biosafety review, and promoting compliance with the NIH Guidelines.     

Mitochondrial DNA polymerase, deoxyribonuclease and ribonuclease H activities from brain of chick embryo

R-DNA polymerase, D-DNA polymerase, DNase and RNase H activities in mitochondria from chick embryonic brain were studied by ion-exchange chromatography. Two main fractions were separated according to their chromatographic behaviour: a fraction M Ib which is eluted with the washing buffer from two successive DEAE-cellulose columns and a fraction M IV which is eluted at 400 mM KC1 from a phosphocellulose column. Although the two fractions contain both the DNA polymerase and the degrading activities, all the specific activities are higher in fraction M IV than in fraction M Ib. Heat inactivation experiments have shown that R-DNA polymerase is inactivated in both fractions, whereas RNase H and DNase are not affected. Thus, degrading activities and R-DNA polymerase activity seem to be catalyzed by different molecular entities. However the fact that in most cases these activities co-chromatograph suggests that the corresponding molecules form rather stable complexes.     

DNA-mediated transformation of mammalian cells in culture. Increased transforming efficiency following sonication

A series of 100 experiments was completed to determine if DNA is capable of transforming the genotype of a murine lymphoma (P388) in cell culture. The test system was concerned with the transformation of cells from 8-azaguanine (AZG) sensitivity to resistance. By the use of this marker, it was determined that transformation by DNA did occur, and that the efficiency of transformation was greatly increased by sonication of the DNA. A statistical analysis of 100 experiments demonstrated that the increase in the number of resistant cells after treatment with sonicated resistant DNA (R-DNA) was statistically significant (χ² > 4.25, 0.05 > p > 0.02) in 66% of the experiments. DNA from sensitive parental cells and DNA from other sources produced no effect while DNase and UV treatment abrogated effective transformation by either sonicated or nonsonicated R-DNA. RNase was without effect. Sucrose gradient analysis of sonicated and nonsonicated R-DNA demonstrated that the peaks which correspond to the highest specific transforming activity are not altered by sonication and do not coincide with the OD₂₆₀ peaks, in spite of the fact that sonication shifted the peak of maximum OD₂₆₀ to a slower sedimenting region of the gradient. The major portion, however, of the transforming material did shift after sonication to the slower sedimenting region of the gradient and did coincide with the OD₂₆₀ peak. The hereditary stability of the transformed cells was established by cloning a representative number of transformants, growing them in the absence of AZG for an extended period and then testing their ability to grow in graded concentrations of AZG. In addition, DNA extracted from transformants successfully transformed sensitive cells.     

An evaluation of the role and effectiveness of institutional biosafety committees in providing oversight and security at biocontainment laboratories

Institutional biosafety committees (IBCs) have been charged with the oversight and review of biosafety at thousands of biocontainment labs nationwide, hundreds of which are high-level BSL-3 and BSL-4 labs. In light of the recent rapid proliferation of BSL-3 and BSL-4 facilities and the increases in research in the areas of biodefense, select agents, recombinant DNA, and synthetic biology and dual-use research, questions have been raised about whether IBCs are fulfilling their oversight responsibilities. This article reviews information on the responsibilities and expectations of IBCs as currently constituted and provides an analysis of IBC performance from survey data of hundreds of research institutions over the past several years. The findings highlight serious ongoing problems with IBCs' adherence to NIH Guidelines. This raises questions about the current voluntary governance framework as an effective system to monitor and oversee U.S. research facilities, including high-containment facilities, and their research activities. The findings strongly suggest the need for immediate improvement or replacement of the IBC system.     

美国国立卫生研究院概况

美国国立卫生研究院(the National Institutes of Health,NIH)是美国主要的医学与行为学(medical and behavioral research)研究机构,拥有27个研究所及研究中心和1个院长办公室(office of the director,OD),任务是探索生命本质和行为学方面的基础知识,并充分运用这些知识延长人类寿命,以及预防、诊断和治疗各种疾病和残障。NIH不仅拥有自己的实验室从事医学研究,还通过各种资助方式和研究基金全力支持各大学、医学院校、医院等的非政府科学家及其他国内外研究机构的研究工作,并协助进行研究人员培训,促进医学信息交流。世界一流的科学家在NIH的支持下,自由探索科学问题,取得了辉煌的成就,极大地改善了人类的健康和生存状况。本文旨在介绍NIH的概况、基金管理模式、经费预算等,希望对我国的医学研究事业有所借鉴。     

Isolation of human germ-line DNA suitable for recombinant DNA studies

Dna from human sperm cells can be isolated with size and purity sufficient for use in recombinant DNA research. The DNA averages 100 000 base pairs (bp) in size (about 70.10(6) daltons) and is free of somatic cell, bacterial, and viral DNA. It can therefore be cloned under P2 + EK2 conditions as stipulated in the 1978 NIH Guidelines for recombinant DNA research.     

Deoxyribonucleic Acid Polymerase Activities in Normal and Leukovirus-Infected Chicken Embryo Cells

Chicken embryo cells normally contain, in addition to deoxyribonucleic acid (DNA)-dependent DNA (D-DNA) polymerases, a novel "R-DNA-polymerase" which specifically copies polyriboadenylic acid strands. This R-DNA polymerase cannot copy natural ribonucleic acid or polyribocytidylic acid strands to a significant extent. Infection of cells with the leukovirus RAV-2 leads to the intracellular formation of large amounts of the viral RNA-dependent DNA polymerase whose properties differ from the cell R-DNA polymerase. Chicken cells transformed by a Rous sarcoma virus mutant which produce noninfectious alpha-type Rous sarcoma virus (f), a leukovirus known to be deficient in the viral RNA-dependent DNA polymerase, do not contain detectable viral RNA-dependent DNA polymerase, whereas the cellular R-DNA polymerase is found in normal amounts. There seems to be no relationship between the cellular R-DNA polymerase and the RNA-dependent DNA polymerase of the avian leukoviruses.     

Identification of human satellite DNA sequences associated with chemically resistant nonhistone polypeptide adducts

A fraction of DNA fragments of highly purified and completely unfolded eukaryotic DNA inevitably remains associated with chemically resistant nonhistone DNA-polypeptide complexes. This fraction can be isolated by nitrocellulose filtration because the polypeptide-associated DNA fragments are retained on nitrocellulose filters while bulk DNA passes through the filters. The fraction of AluI-fragmented DNA from human placenta retained on filters as a result of the binding factors (R-DNA, 12%) represents a subset of genomic sequences with a sequence complexity different from unfractionated DNA and DNA recovered in the filtrate (F-DNA). DNA sequences prevalent in the retained fraction were detected by differential plaque hybridization of a recombinant gt10 library with radiolabeled F- and R-DNA fractions. Several recombinant phages showing much stronger hybridization signals with the R-DNA probe than with the F-DNA probe were selected, plaque-purified and analyzed. Analysis of the inserts of such clones showed that repetitive DNA sequences of the alphoid dimeric and tetrameric family, satellite III and satellite III-like sequences are highly enriched in the retained fraction, which indicates that these sequences specifically attract the polypeptides involved in the tightly bound and resistant complexes. This property of repetitive sequences is of interest since tandemly repetitive sequences have been suggested to code for locus-specific fixation and stabilization of the chromatin fiber in the cell nucleus.by L. ManuelidisThis work contains parts of the Ph.D. thesis of M.P. (University of Giessen).     

The art and design of functional metagenomic screens

This article summarizes general design principles for functional metagenomics. The focus is on Escherichia coli as an expression host, although alternative host-vector systems are discussed in relation to optimizing gene recovery in activity-based screens. Examples of DNA isolation and enrichment approaches, library construction and phenotypic read-out are described with special emphasis on the use of high throughput technologies for rapid isolation of environmental clones encoding phenotypic traits of interest.     

U.S. National Institutes of Health Core Consolidation–Investing in Greater Efficiency

The U.S. National Institutes of Health (NIH) invests substantial resources in core research facilities (cores) that support research by providing advanced technologies and scientific and technical expertise as a shared resource. In 2010, the NIH issued an initiative to consolidate multiple core facilities into a single, more efficient core. Twenty-six institutions were awarded supplements to consolidate a number of similar core facilities. Although this approach may not work for all core settings, this effort resulted in consolidated cores that were more efficient and of greater benefit to investigators. The improvements in core operations resulted in both increased services and more core users through installation of advanced instrumentation, access to higher levels of management expertise; integration of information management and data systems; and consolidation of billing; purchasing, scheduling, and tracking services. Cost recovery to support core operations also benefitted from the consolidation effort, in some cases severalfold. In conclusion, this program of core consolidation resulted in improvements in the effective operation of core facilities, benefiting both investigators and their supporting institutions.     

A generalized model of plasmid replication

A simple model is developed that permits the determination of cellular extrachromosomal DNA content for a large number of host-plasmid systems. The model incorporates host, vector, and environmental influences on plasmid replication through the use of empirical expressions. The model successfully predicts the plasmid content of many host-vector systems in a balanced growth situation, both in continuous and batch cultures. Techniques for determining model parameters are also presented. The model parameters, which characterize the effect of plasmid on its own synthesis, are given physical interpretation through the development of a structured model for a particular class of plasmids and subsequent comparison of the predictions of the two models. The simplicity of the model expressions should, in the absence of: (1) discriminatory criteria such as plasmid concentration under transient growth conditions or (2) detailed knowledge on molecular mechanisms of plasmid replication, prove useful in the study of host-vector systems for genetic engineering applications.     

Microfluidics Approaches in Modern Developmental Biology

Modern automated microsystems based on microhydrodynamic (microfluidic) technologies— labs on chips—make it possible to solve various basic and applied research problems. In the last 15 years, the development of these approaches in application to the problems of modern quantitative (systems) development biology has been observed. In this field, high-throughput experiments aimed at accumulating ample quantitative data for their subsequent computer analysis are important. In this review, the main directions in the development and application of microfluidics approaches for solving problems of modern developmental biology using the classical model object, Drosophila embryo, as an example is discussed. Microfluidic systems provide an opportunity to perform experiments that can hardly be performed using other approaches. These systems allow automated, rapid, reliable, and proper placing of many live embryos on a substrate for their simultaneous confocal scanning, sorting them, or injecting them with various agents. Such systems make it possible, in particular, to create controlled gradients of microenvironmental parameters along a series of developing embryos or even to introduce discontinuity in parameters within the microenvironment of one embryo, so that the head half is under other conditions compared to the tail half (at continuous scanning). These approaches are used both in basic research of the functions of gene ensembles that control early development, including the problems of resistance of early patterns to disturbances, and in test systems for screening chemical agents on developing embryos. The problems of integration of microfluidic devices in systems for automated performance of experiments simultaneously on many developing embryos under conditions of their continuous scanning using modern fluorescence microscopy instruments will be discussed. The methods and approaches developed for Drosophila are also applicable to other model objects, even mammalian embryos.     

Replication of the R Factor Rts1 in Proteus mirabilis

The replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density-gradient centrifugation. The proportion of Rts1 deoxyribonucleic acid (DNA) relative to the host chromosomal DNA (% R-DNA) was 7% in both exponential and stationary growth phases in Penassay Broth and supplemented M9 minimal medium at 30 C. The chromosomal DNA content per cell varied over a threefold range in the different growth media. In agreement with previous genetic observations, the replication of Rts1 was found to be temperature-sensitive and Rts1 DNA was diluted from the cells during exponential growth at 42 C. (14)N-(15)N medium transfer experiments have shown that individual copies of Rts1 are selected at random for replication during the duplication of the multicopy episome pool.     

Deletion of late genes of the prophage lambda

The deletions in tandem prophage lambda appear with high frequency (to 10%) in rec A- strain of Escherichia coli. The deletions were shown by marker rescue and hybridization of fragments of DNA on nitrocellulose filters with nick-translated phage lambda DNA localized only in prophage area. Right and left att sites are not involved. The majority of defective lysogens had all regulatory regions and deletions of late structural genes. These strains may be used for construction of the host-vector systems with the strongest promoter p'R of phage lambda.     

Bacteria are small but not stupid: cognition, natural genetic engineering and socio-bacteriology

Forty years' experience as a bacterial geneticist has taught me that bacteria possess many cognitive, computational and evolutionary capabilities unimaginable in the first six decades of the twentieth century. Analysis of cellular processes such as metabolism, regulation of protein synthesis, and DNA repair established that bacteria continually monitor their external and internal environments and compute functional outputs based on information provided by their sensory apparatus. Studies of genetic recombination, lysogeny, antibiotic resistance and my own work on transposable elements revealed multiple widespread bacterial systems for mobilizing and engineering DNA molecules. Examination of colony development and organization led me to appreciate how extensive multicellular collaboration is among the majority of bacterial species. Contemporary research in many laboratories on cell-cell signaling, symbiosis and pathogenesis show that bacteria utilise sophisticated mechanisms for intercellular communication and even have the ability to commandeer the basic cell biology of 'higher' plants and animals to meet their own needs. This remarkable series of observations requires us to revise basic ideas about biological information processing and recognise that even the smallest cells are sentient beings.     

Genetic containment of forest plantations

Dispersal of pollen, seeds, or vegetative propagules from intensively bred, exotic, or recombinant DNA modified forest plantations may cause detrimental or beneficial ecological impacts on wild or managed ecosystems. Insertion of genes designed to prevent or substantially reduce dispersal could reduce the risk and extent of undesired impacts. Containment measures may also be required by law or marketplace constraints, regardless of risks or benefits. We discuss: (1) the context for when genetic containment or mitigation systems may be needed; (2) technology approaches and mechanisms; (3) the state of knowledge on genes/genomics of sexual reproduction in forest trees; (4) stability of transgene expression during vegetative growth; (5) simulation studies to define the level of containment needed; and (6) needed research to deliver effective containment technologies. We illustrate progress with several examples from our research on recombinant DNA modified poplars. Our simulations show that even partial sterility can provide very substantial reductions in gene flow into wild trees. We conclude that it is impossible to define the most effective containment approaches, nor their reliability, based on current genomic knowledge and technological tools. Additional genomic and technological studies of a wide variety of options are needed. Studies in field environments are essential to provide data relevant to ecological analysis and regulatory decisions and need to be carried out in phylogenetically diverse representatives of the economically most important taxa of forest trees.

Gene cloning and expression in lactic streptococci

Abstract Recent developments have made the mesophilic lactic streptococci, widely used in dairy fermentations, accessible to genetic manipulation. Several host-vector systems have been described which currently are used in the cloning and expression of homologous and heterologous genes. The essential elements of these systems, the various cloning strategies and the first successful cloning experiments are described with emphasis on the molecular organization of proteinase genes. In addition, the organization and nucleotide sequence of signals which are involved in gene expression in lactic streptococci are summarized.     

Parameters associated with cloning in Actinobacillus actinomycetemcomitans

Characterization of virulence traits in Actinobacillus actinomycetemcomitans requires the application of recombinant DNA techniques. To develop appropriate genetic tools it is necessary to identify suitable host-vector systems. The current study assessed cloning parameters in A. actinomycetemcomitans for two previously described vectors, pDMG4 and pMMB67. It was determined that the maximum size of recombinant molecules that could be transferred to A. actinomycetemcomitans strain ATCC29522 via electroporation was 33 kb. The size limit for transformation of the same strain with ligation mixtures (direct cloning), however, was limited to 23-24 kb. Additional experiments included electroporation of various A. actinomycetemcomitans strains with plasmid DNA isolated from Escherichia coli and different A. actinomycetemcomitans sources. Differences in transformation efficiencies suggested the presence of a restriction modification system for pDMG4 in some strains of A. actinomycetemcomitans. Cloning of portions of the enterococcal plasmid pJH1 into A. actinomycetemcomitans resulted in the insertion of the intact vector into the chromosome.