Recombinant DNA Experiments Research Articles

Generation and characterization of a novel ovariole cell line derived from Spodoptera frugiperda in China with sensitivity to both SfMNPV and AcMNPV

Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), belonging to the species Alphabaculovirus spofrugiperdae, has been recently registered as an insecticide in China. This virus has a specific effect on the global major agricultural pest Spodoptera frugiperda. To gain insights into viral infection, replication processes, and the complex formation of viral particles, in vitro studies using cell lines are essential tools. Although the IPLB-Sf9 and IPLB-Sf21 cell lines derived from S. frugiperda are widely used for studies on the infection and replication mechanisms of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), their capacity to produce viral polyhedra after SfMNPV infection is not optimal. To address this limitation, a novel cell line named IOZCAS-Sf-1 has been developed from a S. frugiperda population sourced Yunnan, China. The mitochondrial COX1 gene analysis confirmed the species origin of the IOZCAS-Sf-1 cell line. Furthermore, a comparative study was carried out to contrast the COX1 gene sequence of this novel cell line with that of IPLB-Sf9, highlighting the distinctions between the two. Importantly, the IOZCAS-Sf-1 cells exhibited a remarkable ability to generate polyhedra when infected with AcMNPV and SfMNPV, respectively. Consequently, this cellular lineage is considered a promising and valuable resource. It serves not only to investigate the molecular mechanisms of viral replication and its impact on host cells, but also to explore the transfection efficiency of SfMNPV DNA. This exploration further expands into its potential application in recombinant DNA experiments, laying a theoretical groundwork for the advancement of more effective biopesticides and sustainable agricultural practices.

Cloning and Transformation with Plasmid Vectors.

Plasmids occupy a place of honor in molecular cloning: They were used in the first recombinant DNA experiments and, 40 or more years later, they remain as the carriage horses of molecular cloning. After almost half a century of sequential improvement in design, today's plasmid vectors are available in huge variety, are often optimized for specific purposes, and bear only passing resemblance to their forebears. Here, various features of plasmid vectors and methods for transforming E. coli cells are introduced.

Chimeric hPIV2/Corona-Spike Nasal Vaccine Robustly Protects the Upper and Lower Airways Against SARS-CoV-2

We developed an intranasal vaccine against SARS-CoV-2 using the replication-incompetent human parainfluenza virus type 2 (hPIV2) vector BC-PIV, which can deliver ectopic gene as stable RNA and ectopic protein on the envelope. BC-PIV expressing the full-length prefusion-stabilized spike gene of SARS-CoV-2, S2PM, possessed a corona-like viral envelope. Intranasal vaccination of mice with BC-PIV/S2PM induced high levels of neutralizing IgG and mucosal IgA antibodies against the spike protein. While BC-PIV showed hemagglutinating activity, BC-PIV/S2PM lacked such activity, in accordance with the presence of the massive spike protein on the viral surface. Furthermore, single-dose intranasal vaccination of hamsters with BC-PIV/S2PM completely protected the lungs from SARS-CoV-2 at 11 weeks post-immunization, and prime-boost vaccination conferred virtually complete protection of the nasal turbinates against SARS-CoV-2 challenge at 11 weeks post-priming. Thus, this chimeric hPIV2/spike intranasal vaccine is one of the strong candidates for an ultimate vaccine against SARS-CoV-2 to curtail virus transmission.Funding: This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology in Japan (17K19652, 20K21614), by a Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) (JP19fk0108113), Mie University (for research institutes of excellence), Mie Prefecture, Junior Chamber International Yokkaichi, and MediciNova, Inc.Conflict of Interest: J.O., M.F., M.Im., R.O., S.Y., Y.Kaw., and T.N. are patent applicants for recombinant BCPIV vaccine against SARS-CoV-2. M.F. is a founder of BioComo, Inc., and J.O. is an employee of BioComo, Inc. J.O., M.F., M.M., and T.N. have shares of stock in Biocomo, Inc. M.M. is a scientific advisor of JEOL Ltd. T.N. is a scientific advisor of MediciNova, Inc. The other authors declare no competing interests.Ethical Approval: Recombinant DNA experiments with SARS-CoV-2 S gene fragments were approved by the Ministry of Education, Culture, Sports, Science and Technology in Japan (Approved No. 2019-728, 729; 2020-362, 373, 948). The animal studies were approved by the Animal Care Committees of Mie University(Approved No. 23-33) and the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (Approved No. PA19-75), and all methods were performed under institutional regulations of animal experiments in accordance with the current national guidelines. Animal experiments using SARS-CoV-2 S gene fragments orSARS-CoV-2 were also approved by the Ministry of Education, Culture, Sports, Science and Technology in Japan (Approved No. 2019-728, 729; 2020-362, 373, 2020-948).

CRISPR as agent: a metaphor that rhetorically inhibits the prospects for responsible research

In 2015, a group of 18 scientists and bioethicists published an editorial in Science calling for “open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome” and recommending that steps be taken to strongly discourage “any attempts at germline genome modification” in humans with this powerful new technology. Press reports compared the essay to a letter written by Paul Berg and 10 other scientists in 1974, also published in Science, calling for a voluntary deferral of certain types of recombinant DNA experimentation. A rhetorical analysis of the metaphors in these two documents, and in the summary statements that came out of the respective National Academy of Sciences conferences they instigated, shows that while they have a lot in common, they are different in at least one important way. The more recent texts deploy conceptual metaphors that portray the biotechnology in question as an autonomous agent, subtly suggesting an inevitability to its development, in contrast to the earlier texts, which portray the scientists who are using the technology as the primary agents who take action. Rhetorical moves depicting biotechnology as an agent in the 2015 texts hint at contemporary skepticism about whether humans can restrain the forward momentum of science and technology in a global context, thus inhibiting scientists from imagining a consequential role for themselves in shaping the future of responsible research.

History and future of genetically engineered food animal regulation: an open request.

Modern biotechnology resulted from of a series of incremental improvements in the understanding of DNA and the enzymes that nature evolved to manipulate it. As the potential impact of genetic engineering became apparent, scientists began the process of trying to identify the potential unintended consequences. Restrictions to recombinant DNA experimentation were at first self-imposed. Collaborative efforts between scientists and lawyers formalized an initial set of guidelines. These guidelines have been used to promulgate regulations around world. However, the initial guidelines were only intended as a starting point and were motivated by a specific set of concerns. As new data became available, the guidelines and regulations should have been adapted to the new knowledge. Instead, other social drivers drove the development of regulations. For most species and most applications, the framework that was established has slowly allowed some products to reach the market. However, genetically engineered livestock that are intended for food have been left in a regulatory state of limbo. To date, no genetically engineered food animal is available in the marketplace. A short history and a U.S.-based genetic engineer's perspective are presented. In addition, a request to regulatory agencies is presented for consideration as regulation continues to evolve. Regulators appear to have shown preference for the slow, random progression of evolution over the efficiency of intentional design.

Genentech: The Beginnings of Biotech by Sally Smith Hughes (review)

Reviewed by: Genentech: The Beginnings of Biotech by Sally Smith Hughes Lisa Onaga (bio) Genentech: The Beginnings of Biotech. By Sally Smith Hughes. Chicago: University of Chicago Press, 2011. Pp. xii+ 213. $25. The story line of Genentech’s founding in 1976 and the early days of biotechnology have been rehearsed by numerous individuals, from journalists to scientists to venture capitalists. Like DNA, these stories are also prone to replication and subject to change when retold, with small pieces missing or slightly changed. Like myths, these stories are susceptible to oversimplification. One counter to such challenges to the historical record has been the practice of collecting oral histories and building archives of recent science. Historians have long been indebted to Sally Smith Hughes for her oral history interviews with key people in the biotechnology field, and many of these, archived at the Regional Oral History Office of the Bancroft Library at the University of California, Berkeley, reflect her unprecedented access to key scientists and individuals involved with the Genentech Corporation. Along with the private papers of Bob Swanson and the Genentech corporate archives, Hughes notably draws on these interviews to craft this long-awaited monograph, Genentech: The Beginnings of Biotech. Multiple approaches could have been taken to describe the emergence of biotechnology, but from the start, Hughes focuses her lens quite tightly on the careers and activities of the Genentech cofounders, Herbert Boyer and Bob Swanson. The temporal scope of analysis ends with Genentech’s initial public offering in 1980, offering a clue as to how the author makes sense of the gradual formation of a biotechnology “milieu” (pp. 16, 31, 165). The central issue that motivates this book is a question about the nature of biotechnology itself. To answer this, Hughes invites readers to a behind-the-scenes view of how Boyer, Swanson, and others came to work together and navigate the terrain of venture capital, recombinant DNA politics, risk-taking, and naïve missteps that were integral to the formation of Genentech. According to Hughes, the eventual new normal, in which bioscientists overcame hesitancy to conduct research in biotech start-up firms that could be done otherwise in academia, was made possible by the trials, errors, and successes of Genentech that in turn emboldened a generation of bio-entrepreneurs. Hughes’s observations about the biotech venture help disturb any assumptions that the Cohen-Boyer recombinant DNA experiments paved a self-fulfilling path to incorporation. By giving readers access to some of the timely decision-making processes that took place in a period of declining federal funding for basic scientific research, she makes clear why joining Genentech was appealing for some. The book impresses upon readers that the social environment of biotech forged at the time appeared as a virgin [End Page 511] territory of science, technology, and industry that Genentech needed to chart in order to stay intact. The three central chapters describe key research developments that constitute Genentech’s synthesis story: the proof-of-concept research on the unmarketable growth-inhibiting hormone somatostatin, human insulin, and human growth hormone. To demonstrate the emergent culture of biotech, Hughes highlights important material that makes this account of Genentech ultimately a human story about the different personalities, male culture, and risk-taking behaviors that counterweighted the many anxieties of the start-up company, from finding talent, lab space, and funding to dealing with research moratoria, managing public images, staying within the good graces of universities and the NIH, and handling legal negotiations. It is in these discussions, particularly at the end of the fifth chapter when the women biotechnology researchers are given some attention, that the broader implications of Genentech for understanding the dynamics of labor and work in biotech firms stand out. The involvement of Keiichi Itakura, a scientist of Japanese heritage, in the early Genentech experiments at City of Hope National Medical Center, and later international legal disputes hint at further opportunities for understanding the depth of the firm’s international significance. (A Japanese translation of Genentech by Hiroe Chiba was published by Ittosha in 2013.) The “recombinant culture” (p. 132) that Hughes refers to was characterized by academic values interbred with corporate form, as at Genentech, a...

Historical Perspectives Pertaining to the NIH Recombinant DNA Advisory Committee

Science is host to a constantly emerging series of new paradigms, and it is this characteristic that makes science both interesting and dynamic. As a part of this continuum, it became possible to create recombinant DNA molecules. Immediately it was recognized that there was a potential for serious adverse events associated with this new technology. Following two scientific conferences at Asilomar, California, the National Institutes of Health moved quickly to create the Recombinant DNA Advisory Committee (RAC). For approximately 38 years the RAC has served as an open forum for review of various recombinant DNA experiments, and for the last 23 years it has played a pivotal role in the oversight of human gene therapy. The RAC's existence obviated the need for more restrictive governmental legislation and has supported the development of genetic interventions that are leading to actual human therapies.

Inhibition of Nuclear Translocation of Apoptosis-Inducing Factor Is an Essential Mechanism of the Neuroprotective Activity of Pigment Epithelium-Derived Factor in a Rat Model of Retinal Degeneration

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

Comparative Analysis of Two UDP-glucose Dehydrogenases in Pseudomonas aeruginosa PAO1

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K(m) values of PA2022 and PA3559 for UDP-glucose are approximately 0.1 and 0.4 mM, whereas the K(m) values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg(2+), which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.

Advancing the Frontiers

![Figure][1] There has been much discussion recently about ways to stimulate more “high-risk-high-payoff” research: projects that have the potential to make major leaps in scientific understanding. In the United States, the National Science Board, for example, has had a task force dedicated to this issue for over a year, and the National Institutes of Health's Roadmap includes efforts to transform fundamental and clinical biomedical research. One approach to advancing these frontiers has proven quite successful over the years. It is the model used by the Gordon Research Conferences (GRCs), which this year celebrate their 75th anniversary; their 2006 program appears in this issue of Science . The GRCs, which began in 1931 as a chemistry meeting conceived and organized by Neil Gordon, now encompass some 180 conferences each year at 17 different sites in the United States and abroad. Over 20,000 international scientists will participate in these intense meetings that span the whole spectrum of science, science education, and science policy. The American Association for the Advancement of Science (AAAS) has had a close association with the GRCs since 1938, when it took over managing the conferences—an arrangement that lasted until 1956, when the GRCs became independent. A fascinating compendium[*][2] of personal reminiscences about the conferences and their scientific impacts, contributed by a diverse group of 80 well-respected scientists, reveals why these conferences are so popular and successful and why they have persisted on a regular basis for so long. Reduced to its core, the success of these meetings amounts to the way in which their format has promoted transformative thinking and project development. Maxine Singer, for example, writes about the importance of the 1973 Nucleic Acids meeting in stimulating thinking about the implications of recombinant DNA (cloning) experiments. Other authors cite the central role of the GRCs in the emergence of such multidisciplinary fields as bioinorganic chemistry, organic electronics, and mammary gland biology. ![Figure][1] CREDIT: GORDON RESEARCH CONFERENCES A core lesson from the GRCs is that even in this age of electronic communication technologies, there is no substitute for putting a small group of people together face to face and keeping them in close contact for a few days. The relatively isolated sites used for the GRCs and the fact that each conference is usually restricted to about 100 attendees encourages people to talk to each other with both informality and candor. And the conference agenda allows for plenty of unstructured discussion time and promotes long conversations about frontier science. Many of the important unanswered scientific questions are multidisciplinary in character. This feature of contemporary research was amply demonstrated in Science 's 2005 list of the top 25 questions for the next 25 years.[†][3] To promote the kind of thinking needed for problem-based rather than discipline-based science, one needs to bring experts together from all potentially relevant fields and create an environment in which they may speak freely and frankly with one another. That is exactly the kind of conversation that GRC attendees are engaged in. All discussions are off the record, and all conference communications are considered private. This fosters safe spaces for posing “risky” ideas and engaging in creative and occasionally speculative communal thinking. As Norman Hackerman emphasizes in the GRC compendium, “The greatest advantage of these meetings was that attendees were able to participate without worrying about being proved wrong in publication …” On the nonhierarchical nature of the meetings, Roy Vagelos reflects, “There I was [at his first Lipid Metabolism conference], a pipsqueak only a few years out of a postdoctoral fellowship, speaking alongside these giants of biochemistry.” Not surprisingly, some of the giants later became Vagelos' collaborators. The GRCs are only one way to encourage transformative thinking and research, but their track record suggests that we may need more venues like them. Scientists sometimes lament that peer review may be biased in favor of cautious and “safe” research, unsupportive of departures from mainstream thinking. By creating a relatively unthreatening, unconstrained atmosphere, the GRCs provide a refreshing opportunity to try out new ideas on one's colleagues, brainstorm about difficult and complex issues, and think about possible solutions. Not a bad strategy at all. [1]: pending:yes [2]: #fn-1 [3]: #fn-2

Escherichia coli host strains.

To successfully perform molecular genetic techniques it is essential to have a full understanding of the properties of the various Escherichia coli host strains commonly used for the propagation and manipulation of recombinant DNA. E. coli is an enteric rod-shaped Gram-negative bacterium with a circular genome of 4.6 Mb (1). It was originally chosen as a model system because of its ability to grow on chemically defined media and its rapid growth rate. In rich media, during the exponential phase of its growth, E. coli doubles every 20–30 min; thus, during an overnight incubation period, a single selected organism will double enough times to yield a colony on an agar plate, or 1–2 billion cells per milliliter of liquid media. The ease of its transformability and genetic manipulation has subsequently solidified the role of E. coli as the host of choice for the propagation, manipulation, and characterization of recombinant DNA. In the past 60 yr E. coli has been the subject of intensive research and more is now known about these bacilli than any other organisms on earth. A wide variety of E. coli mutants have been isolated and characterized. Almost all strains currently used in recombinant DNA experiments are derived from a single strain: E. coli K-12, isolated from the feces of a diphtheria patient in 1922 (2). This chapter will discuss characteristics of E. coli host strains that are important for recombinant DNA experiments in order to aid in the choice of a suitable host and circumvent possible problems that may be encountered. Common mutations and genotypes that are relevant to recombinant DNA experiments are summarized in Table 1. A complete listing of genetically defined genes has been compiled by Berlyn et al. (3).

Positive Selection Vectors

ABSTRACT: This review describes information concerning positive selection vectors on their mechanism, classification, property, and limitation. A total of 72 positive selection vectors collected were discussed. Positive selection vectors can reduce background and directly screen transformants containing cloned DNA fragments. The mechanisms to perform positive selection include insertional inacti-vation and the replacement of functional genes of the vectors. In general, the former is much more convenient than the latter. The functional genes are controlled either by their promoters or by heter-ologous promoters introduced. On the basis of the structures, positive selection vectors could be classified into five groups. The positive selection vectors are commonly based on the mechanisms of lethal genes and the sensitivity of compounds. The vectors, with molecular weights ranging from 2.6 to 17.0 kb, have diverse genetic markers and wide host ranges, including Escherichia coli, Bacillus, Streptomyces, lactic acid bacteria, yeasts, and mammalian cells. Although some limitations exist for using some positive selection vectors, they are useful in recombinant DNA experiments.

Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression.

The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive bacteria remain uncommon. We have developed a Gram-positive cloning vector that utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant plasmids. To facilitate introduction of foreign DNA, a multiple cloning site (MCS) was inserted distal to the region coding for the putative signal peptide of phoZ. Alkaline phosphatase expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range plasmid, to produce pDC123. In pDC123, phoZMCS was transcriptionally linked to the chloramphenicol acetyl transferase (cat) gene under the control of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign DNA into the MCS of phoZMCS produced a white colonial phenotype in E. coli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cpsE gene from the plasmid pCER111, which carries a 9.0-kb fragment of the GBS capsular polysaccharide synthesis locus. The plasmid pDC123 containing cpsE was isolated by direct electroporation into GBS strain A909 with selection of transformants containing recombinant plasmids achieved by 'blue/white' screening, without the use of an intermediate host. This new cloning vector should improve the efficiency of performing recombinant DNA experiments in Gram-positive bacteria.

Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases.

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.

Norms for patents concerning human and other life forms.

The rationale of patients on transgenic organisms leads to the startling notion of the human qua infringement. The moral reasons by which we may tenably reject such notion are not conclusive as to human life forms outside the body. A close look at recombinant DNA experimentation reveals ingenious processes, but not entities that the body lacks. Except for artificial genes, the genes of biotechnology are found on chromosomes, albeit nonconsecutively, and their uninterrupted transcripts appear in messenger RNA. An enhanced form of protection for ingenious processes, the "human methods patent," is proposed and defended as a replacement for product patents. The proposed patent would pertain to biotechnology manufacturing and genetic intervention in somatic and germ cells. A counterpart could govern nonhuman life forms. It is argued that compulsory licensing protections should be a condition of such patent. Contrary to the conservative assumption that statutory sobriquets suffice, the reckoning of what qualifies as a patentable ingenious process will continue to require systematic scientific guidance.

Rapid method for the purification of DNA from subgingival microorganisms.

A method is described which facilitates the rapid purification of high molecular weight chromosomal DNA from gram positive and gram negative bacteria grown on solid media. A total of 32 reference strains and fresh isolates were examined in this study. The purification procedure involved lysis of cells with SDS in the presence of proteinase K, followed by removal of cellular polysaccharides and proteins with hexadecyltrimethyl ammonium bromide (CTAB) and phenol:chloroform:isoamyl alcohol. Preparations were incubated with RNase and, after removal of the enzyme, DNA was precipitated with ethanol. Several hundred micrograms of DNA could be prepared within 5 h from cells grown on 1-2 agar plates. None of the final preparations contained RNA; protein was detected in 12/32 preparations. The resultant DNA proved suitable for restriction enzyme digestion and biotin-labelling by a random primer technique. DNA probes constructed from these preparations were capable of detecting 100 pg of homologous target DNA fixed to nitrocellulose. Cross reactions between closely related species displayed weaker signal intensities than, and, thus, were easily distinguished from, true positive reactions between homologous species. DNA obtained by this procedure may also be suitable for DNA-DNA homology studies, recombinant DNA experiments and molecular fingerprinting.

Isolation and Characterization of Marine Caulobacters and Assessment of Their Potential for Genetic Experimentation

A total of 25 marine caulobacters were isolated from littoral marine sources. Several aspects of their physiology and morphology were examined, as well as their suitability for genetic manipulation in laboratory cultivation. Caulobacters were readily isolated from all sources, including samples from areas containing pollution-related organic compounds. All isolates grew best in media containing seawater, but eight strains grew if sea salts were replaced with NaCl alone, three strains grew at 1/10 the normal sea salt concentration, and one isolate grew, albeit poorly, in freshwater medium. Of the marine isolates, 12 strains grew under anaerobic conditions, indicating that some caulobacters are not obligately aerobic bacteria, as they are currently categorized. Although some freshwater caulobacters are able to oxidize manganese, this capability was not found in these marine caulobacters. Of the marine isolates, 10 strains were resistant to mercury chloride concentrations 10- to 20-fold greater than that tolerated by sensitive bacteria. However, a mercury reductase gene comparable with that found in R100-type plasmids was not detected by gene hybridization. With respect to the potential for genetic experimentation, most strains grew rapidly (3- to 4-h generation time at 30 degrees C), producing colonies on solid media in 2 to 3 days. The isolates were sensitive to antibiotics commonly used in recombinant DNA experiments, and spontaneous drug-resistant mutants were selectable. Conjugal transfer of plasmids from Escherichia coli to several marine caulobacters was demonstrated for four broad-host-range plasmid incompatibility groups, by using both self-transmissible plasmids and cloning-oriented plasmids that require a helper plasmid. Conjugal transfer of broad-host-range plasmids between freshwater and marine caulobacters was also demonstrated in both directions. Native plasmids of approximately 100- to 150-kilobase sizes were found in 2 of the 25 marine Caulobacter strains. The native plasmids were present in relatively high copy number and appeared stable in laboratory culture. In short, the marine caulobacters appeared appropriate as candidates for genetic manipulation and the expression of selected genes in the marine environment.

Industrial applications of recombinant DNA technology

Some of the essential requirements for carrying out recombinant DNA experiments, potential industrial applications of recombinant DNA technology, and three specific current applications.

A program for the graphic representation and manipulation of DNA sequences.

We have developed a program for the graphic representation and manipulation of DNA sequences. The program (named CARTE from the French for 'map') is intended as a tool in the planning and analysis of recombinant DNA experiments. DNA sequences are represented as standard restriction maps, using any desired combination of restriction enzymes. Features of interest, such as promoters or coding sequences, can be highlighted. The sequence can be manipulated to mimic cloning, using deletions, insertions or replacements at specified sites. This process is facilitated by the simultaneous display of a graphic map of the entire sequence, a detailed picture of the work in progress, and a menu of functions.

Analysis and optimization of recombinant DNA joining reactions

The statistical segment length of duplex DNA was determined in phage T4 ligase (poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMP forming), EC 6.5.1.1) buffer (50 m m-Tris · HCl (pH 7.8), 20 m m-dithiothreitol, 10 m m-MgCl 2, 1 m m-ATP) at 12 °C to be 1030(± 116) Å. This result was obtained by electron microscopic examination of the molecular distributions generated by T4 ligase-mediated joining of EcoRI-cleaved pBR322 DNA. This value of the statistical segment length was utilized in an extension of the Jacobson-Stockmayer theory on the probability of intramolecular cyclization in order to optimize DNA joining reactions that are of great utility in recombinant DNA experiments. Five cloning systems were analyzed: circular plasmid vectors that had been linearized with one or two restriction endonucleases, circular plasmids that had been tailed with deoxyhomopolymers before joining, lambda-type cloning vectors and cosmids. The results are tabulated for convenient use in molecular cloning experiments.