Showing papers in "Nature Biotechnology in 1983"
TL;DR: In this paper, a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli was developed, which can utilize any gram negative bacterium as a recipient for conjugative DNA transfer.
Abstract: We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli. The system consists of two components: special E. coli donor strains and derivatives of E. coli vector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly used E. coli vectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.
7,278 citations
TL;DR: The molecular cloning and characterization of the gene encoding exo–cellobiohydrolase I of Trichoderma reesei strain L27 is reported and it is found to contain a 10 bp sequence, CAGCT–GACTG, that is homologous to a sequence necessary for splicing of introns in yeast2.
Abstract: The molecular cloning and characterization of the gene encoding exo–cellobiohydrolase I (CBHI) of Trichoderma reesei strain L27 is reported. Two adjacent HindIII genomic fragments of 1.16 kb and 2.3 kb were identified using differential hybridization techniques and were sub–cloned into plasmid pBR322. The identification of the gene encoding CBHI was determined by hybrid selection and confirmed by DNA sequence analysis. There are two introns in the genomic DNA that were identified by comparing the coding sequence with the published amino acid sequence1 and confirmed by sequencing of cDNA clones. Both introns were found to contain a 10 bp sequence, CAGCT–GACTG, that is homologous to a sequence necessary for splicing of introns in yeast2.
408 citations
TL;DR: Much more research is needed to exploit efficiently all the activities of the Interferon system, and most of the presentations at the Rotterdam meeting focused on development of interferon technology rather than its specific applications.
Abstract: THE BIOLOGY OF THE INTERFERON SYSTEM T he Netherlands Organization for Applied Scientific Research (TNO) sponsored its second conference on \"The Biology of the Interferon System\" on 18-22 April 1983 in Rotterdam. Although it came only six months after the Third International Congress for Interferon Research, the meeting was both successful and informative. An unannounced item on the agenda was the election of a committee to review and revise the bylaws of the International Society for Interferon Research (ISIR), a new scientific association. One important task for ISIR will be to encourage better coordination between European and American researchers and limitation of meetings to one large symposium each year. Too much of a good thing may become a bad thing; obtaining scientific results requires some time to work at the bench. Interferon (IFN) is, in many ways, an exemplary biotechnology product. Its discovery 25 years ago spawned great hopes for a major breakthrough in human therapeutics and considerable profits for interested industries. A major hurdle for industrial development, the limited availability of pure material, has largely been overcome in the past few years. K. Cantell developed a convenient method for production of human interferons from buffy coats, a component of blood, that permitted scaling up of production to the limit of the availability of blood donated in Finnish transfusion centers. In 1980, C. Weissman and his collaborators first achieved expression of an interferon gene in E . coli. These developments stimulated the interferon field and marked the beginning of the industrial era of the interferon system. (The expression \"the interferon system\" alludes to the multiple potentialities of interferon. Coined in 1979, it refers to the fact that different classes of interferon exist that exert different biological activities via a number of different pathways.) Much more research is needed to exploit efficiently all the activities of the interferon system, and most of the presentations at the Rotterdam meeting focused on development of interferon technology rather than its specific applications. For example,
289 citations
TL;DR: This review focuses on the bacterial transposon Tn5 which encodes resistance to kanamycin and related aminoglycoside antibiotics and how it is proving to be a powerful new tool for in vivo genetic engineering in many bacterial species.
Abstract: The current interest in transposable elements stems from their ubiquity and their characteristic ability to insert into many sites in the genomes of their host organisms. They are responsible for a large fraction of spontaneous mutations and chromosome rearrangements. They alter the expression of genes near their sites of insertion. They facilitate the flow of genes encoding traits such as antibiotic resistance and pathogenicity between different bacterial species. This review focuses on the bacterial transposon Tn5 which encodes resistance to kanamycin and related aminoglycoside antibiotics: (i) How it is proving to be a powerful new tool for in vivo genetic engineering in many bacterial species; (ii) Our understanding of the mechanism and control of its transposition; and (iii) Current concepts of how it and other mobile elements may have evolved and contributed to the evolution of bacterial populations.
247 citations
TL;DR: Evidence that MINI–Ti, when transferred to Agrobacterium by cointegration with a shuttle vector, in the presence of helper plasmid pAL4404 containing VIR genes, can incite nopaline positive tumors is presented, evidence of T–DNA transfer.
Abstract: Agrobacterium tumefaciens has the natural ability to insert a specific part of its Ti (tumor–inducing) plasmid, called T–DNA, into the chromosomal DNA of host plant cells, causing tumorous growth. Foreign DNA artificially introduced into T–DNA is inserted into the plant genome by this natural vector system. Genes on the Ti plasmid required for T–DNA transfer, called VIR (virulence) genes, are situated outside T–DNA. We have separated T–DNA from the VIR functions onto a MINI–Ti plasmid that replicates in E. coli and contains full–length T–DNA (including the 25 basepair border signals) from pTi T37, a nopaline–type Ti plasmid. We present evidence that MINI–Ti, when transferred to Agrobacterium by cointegration with a shuttle vector, in the presence of helper plasmid pAL4404 containing VIR genes, can incite nopaline positive tumors, evidence of T–DNA transfer. It is not clear whether MINI–Ti functions in trans to the VIR genes, or whether spontaneous cointegration of pAL4404 with MINI–Ti/pRK (the shuttle form of MINI–Ti) accounts for the tumor–inducing ability of the bacteria. The MINI–Ti approach simplifies procedures for exploiting T–DNA as a gene vector for plants.
236 citations
TL;DR: It is shown by hybrid messenger selection and partial DNA sequencing that one of the clones contains a gene coding for the major cellulolytic enzyme of T. reesei, cellobiohydrolase I.
Abstract: The filamentous fungus Trichoderma reesei produces a number of inducible cellulolytic enzymes. By differential hybridization of cDNA probes made from induced and repressed messenger RNA populations to a λ phage gene bank, we have isolated a number of genes strongly expressed during production of cellulolytic enzymes. We have shown by hybrid messenger selection and partial DNA sequencing that one of the clones contains a gene coding for the major cellulolytic enzyme of T. reesei, cellobiohydrolase I.
200 citations
TL;DR: A full–length cDNA clone encoding the precursor to the small subunit of wheat ribulose–1,5–bisphosphate carboxylase is isolated and characterized and it is deduced that the precursor contains the mature small sub unit and an amino–terminal transit sequence.
Abstract: We have isolated and characterized a full–length cDNA clone encoding the precursor to the small subunit of wheat ribulose–1,5–bisphosphate carboxylase. From the nucleotide sequence, we deduce that the precursor contains the mature small subunit of 128 amino acid residues and an amino–terminal transit sequence of 47 amino acid residues. Southern blot analysis reveals that the small subunit precursor is encoded by a multigene family. One member of the family has been isolated. Nucleotide sequence and R–loop analyses show that it contains only one intron which separates the coding sequence of the transit peptide from that of the mature protein. This gene is expressed in vivo, as shown by mRNA hybridization to a specific probe from its 3′ non–coding region.
183 citations
TL;DR: The current status of efforts to target cytotoxic agents to tumor cells using antitumor antibodies and particulate drug carriers such as liposomes and microspheres is reviewed.
Abstract: The current status of efforts to target cytotoxic agents to tumor cells using antitumor antibodies and particulate drug carriers such as liposomes and microspheres is reviewed. Emphasis is given to the role of anatomic and physiologic factors in determining the disposition, fate, and therapeutic efficacy of targeted drug delivery systems and to the importance of tumor cell heterogeneity as a major obstacle to effective cancer therapy.
156 citations
TL;DR: Two structural genes, celA and celB, coding respectively for the endoglucanases A and B of Clostridium thermocellum, were subcloned from cosmids of a gene bank into plasmid pBR322, expressing in E. coli independently of their orientation in the cloning vector.
Abstract: Two structural genes, celA and celB, coding respectively for the endoglucanases A and B of Clostridium thermocellum, were subcloned from cosmids of a gene bank into plasmid pBR322. Both genes are carried in a DNA sequence of about 2.7 kb. They share no homology and are not contiguous on the C. thermocellum chromosome. They are expressed in E. coli independently of their orientation in the cloning vector.
136 citations
TL;DR: Amino terminal protein sequencing of the first 30 amino acid residues of CBHI and EGI indicates significant homology, however, high pressure liquid chromatography analysis of tryptic peptides revealed little or no homology between these two proteins.
Abstract: Trichoderma reesei strain L27 produces multiple 1,4–β–D–glucan cellobiohydrolases, endo–1,4–β–D–glucanases, and 1,4–β–D–glucosidases. The major cellobiohydrolase (CBHI) and endoglucanase (EGI) have been purified to homogeneity and a β–glucosidase (BGI) has been substantially purified. Specific antibodies have been raised against these proteins. Amino terminal protein sequencing of the first 30 amino acid residues of CBHI and EGI indicates significant homology. High pressure liquid chromatography (HPLC) analysis of tryptic peptides, however, revealed little or no homology between these two proteins.
135 citations
TL;DR: Regulation of maturation would facilitate future efforts to manipulate somatic embryos for large scale propagation in batch cultures, mechanized planting, artificial induction of dormancy, and incorporation into artificial seeds.
Abstract: The ability to control plant somatic embryogenesis is a necessary prelude to its development as an efficient biotechnological tool. The influence of different suspension culture techniques on the maturation of caraway (Carum carvi) somatic embryos and the effect of growth hormones in controlling development were studied. The three types of culture vessels (tumble tubes, test tubes, and Erlenmeyer flasks), each providing contrasting techniques of agitation, generated populations differing significantly in the frequencies of normal and abnormal embryos. Abscisic acid (ABA), at the appropriate concentrations, effectively normalized development in all systems, inhibiting abnormal proliferations and precocious germination and fostering normal maturation. For those cultures where embryos failed to develop on unsupplemented medium, zeatin in combination with ABA fostered growth and normal maturation. Carrot (Daucus carota) somatic embryo development could be similarly controlled. Such regulation of maturation would facilitate future efforts to manipulate somatic embryos for large scale propagation in batch cultures, mechanized planting, artificial induction of dormancy, and incorporation into artificial seeds.
TL;DR: Sod webworm susceptible and resistant cultivars, selections, and single–plant progenies of perennial rye–grass were analyzed for the presence of an endophytic fungus and the significance of this endophyte–enhanced resistance to insects in the breeding and maintenance of turfgrass and forage cultivars is discussed.
Abstract: Sod webworm (Crambus spp) susceptible and resistant cultivars, selections, and single–plant progenies of perennial rye–grass (Lolium perenne L) were analyzed for the presence of an endophytic fungus This was done using both the ELISA technique and microscopic examination of remnant seed samples All entries resistant to sod webworms in field trials contained high levels of fungal endophyte, whereas susceptible entries did not contain the endophyte The significance of this endophyte–enhanced resistance to insects in the breeding and maintenance of turfgrass and forage cultivars is discussed
TL;DR: In this article, a rational strategy for composting is developed based on the interacting factors of microbial heat generation, temperature, ventilation, and vaporization of water, and an interactive ventilation control system is used to remove heat as needed to prevent a temperature increase to activity.
Abstract: A rational strategy for composting is developed based on the interacting factors of microbial heat generation, temperature, ventilation, and vaporization of water. Because the heat generation results from decomposition of organic material, its rate should be maximized. Implementation is by means of an interactive ventilation control system to remove heat as needed to prevent a temperature increase to activity–limiting levels. The threshold to limitation is approximately 60°C. Since heat generation drives vaporization, the material dries as composting progresses. In contrast, current processes typically operate at 80°C; consequently, waste is decomposed slowly and little water is vaporized.
TL;DR: The three general approaches are: stimulation of the endogenous microbial population; injection of microorganisms with proven ability to perform well in situ; and the use of microbial products, such as xanthan gum, produced by Xanthomonas campestris.
Abstract: Microorganisms and microbial products can be used to recover oil from reservoirs. To be successful, the complexity of oil and the physical constraints in the reservoir must be taken into account. The three general approaches are: stimulation of the endogenous microbial population; injection of microorganisms with proven ability to perform well in situ; and the use of microbial products, such as xanthan gum, produced by Xanthomonas campestris.
TL;DR: Gellan gum, the extracellular polysaccharide produced by Pseudomonas elodea, can be used in microbiological media and in gelled and structured food products, making it useful in agricultural applications, particularly flowable pesticides and liquid fertilizers.
Abstract: In recent years, significant progress has been made in discovering and developing new bacterial polysaccharides that possess novel and highly functional properties. For example, the relationship between the unique properties of xanthan gum—the first microbial polysaccharide of commercial significance—and its use in major food, industrial, and oil field applications is discussed. In addition, gellan gum, the extracellular polysaccharide produced by Pseudomonas elodea, can be used in microbiological media and in gelled and structured food products. Three other industrially useful bacterial polysaccharides have been developed. S–130, the extracellular, high viscosity polysaccharide produced by a strain of Alcaligenes, has excellent suspending and heat stability properties useful in oil field drilling, workover and completion, and enhanced oil recovery fluids. S–194 has excellent suspending properties and unusual compatibility with salts, making it useful in agricultural applications, particularly flowable pesticides and liquid fertilizers. S–198 has excellent stability to shear and has potential application in the developing market of water–based lubricants.
TL;DR: A set of broad host range cloning vectors has been constructed from the IncW plasmid pSa, which encodes resistance to kanamycin and spectinomycin–streptomycin and contains the chloramphenicol resistance gene of pSa.
Abstract: A set of broad host range cloning vectors has been constructed from the IncW plasmid pSa These vectors have been constructed from the transfer defective deletion derivative pSa151, which encodes resistance to kanamycin and spectinomycin–streptomycin Two of the vectors also contain the chloramphenicol resistance gene of Tn9, and a third contains the chloramphenicol resistance gene of pSa One of the vectors contains the cos sequence of bacteriophage λ and can be used with in vitro packaging systems in the construction of large recombinant plasmids Two of these vectors can be mobilized and transferred in the presence of a pBR322 derivative containing the transfer genes of pSa Together these vectors contain cloning sites for SstII, HindIII, EcoRI, KpnI, PvuII, BamHI, SmaI, and Bg1II, and recombinants at certain of these sites can be detected by insertional inactivation of a drug resistance phenotype The broad host range properties of the origin of replication of pSa allow the use of these vectors in a variety of gram–negative bacteria
TL;DR: A set of E. coli transformants containing plasmids pD316, pD365 and their derivatives secrete about 30% of their endoglucanase activity into the medium, 30% into the periplasmic space, while 40% remains in the cytoplasm.
Abstract: A set of E. coli transformants (2,500) containing plasmid pBR322 with Thermomonospora YX DNA inserts larger than 3 kb was prepared. The transformants were screened directly for the ability to hydrolyze carboxymethyl cellulose. Two colonies exhibiting cellulase activity were found. One colony contains a plasmid (pD365) that contains a 7.6 kb insert, including the entire gene coding for the major endoglucanase activity of Thermomonospora YX. The other colony (D315) had two plasmids in it: pD316 contains a 8.5 kb insert which includes at least 80% of the major endoglucanase gene; pD315 contains a 6.6 kb insert and does not express cellulase activity. Restriction maps were prepared for each plasmid and the endoglucanase gene was localized to a 2.3 kb SalI fragment in pD316 and to a 3.7 kb EcoRI fragment in pD365. These fragments were subcloned into plasmids containing active promoters regulated by the lactose repressor. The new strains each made about 50 times more cellulase than the original strains. E. coli transformants containing plasmids pD316, pD365 and their derivatives secrete about 30% of their endoglucanase activity into the medium, 30% into the periplasmic space, while 40% remains in the cytoplasm. Thermomonospora YX DNA restriction digests were fractionated by agarose gel electrophoresis and the fragments which hybridized to pD315 and pD316 were detected by nitrocellulose blotting techniques. The results indicated that each plasmid contains a single contiguous segment of Thermomonospora YX DNA and that the pD315 and pD316 inserts are linked in the Thermomonospora YX genome.
TL;DR: This review focuses on oxidoreductases, the large class of enzymes catalyzing biological oxidation/reduction reactions, which can be visualized in areas as diverse as polymer synthesis, pollution control, and oxygenation of hydrocarbons.
Abstract: The variety of chemical transformations catalyzed by enzymes make these catalysts a prime target for exploitation by the emerging biotechnology industries. Over the last two decades, intense research in the area of enzyme technology has provided many approaches that facilitate the practical application of enzymes. This review focuses on oxidoreductases, the large class of enzymes catalyzing biological oxidation/reduction reactions. Since so much industrial chemistry involves oxidation/reduction processes, the use of oxidoreductases to carry out synthetic transformations is a major area of interest. Of particular significance in this regard are oxidoreductase–mediated asymmetric syntheses of amino acids, steroids, and other pharmaceuticals, and of a host of specialty chemicals. Another area of major importance is the current extensive use of oxidoreductases in clinical diagnosis and other analytical applications. Future applications for oxidoreductases can be visualized in areas as diverse as polymer synthesis, pollution control, and oxygenation of hydrocarbons.
TL;DR: The unique gene combinations made possible by protoplast fusion ensure that new plant varieties will soon be derived from somatic hybridization.
Abstract: Plant protoplasts can be fused and the fusion products cultured to produce somatic hybrid plants. This technique has been used to produce germplasm previously unavailable to the plant breeder. Several researchers have emphasized production of hybrids between distantly related, sexually incompatible species, but many of these hybrids are sterile, precluding incorporation into a breeding program. Hence, to transfer traits such as disease and herbicide resistance, emphasis has shifted to production of hybrids between more closely related species. Novel variation has been observed in such somatic hybrids due to segregation of mixed organelles, cytoplasmic and nuclear gene recombination, and somaclonal variation. The unique gene combinations made possible by protoplast fusion ensure that new plant varieties will soon be derived from somatic hybridization.
TL;DR: Agrobacteria, with their so–called Ti plasmids, provide us with a natural gene vector that can readily be modified to fulfill most of the requirements of an ideal gene vector for plants.
Abstract: The fundamental goals of present day genetic engineering are in fact identical to those of traditional plant breeding: to engineer plants that are more valuable as crop plants, as biomass or as sources of important pharmaceuticals and enzymes. Central to many schemes designed to genetically modify plants is the availability of an efficient host gene vector system. Ideally the gene vector should provide an easy and reproducible system for the introduction of isolated genes—or groups of genes—into the genome of plant cells. The foreign genes should be appropriately expressed in the genetically modified (transformed) plant cells and stably transmitted, both somatically and sexually, to the offspring of the initially transformed cells. An ideal gene vector should also make it possible to introduce, maintain, and express any gene in plant cells independent of its origin, whether it is from plants, animals, bacteria, fungi or viruses. Finally one would wish the gene vector to have a very broad host range in order to be able to engineer the widest possible range of different plants. Agrobacteria, with their so–called Ti plasmids, provide us with a natural gene vector that can readily be modified to fulfill most of the requirements of an ideal gene vector for plants.
TL;DR: DNA probes are today's most sophisticated and sensitive technology for identifying genes, and with Wall Street analysts predicting that probes will be next biotechnology glamour child, they may garner substantial shares of several lucrative markets.
Abstract: DNA probes are today's most sophisticated and sensitive technology for identifying genes. Recent technological advances are opening new commercial avenues in diagnosing infectious diseases, identifying food contaminants, and engineering genes. Industrial researchers expect to market half a dozen DNA probe diagnostics during the next year. With Wall Street analysts predicting that probes will be next biotechnology glamour child, the tests may garner substantial shares of several lucrative markets.
TL;DR: It is shown that acrylate is an intermediate in the direct reduction of lactate to propionate in Clostridium propionicum in the presence of 3–butynoic acid, an inhibitor of acyl–CoA dehydrogenase.
Abstract: We present evidence that acrylate is an intermediate in the direct reduction of lactate to propionate in Clostridium propionicum. In the presence of 3–butynoic acid, an inhibitor of acyl–CoA dehydrogenase, acrylate was formed from D–lactate and detected by gas–liquid chromatography and mass spectrometry. This represents the first direct demonstration of acrylate accumulation from lactate in C. propionicum.
TL;DR: In this paper, mass spectrometers were used for on-line data acquisition in yeast fermentation, and the data reduction was accomplished by desk computer. But they were not used to analyze gas and liquid phases.
Abstract: It is shown here how mass spectrometry (MS) can be used for on–line data acquisition in fermentation. MS was applied in this work to analyze gas and liquid phases. Gas phase analysis allows fast and accurate measurement of all gases of interest (O2, N2, CO2, Ar, He etc.). Liquid phase analysis is possible with a steam sterilizable membrane probe and permits direct analysis of dissolved gases (O2, CO2, N2) and various volatiles. Automatic switching between gas inlet and membrane probe and the data reduction was accomplished by desk computer. Continuous yeast fermentation was monitored over long periods to study long–term stability, which seems to be adequate for fermentation processes. *Parts of this work were presented at the 2nd European Congress Biotechnology in Eastbourne, U.K., 1981, and at the 9th International Mass Spectrometry Conference in Vienna, A, 1982. **Symbols used in this paper appear preceding the reference section.
TL;DR: An inverse correlation is inferred between aflatoxin production and the presence of double–stranded RNA, which has been reported in A. flavus, and cycloheximide has an inhibitory effect on 80S ribosome–directed protein synthesis as well as on double–Stranded RNA virus synthesis in fungi.
Abstract: Cycloheximide Induction of Aflatoxin Synthesis in a Nontoxigenic Strain of Aspergillus Flavus
TL;DR: The feasibility of utilizing fibroblasts for preparation of plasminogen activator was demonstrated on a laboratory scale using batchwise and continuous processes designed to minimize the effects of the feedback control.
Abstract: The capacity of diploid human lung fibroblasts to produce plasminogen activator was found to vary about 20–fold depending on culture conditions, particularly on the serum used to supplement the growth medium and on the substratum to which the cells are attached. Fibroblasts adapted to produce large amounts of plasminogen activator, by growing for several generation times under conditions which enhance productivity of plasminogen activator, continue to synthesize and secrete the enzyme when transferred to a serum–free medium. Enzyme synthesis is, however, regulated by a negative feedback control exerted by the extracellular enzyme. The feasibility of utilizing fibroblasts for preparation of plasminogen activator was demonstrated on a laboratory scale using batchwise and continuous processes designed to minimize the effects of the feedback control.
TL;DR: Results suggest that selection for NO3− utilization could be a useful screen in the development of yeast strains with improved ability to ferment xylose.
Abstract: Xylose is a major constituent of hardwood and agricultural residues. Its effective utilization is essential to the economic practicability of any process seeking to use these materials for chemical production. A yeast, Pachysolen tannophilus, has been shown to ferment xylose to ethanol, acetic acid and xylitol, but the rate is slow and yields of ethanol are low. Growth on NO3− is known to stimulate synthesis of pentosephosphate pathway enzymes, and these studies showed that NO3− stimulates the aerobic production of ethanol from xylose by Pachysolen. Anaerobic production of ethanol, however, was inhibited by NO3−. Inhibition required induction by growth on NO3−, and inhibition was repressed by NH4+, indicating that nitrate reductase might be involved. Growth rates and product ratios were affected significantly by the nitrogen source used. These results suggest that selection for NO3− utilization could be a useful screen in the development of yeast strains with improved ability to ferment xylose. *Maintained in cooperation with the University of Wiscosin, Madison, Wis.
TL;DR: The isolation of a 2.7 kb chromosomal DNA fragment from Bacillus megaterium is described which, when inserted in a multi–copy vector and transferred into one of these highly productive strains, results in a further increase in productivity.
Abstract: We have isolated bacillus strains highly productive for penicillin acylase using conventional mutagenesis and screening techniques. This report describes the isolation of a 2.7 kb chromosomal DNA fragment from Bacillus megaterium which, when inserted in a multi–copy vector and transferred into one of these highly productive strains, results in a further increase in productivity. This fragment did not direct the synthesis of active enzyme when transformed into Bacillus subtilis, which does not naturally produce penicillin acylase. This report also describes the cloning of a 6.2 kb B. megaterium DNA fragment containing at least five of the structural genes of the tryptophan biosynthesis pathway. All of these genes are expressed at a functional level in B. subtilis.
TL;DR: By following a logical set of selection rules, a dodecadeoxyribonucleotide with potentially unique coding properties was designed and synthesized and used to construct stable plasmids with 30–90 copies of a palindromic sequence that would not otherwise persist in E. coli.
Abstract: By following a logical set of selection rules, a dodecadeoxyribonucleotide with potentially unique coding properties was designed and synthesized. This dodecamer is palindromic throughout 2/3 of its length, it self–anneals to generate multimers with 4bp single–stranded extensions, and, upon treatment with DNA ligase, it can be covalently joined to any double–stranded DNA molecule previously digested with EcoRI. The presence within our polymerized dodecamer of cleavage sites for endonucleases HaeIII and Bal I facilitated the monitoring of polymerization, ligation, and cloning. The polymerized dodecamer was positioned downstream from the lac UV5 promoter in a frameshift plasmid, pPCφ1, chosen to ensure that a particular translational reading frame would be utilized. Cloned, synthetic palindromic DNA was stable in vivo, but only when its size did not exceed 120 bp. The apparent lack of viability of cells harboring plasmids with inserts longer than 120 bp appears to be solely attributable to the palindromic character of the DNA. An alternative approach that has shown promise as a way of increasing the cellular content of a highly palindromic sequence is to interrupt the sequences with nonpalindromic DNA. This strategy was used to construct stable plasmids with 30–90 copies of a palindromic sequence that would not otherwise persist in E. coli.