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Large-scale synthesis of PEG-NADH
TIBTECH - A U G U S T 1987 [Vol. 5]
Large-scale synthesis of PEG-NADH Water-soluble macromolecular NADH derivatives have been used in studies of coenzymes properties and in biochemical reactors. To extend this use to largescale reactors, an improved synthetic method has been developed. Up to 1 kg of carboxylated polyethylene glycol (PEG, MA 20 000) can be prepared in the laboratory by a method involving chlorination by thionylchlor-
ide and subsequent amination and carboxylation under mild conditions. A preparation of N(1)-(2-aminoethyl)-NAD was produced from 200 g NAD in water and ethyleneimine at pH 3.5. Unreacted ethyleneimine was removed to yield a crude mixture of unreacted NAD (25%), N(1)-(2-ammoethyl)-NAD (67%) and NAD-containing by-products (8%). The coupling of the PEG and NAD
Gene transfer in myxobacteria The myxobacteria is a family of Gramnegative bacteria which secrete a large number of molecules such as antibiotics and proteins. They are also of interest as models for studies on microbial differentiation. Genetic manipulation studies until recently had been restricted to a few strains of Myxococcus xanthus which has been induced to express foreign DNA such as that coding for a periplasmic
phosphatase of E. co]i or extracellular enzymes of the plant pathogen, Erwinia chrysan th emi. It has recently been found that the broad range plasmid RP4 is maintained stably in M. xanthus upon integration into the host chromosome. Derivatives of the plasmid which demonstrate very high frequencies of intregration have now been used as shuttle vectors for DNA transfer among several different
Determining plasmid copy number For many processes involving the use of organisms containing genetically engineered plasmid, it will be useful to determine simply and accurately the plasmid copy number. Density gradient centrifugation methods measure only covalently closed circular plasmid DNA, direct gel electrophoresis may be inexact, and filter hybridization requires partial purification of DNA. A recent paper has described a method of determining plasmid copy number involving nucleic acid sandwich hybridization. The principle of the method is that two different restriction fragments from the plasmid are cloned. One of these serves as a capturing agent; that is, the cloned fragment (both polarities) is fixed onto nitrocellulose filters and then exposed to a preparation containing the target DNA - the plasmid in this case. The preparation is crude - bacterial or yeast lysates boiled in alkaline SDS to release, nick and denature the DNA. Once the target DNA is bound, a second cloned restriction fragment, radiolabelled this time, is used to locate the bound target DNA.
The assay thus detects only DNA fragments with homologies to both restriction fragments. To determine copy
derivatives took place at room temperature and pH 4.7 in the presence of a carbodiimide derivative. Uncoupled coenzyme material was removed by cation exchange. After chemical reduction of the NAD derivatives to NADH derivatives, the N(1)-NADH derivative was converted to an N6-NADH derivative in a Dimroth rearrangement reaction. The overall yield for preparation of technical grade PEG-N6-(2-aminoethyl)NADH was 33.5%. Buckmann, A. F., Morr, M. and Kula, M. R. (1987) Biotechnol. App]. Biochem. 9,258-268: Gesellschaft fiir Biotechnologische Forschung mbH (GBF), Mascheroder Weg. 1, D-3300 Braunschweig, West Germany.
species of myxobacteria. Among the myxobacteria to which DNA can be transferred are various antibiotic producing strains. Saulnier, P., Jaoua, S., Breton, A.M., Reichenbach, H., Guespin-Michel, J.F. (1987) Proc. 4th European Congress on Biotechno]ogy, Vol. 1 (Neijssel, O.M., van der Meer, R.R. and Luyben, K. Ch. A. M., eds), p. 387-388, Elsevier: Laboratoire de G6n6tique Microbienne, Universit6 de Technologie de Comp~igne, BP 233, 60206 Comp~igne, France. number, an assay for genomic sequences is carried out on the DNA preparations. Korpela, K., Buchert, P. and Soderlund, H. (1987)]. Biotechnol. 5,267-277: Orion Genetic Engineering Laboratory, Orion Corp. Ltd., Helsinki, Finland.
Replication origins of plant organelle DNA A number of potential origins of replication have been isolated from chloroplast and mitochondrial DNA of Petunia hybrida. Seven potential origins were obtained: each of them contained regions with high AT content, numerous direct and inverted repeats and at least one yeast ARS (autonomously replicating sequence) consensus sequence. In addition, the chloroplast-derived sequences contained regions homologous to conserved sequences found in the alga, Chlamydomonas reinhardii. The regions from Chlamydomonas promote autonomous replication in yeast and in Ch]amydomonas itself. One of the Petunia hybrida mitochondial ARS also contained the C. reinhardii ARS region and other replica-
tion origin features such as a potential stem-and-loop structure, two GC-rich stretches and a potential gyrase binding site. However, one of the chloroplast ARS regions seems to be the best candidate for an origin of replication: it contains a 100 base pair AT-rich region which show extensive homology with the replication origin of Eug]ena gracilis and it is preferentially labelled in an in vitro DNA synthesizing system. de Haas, J. M., Kool, A. J. and Nijkamp, H. J. J. (1987) Proc. 4th European Congress on Biotechnology, Vol. 1 (Neiissel, O. M., van der Meer, R. R. and Luyben, K. Ch. A.M., eds), p. 449, Elsevier: Department of Genetics, Free University, de Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
O 1987, Elsevier Publications, Cambridge 0166- 9430/87/$02.00
Large-scale synthesis of PEG-NADH Water-soluble macromolecular NADH derivatives have been used in studies of coenzymes properties and in biochemical reactors. To extend this use to largescale reactors, an improved synthetic method has been developed. Up to 1 kg of carboxylated polyethylene glycol (PEG, MA 20 000) can be prepared in the laboratory by a method involving chlorination by thionylchlor-
ide and subsequent amination and carboxylation under mild conditions. A preparation of N(1)-(2-aminoethyl)-NAD was produced from 200 g NAD in water and ethyleneimine at pH 3.5. Unreacted ethyleneimine was removed to yield a crude mixture of unreacted NAD (25%), N(1)-(2-ammoethyl)-NAD (67%) and NAD-containing by-products (8%). The coupling of the PEG and NAD
Gene transfer in myxobacteria The myxobacteria is a family of Gramnegative bacteria which secrete a large number of molecules such as antibiotics and proteins. They are also of interest as models for studies on microbial differentiation. Genetic manipulation studies until recently had been restricted to a few strains of Myxococcus xanthus which has been induced to express foreign DNA such as that coding for a periplasmic
phosphatase of E. co]i or extracellular enzymes of the plant pathogen, Erwinia chrysan th emi. It has recently been found that the broad range plasmid RP4 is maintained stably in M. xanthus upon integration into the host chromosome. Derivatives of the plasmid which demonstrate very high frequencies of intregration have now been used as shuttle vectors for DNA transfer among several different
Determining plasmid copy number For many processes involving the use of organisms containing genetically engineered plasmid, it will be useful to determine simply and accurately the plasmid copy number. Density gradient centrifugation methods measure only covalently closed circular plasmid DNA, direct gel electrophoresis may be inexact, and filter hybridization requires partial purification of DNA. A recent paper has described a method of determining plasmid copy number involving nucleic acid sandwich hybridization. The principle of the method is that two different restriction fragments from the plasmid are cloned. One of these serves as a capturing agent; that is, the cloned fragment (both polarities) is fixed onto nitrocellulose filters and then exposed to a preparation containing the target DNA - the plasmid in this case. The preparation is crude - bacterial or yeast lysates boiled in alkaline SDS to release, nick and denature the DNA. Once the target DNA is bound, a second cloned restriction fragment, radiolabelled this time, is used to locate the bound target DNA.
The assay thus detects only DNA fragments with homologies to both restriction fragments. To determine copy
derivatives took place at room temperature and pH 4.7 in the presence of a carbodiimide derivative. Uncoupled coenzyme material was removed by cation exchange. After chemical reduction of the NAD derivatives to NADH derivatives, the N(1)-NADH derivative was converted to an N6-NADH derivative in a Dimroth rearrangement reaction. The overall yield for preparation of technical grade PEG-N6-(2-aminoethyl)NADH was 33.5%. Buckmann, A. F., Morr, M. and Kula, M. R. (1987) Biotechnol. App]. Biochem. 9,258-268: Gesellschaft fiir Biotechnologische Forschung mbH (GBF), Mascheroder Weg. 1, D-3300 Braunschweig, West Germany.
species of myxobacteria. Among the myxobacteria to which DNA can be transferred are various antibiotic producing strains. Saulnier, P., Jaoua, S., Breton, A.M., Reichenbach, H., Guespin-Michel, J.F. (1987) Proc. 4th European Congress on Biotechno]ogy, Vol. 1 (Neijssel, O.M., van der Meer, R.R. and Luyben, K. Ch. A. M., eds), p. 387-388, Elsevier: Laboratoire de G6n6tique Microbienne, Universit6 de Technologie de Comp~igne, BP 233, 60206 Comp~igne, France. number, an assay for genomic sequences is carried out on the DNA preparations. Korpela, K., Buchert, P. and Soderlund, H. (1987)]. Biotechnol. 5,267-277: Orion Genetic Engineering Laboratory, Orion Corp. Ltd., Helsinki, Finland.
Replication origins of plant organelle DNA A number of potential origins of replication have been isolated from chloroplast and mitochondrial DNA of Petunia hybrida. Seven potential origins were obtained: each of them contained regions with high AT content, numerous direct and inverted repeats and at least one yeast ARS (autonomously replicating sequence) consensus sequence. In addition, the chloroplast-derived sequences contained regions homologous to conserved sequences found in the alga, Chlamydomonas reinhardii. The regions from Chlamydomonas promote autonomous replication in yeast and in Ch]amydomonas itself. One of the Petunia hybrida mitochondial ARS also contained the C. reinhardii ARS region and other replica-
tion origin features such as a potential stem-and-loop structure, two GC-rich stretches and a potential gyrase binding site. However, one of the chloroplast ARS regions seems to be the best candidate for an origin of replication: it contains a 100 base pair AT-rich region which show extensive homology with the replication origin of Eug]ena gracilis and it is preferentially labelled in an in vitro DNA synthesizing system. de Haas, J. M., Kool, A. J. and Nijkamp, H. J. J. (1987) Proc. 4th European Congress on Biotechnology, Vol. 1 (Neiissel, O. M., van der Meer, R. R. and Luyben, K. Ch. A.M., eds), p. 449, Elsevier: Department of Genetics, Free University, de Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
O 1987, Elsevier Publications, Cambridge 0166- 9430/87/$02.00