Size and Base Composition of RNA in Supercoiled Plasmid DNA

Williams, Peter H.; Boyer, Herbert W.; Helinski, Donald R.

doi:10.1073/pnas.70.12.3744

Cited by 25 publications

(10 citation statements)

References 32 publications

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“…A stage of transcription was separable from a subsequent stage of DNA synthesis. A dramatic verification of RNA priming by RNA polymerase action has come with the RNA piece still in place (13). The initial choice of M13 replication (SS to RF conversion) as an object for examining rifampicin inhibition was fortunate, inasmuch as a test with 0X174 proved negative (8).…”

Section: Multienzyme Systems Of Dna Replicationmentioning

confidence: 99%

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Multienzyme Systems of DNA Replication

Schekman

Weiner

Kornberg

1974

Science

234

120

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Replication is accomplished by multienzyme systems whose operations are usefully considered in respect to three stages of the process: initiation, elongation, anid termination. 1) Initiation entails synthesis of a short RNA fragment that serves as primer for the elongation step of DNA synthesis. This stage, probed by SS phage DNA templates, reveals three distinctive and highly specific systems in E. coli. The Ml3 DNA utilizes RNA polymerase in a manner that may reflect how plasmid elements are replicated in the cell. The ØX174 DNA does not rely on RNA-polymerase, but requires instead five distinctive proteins which may belong to an apparatus for initiating a host chromosome replication cycle at the origin. The G4 DNA, also independent of RNA polymerase, needs simply the dnaG protein for its distinctive initiation and may thus resemble the system that initiates the replication fragments at the nascent growing fork. In each case it is essential that in vitro the DNA-unwinding protein coat the viral DNA and influence its structure. 2) Elongation is achieved in every case by the multisubunit, holoenzyme form of DNA polymerase III. Copolymerase III, which is an enzyme subunit, and adenosine triphosphate are required to form a proper complex with the primer template but appear dispensable for the ensuing chain growth by DNA polymerase (33). 3) Termination requires excision of the RNA priming fragment, filling of gaps and sealing of interruptions to produce a covalently intact phosphodiester backbone. DNA polymerase I has the capacity for excision and gapfilling and DNA ligase is required for sealing. What once appeared to be a simple DNA polymerase-mediated conversion of a single-strand to a duplex circle (34) is now seen as a complex series of events in which diverse multienzyme systems function. Annoyance with the difficulties in resolving and reconstituting these systems is tempered by the conviction that these are the very systems used ,by the cell in replicating its chromosome and extrachromosomal elements. Thus, understanding of the regulation of replication events in the cell, their localization at membrane surfaces and integration with cell division, and their coordination with phage DNA maturation and particle assembly will all be advanced by knowledge of the components of the replicative machinery.

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Section: Multienzyme Systems Of Dna Replicationmentioning

confidence: 99%

“…Re.solution and reconstitu(tion of the 13 G4 system. When the foregoing studies (conversion of SS to RF) were extended to a 0X174-like phage, called G4 (16), the results were rewarding in an unexpected way.…”

Section: Resolution Of the 0x174 Systemmentioning

confidence: 99%

Multienzyme Systems of DNA Replication

Schekman

Weiner

Kornberg

1974

Science

234

120

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“…Neutral and alkaline CsCl density gradient analysis was carried out as described by Sakakibara and Tomizawa (30). Separation of complementary DNA strands by poly(UG)-CsCl density gradient centrifugation (31) was carried out as described by Williams et al (32). All gradients were fractionated by drops either directly onto Whatman 3MM filter discs or collected into microtiter trays.…”

Section: Methodsmentioning

confidence: 99%

Nature of R-factor replication in the presence of chloramphenicol.

Crosa¹,

Luttropp²,

Falkow³

1975

Proc. Natl. Acad. Sci. U.S.A.

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Covalently closed circular deoxyribonucleic acid molecules of RSF1030, a nonconjugative Rfactor, initiate and complete rounds of semiconservative replication in the absence of protein synthesis long after the bacterial chromosome has ceased its replication. RSF1030 replication under these conditions is sensitive to the inhibitor of ribonucleic acid synthesis, rifampicin. The product of this replication, a covalently closed DNA molecule, shows, in contrast to those molecules produced during the replication in a logarithmically growing culture of Escherichia coli, a transition to the open circular form upon treatment with ribonucleases or alkali. Analysis of the product resulting from alkali treatment indicates that there is a single break in one strand of the original circular duplex. This alkali-sensitive site occurs with equal probability in either of the complementary strands. These results are interpreted as a requirement for an RNA primer for the initiation of RSF1030 DNA synthesis and as showing that its removal from the covalently closed molecule is inhibited in the absence of protein synthesis.A number of bacterial plasmids have been described that are of a relatively small mass, 1.5 to 10 X 106 daltons, are nonconjugative (non-self-transmissible), and replicate as a multiple copy pool (relaxed replication) in Escherichia coli. The colicinogenic plasmid ColEl (1) is the best known example of this class of plasmids, although an increasing number of analogous plasmid species specifying one or more antibiotic resistance determinants have been encountered in clinical isolates. The R-factor RSF1030 is a nonconjugative plasmid of this class, of molecular weight 5.5 X 106, carrying determinants for ampicillin resistance.In this report we describe the nature of the replication of RSF1030 in the presence of chloramphenicol. This plasmid continues to initiate and complete rounds of semiconservative replication for many hours under conditions of inhibition of protein synthesis. This replication is inhibited by rifampicin, and a significant proportion of DNA molecules of the plasmid contain ribonucleotides as part of their covalently closed double-stranded structure. A dependence of DNA synthesis on RNA synthesis has also been demonstrated in several other systems (2-13), and examples of association of ribonucleotides and deoxyribonucleotides are reported for bacteriophages (6, 7), animal viruses (14, 15), mitochondria (16,17), the E. coli chromosome (18,19), the chromosome of cultured animal cells (20), and the bacterial plasmids ColEl (21) and cloacinogenic factor Clo DF13 (13) replicating in chloramphenicol. A continued semiconservative replication in chloramphenicol and accumulation of oligoribonucleotides in only one site of the circular duplex with equal probability in either strand was previously shown only for the plasmid ColEl (22, 21). MATERIALS AND METHODSBacterial Strains, Media, and Cell Growth. RSF1030 was derived from a strain of E. coli containing the R-factor R111 (23). This strain transferre...

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“…Like recombinant DNA technology fi rst pioneered in the early 1970s at Stanford University and the University of California at San Francisco [34] , the fi rst applications of microbial metabolism were related to human health and medicine. Also in 1932, published fi ve months prior to Kluyver et al .…”

Section: Microbial Metabolism -A Historical Perspectivementioning

confidence: 99%

Industrial Systems Biology

Otero¹,

Nielsen²

2010

Industrial Biotechnology

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The term " industrial biotechnology " fi rst widely appeared in the literature in the early 1980s when genetic engineering, propelled by recombinant DNA technology, was searching for applications beyond health care and medical biotechnology [1, 2] . Today, industrial biotechnology represents a well -defi ned fi eld with signifi cant academic, government, and corporate representation. Formally, industrial biotechnology is the bioconversion, either through microbial fermentation or biocatalysis, of organic feedstocks extracted from biomass or their derivatives to chemicals, materials, and/or energy. Biomass is the result of photosynthetic carbon fi xation by plants to form organic polymers that may be digested, enzymatically or chemically, to carbohydrate, protein, and lipid monomers. Industrial biotechnology, often referred to as " white biotechnology " in Europe [3] , aims to provide costcompetitive, environmentally friendly, self -suffi cient alternatives to existing or newly proposed petrochemical processes.Processes that exploit industrial biotechnology have recently garnered increasing global attention with traditional petrochemical processing under scrutiny as a result of increasing raw material costs, environmental constraints, and decreasing self -suffi ciency.Industrial biotechnology has experienced unprecedented growth with bio -based production processes representing 5% of the total chemical production sales volume. By 2010, several studies have estimated that the total fraction will increase to 20%, representing US$310 billion of a projected total sales volume of US$1600 billion. Industrial biotechnology will continue to capture signifi cant sales volume percentages in the arenas of basic chemicals and commodities (2 -15%), specialty or added -value chemicals (2 -20%), and polymers (1 -15%). However, the greatest percentage gain is likely to occur in the fi ne chemical market (16 -60%), where industrial biotechnology platforms enable complex chemistry that are presently produced via complex synthetic or combinatorial routes [4] .Furthermore, industrial biotechnology is enabling new products, particularly novel therapeutic agents such as polyketides and specialty chemicals not previously identifi ed, such as the diverse polyunsaturated fatty acids and biopolymers produced by microalgae [5] .Industrial biotechnology is by no means a new fi eld, with fermentation processes for antibiotics (penicillin production by Penicillium chrysogenum ; annual market size exceeding US$1.5 billion), vitamins ( L -ascorbic acid production by the Reichstein process and biocatalysis by Gluconobacter oxydans ; annual market size exceeding US$600 million), organic acids (citric acid production by Aspergillus sp.; annual market size exceeding US$1.5 billion), and amino acids ( L -glutamate and L -lysine production by Corynebacterium glutamicum ; annual production exceeding 600 000 tons) well established [5] .In each of these examples, host organisms well suited for production of the target compound were isolated naturall...

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Size and Base Composition of RNA in Supercoiled Plasmid DNA

Cited by 25 publications

References 32 publications

Multienzyme Systems of DNA Replication

Multienzyme Systems of DNA Replication

Nature of R-factor replication in the presence of chloramphenicol.

Industrial Systems Biology

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