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Chromosome Breakage Accompanying Genetic Recombination in Bacteriophage
... The following summary is inspired by the historical review of Haber (Haber 2007). The first model to be published sought to explain recombination between a broken and an intact λ phage molecule ( Figure 11A, (Meselson, Weigle 1961)). They thought that each strand of the broken DNA anneals to an intact complementary DNA, which serves as a template for DNA polymerization. ...
... Renaturation of complementary strand was the first activity discovered for RecA. In 1985, Bryant and The two strands of a broken chromosome fragment can form base pairs with an intact template and promote copying to the end of the template, thus producing a recombined, full-length product (Meselson, Weigle 1961). B. Holliday's 1964 model. ...
The diversity of the viruses infecting bacteria (bacteriophages, or phages for short) is so important that it is difficult to classify them in a pertinent way, and the species notion itself is a matter of debate among specialists. At the root of this diversity, one of the key factors is DNA recombination, which occurs at high levels among phages, and permits gene exchanges among entities that are sometimes very distant. My research has focused on homologous recombination in phages, and in particular on the protein that is key to the process, the recombinase. I have shown, for two different types of recombinases, Rad52-like and Sak4-like, that their fidelity was relaxed, compared to the bacterial recombinase, RecA. Moreover, for Sak4, a protein that had not been studied before, I showed that recombination occurs by single strand annealing, and that it is strictly dependent in vivo on the co-expression of its cognate SSB protein, whose gene is often encoded nearby in phage genomes encoding sak4. Genetic exchanges are therefore greatly facilitated for phages encoding these types of recombinases. Nevertheless, exchanges are not anarchical: recombination is seen up to 22% diverged substrates, but 50% diverged DNA sequences will not recombine. It may be that the species notion should be enlarged for phages, so as to include into a same group all phages exhibiting traces of recent exchanges of genetic material (the so-called mosaicism).
... In the model, (i) replication forks stall upstream of the Tus/Ter barrier and, before they can be resolved by fusion with a converging fork, a subsequent codirectional fork arrives that (ii) displaces the nascent leading strand, which produces a DSB-end at the barrier (Fig. 4F, ii). (iii) RecBCD resects the DSB ends to Chi sites and then loads RecA, which (iv) generates HJs by strand exchange, initiating DSB-repair replication forks (Fig. 4F, iv) (43), a process called joincopy (43) or break-copy (44,45) recombination, or BIR (Fig. 4F, iv). The BIR forks cannot repair the DSB ends unless they converge with an oncoming fork from the permissive side of the barrier (Fig. 4F, vi). ...
Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.
... There was now good reason to suppose that recombination might actually be amenable to biochemical study. It was Matthew Meselson, using phage l, who demonstrated that genetic recombination was accompanied by actual physical exchange of sections of DNA (Meselson and Weigle 1961;Meselson 1964). Recombination therefore necessarily involved breaks in DNA and the models of recombination that were developed are similar to, and can be traced to, an understanding of how cells avoid the lethality associated with the production of double-strand breaks in DNA, a major consequence of ionizing radiation. ...
The double stranded structure of DNA suggested a mechanism for replication. Overlooked was that it also served to maintain genome stability by providing a template for the repair of damage and mistakes in replication...
The persistence of hereditary traits over many generations testifies to the stability of the genetic material. Although the Watson–Crick structure for DNA provided a simple and elegant mechanism for replication, some elementary calculations implied that mistakes due to tautomeric shifts would introduce too many errors to permit this stability. It seemed evident that some additional mechanism(s) to correct such errors must be required. This essay traces the early development of our understanding of such mechanisms. Their key feature is the cutting out of a section of the strand of DNA in which the errors or damage resided, and its replacement by a localized synthesis using the undamaged strand as a template. To the surprise of some of the founders of molecular biology, this understanding derives in large part from studies in radiation biology, a field then considered by many to be irrelevant to studies of gene structure and function. Furthermore, genetic studies suggesting mechanisms of mismatch correction were ignored for almost a decade by biochemists unacquainted or uneasy with the power of such analysis. The collective body of results shows that the double-stranded structure of DNA is critical not only for replication but also as a scaffold for the correction of errors and the removal of damage to DNA. As additional discoveries were made, it became clear that the mechanisms for the repair of damage were involved not only in maintaining the stability of the genetic material but also in a variety of biological phenomena for increasing diversity, from genetic recombination to the immune response.
... This was such an attractive idea that the hypothesis in some form held center stage for nearly thirty years. In 1961 MESELSON andindependently KELLENBERGER, ZICHICHI and disproved BELLING'S attractive hypothesis. They were able to show, with radioactive and density markers, that the parental DNA (chromosome) of phage actually breaks and exchanges segments during some recombinant events. ...
... 24,25,49,51 Her 1961 article on recombination with Weigle 25 was accompanied in PNAS by one with the same conclusions from Matthew Meselson and Jean Weigle. 52 It is sad that although Grete's approach was more original and elegant, 51 Weigle delayed Grete's completed manuscript while waiting for Meselson to finish his experiments, which confirmed what she had already shown. 1 In the end, Meselson's article appeared just before Grete's, and is most cited in genetics textbooks. ...
Grete Kellenberger-Gujer was a Swiss molecular biologist who pioneered fundamental studies of bacteriophage in the mid-20(th) century at the University of Geneva. Her life and career stories are reviewed here, focusing on her fundamental contributions to our early understanding of phage biology via her insightful analyses of phenomena such as the lysogenic state of a temperate phage (λ), genetic recombination, radiation's in vivo consequences, and DNA restriction-modification; on her creative personality and interactions with peers; and how her academic advancement was affected by gender, societal conditions and cultural attitudes of the time. Her story is important scientifically, putting into perspective features of the scientific community from just before the molecular biology era started through its early years, and also sociologically, in illustrating the numerous "glass ceilings" that, especially then, often hampered the advancement of creative women.
... Recombinant DNA technology emerged half a century ago, when so-called 'restriction factors' were observed; which inhibited bacteriophage growth in bacteria, which turned out to be DNA endonucleases [3][4][5][6]. Around this time, DNA ligation was discovered as a basis of genetic recombination [7][8][9], leading to successful assembly of DNA fragments [10][11][12][13][14]. Since these ground-breaking discoveries, classical DNA cloning involved largely serial steps of cutting and pasting isolated fragments together by using restriction enzymes and DNA ligases, into functional DNA molecules (typically plasmids). ...
Multicomponent biological systems perform a wide variety of functions and are crucially important for a broad range of critical health and disease states. A multitude of applications in contemporary molecular and synthetic biology rely on efficient, robust and flexible methods to assemble multicomponent DNA circuits as a prerequisite to recapitulate such biological systems in vitro and in vivo. Numerous functionalities need to be combined to allow for the controlled realization of information encoded in a defined DNA circuit. Much of biological function in cells is catalyzed by multiprotein machines typically made up of many subunits. Provision of these multiprotein complexes in the test-tube is a vital prerequisite to study their structure and function, to understand biology and to develop intervention strategies to correct malfunction in disease states. ACEMBL is a technology concept that specifically addresses the requirements of multicomponent DNA assembly into multigene constructs, for gene delivery and the production of multiprotein complexes in high-throughput. ACEMBL is applicable to prokaryotic and eukaryotic expression hosts, to accelerate basic and applied research and development. The ACEMBL concept, reagents, protocols and its potential are reviewed in this contribution.
... In the early 1950s Luria, Anderson, Ralston and co-workers uncovered cellular processes regulating the host range of bacteriophages [1][2][3]. Subsequent investigations of this phenomenon by Arber, Meselson and co-workers led to the discovery of restriction-modification (R-M) systems, a landmark event in the history of molecular biology [4,5]. While much subsequent work focused on characterizing restriction enzymes as tools for recombinant DNA technology, the biology and biochemistry of the R-M systems proved to be interesting in their own right [6][7][8]. ...
While N⁶‐methyladenosine (m⁶A) is a well‐known epigenetic modification in bacterial DNA, it remained largely unstudied in eukaryotes. Recent studies have brought to fore its potential epigenetic role across diverse eukaryotes with biological consequences, which are distinct and possibly even opposite to the well‐studied 5‐methylcytosine mark. Adenine methyltransferases appear to have been independently acquired by eukaryotes on at least 13 occasions from prokaryotic restriction‐modification and counter‐restriction systems. On at least four to five instances, these methyltransferases were recruited as RNA methylases. Thus, m⁶A marks in eukaryotic DNA and RNA might be more widespread and diversified than previously believed. Several m⁶A‐binding protein domains from prokaryotes were also acquired by eukaryotes, facilitating prediction of potential readers for these marks. Further, multiple lineages of the AlkB family of dioxygenases have been recruited as m⁶A demethylases. Although members of the TET/JBP family of dioxygenases have also been suggested to be m⁶A demethylases, this proposal needs more careful evaluation.
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... hDNA was first detected physically as DNA of intermediate density ('"heavy-light'") produced by recombination between DNA containing heavy isotopes (of C and N) and that containing ordinary (light) isotopes (176). Bromodeoxyuridine-containing DNA has also been used as a source of "heavy"' DNA, but excision-repair activities on this DNA can produce confusion. ...
... This mechanism for DNA repair may be related to the mechanism of genetic exchange. An insight into the mechanisms of genetic recombination was gained through the discovery that h bacteriophage recombinants can be formed by joining fragments of preexisting phage DNA molecules (MESELSON and WEIGLE 1961). As continuity in the base sequence of the phage genome must be preserved, base pairing between overlapping single strands from each parentel molecule is presumably required as a prelude to the formation of a recombinant ( LEVINTHAL 1959). ...
... An obvious difficulty in attempts to answer these questions with phage is that the two processes occur concurrently. Early studies with X showed that some recombinants were formed by the breaking and rejoining of parental chromosomes to produce recombinant chromosomes with little or no DNA replication (Kellenberger et al. 1961;Meselson and Weigle 1961). However, in these studies replication was uninhibited, and so the bulk of the recombinants were composed of newly synthesized DNA. ...
... The action carried out by DNA ligase (the reunion of double-stranded DNA ends) was first realized when genetic recombination was observed between bacteriophages, and that such recombination requires doublestrand DNA breakage and DNA ends rejoining (Meselson and Weigle, 1961). Martin Gellert (1967) first reported the DNA end joining chemical reaction in E.coli cell extract, characterized the reaction and identified several necessary conditions (e.g., presence of ATP and magnesium) for the reaction to proceed. ...
Quantitative analysis of knowledge content of a significant technological innovation is a novel approach to understand the scientific discovery process. Here we describe such an analysis applied to the invention of recombinant DNA technology in the early 1970's. Two focal papers are selected, i.e., Jackson et al., 1972 and Cohen et al., 1973. A knowledge framework called EApc is described to categorize knowledge types and their quantification. The focal papers, along with their reference lists, are used to determine the minimal scientific knowledge necessary for generating the notions central to each focal paper. Attempts are made to trace how each type of knowledge was generated by various research communities. The results are discussed in terms of their potential implications in measuring, evaluating, understanding and managing the scientific research process.
... hDNA was first detected physically as DNA of intermediate density ('"heavy-light'") produced by recombination between DNA containing heavy isotopes (of C and N) and that containing ordinary (light) isotopes (176). Bromodeoxyuridine-containing DNA has also been used as a source of "heavy"' DNA, but excision-repair activities on this DNA can produce confusion. ...
... Copy Choice versus Breakage and Rejoining In considering the mechanism of RNA recombination, I would like to discuss several issues which are pertinent to the formulation of our current model. Unlike DNA recombination, which usually involves double-stranded DNA and can occur by either a breakage-and-rejoining mechanism (73) or, more rarely, a copy choice mechanism (13), only singlestranded RNA has been shown to undergo RNA recombination so far. Thus, the mechanism could be fundamentally different for DNA recombination and RNA recombination. ...
An increasing number of animal and plant viruses have been shown to undergo RNA-RNA recombination, which is defined as the exchange of genetic information between nonsegmented RNAs. Only some of these viruses have been shown to undergo recombination in experimental infection of tissue culture, animals, and plants. However, a survey of viral RNA structure and sequences suggests that many RNA viruses were derived form homologous or nonhomologous recombination between viruses or between viruses and cellular genes during natural viral evolution. The high frequency and widespread nature of RNA recombination indicate that this phenomenon plays a more significant role in the biology of RNA viruses than was previously recognized. Three types of RNA recombination are defined: homologous recombination; aberrant homologous recombination, which results in sequence duplication, insertion, or deletion during recombination; and nonhomologous (illegitimate) recombination, which does not involve sequence homology. RNA recombination has been shown to occur by a copy choice mechanism in some viruses. A model for this recombination mechanism is presented.
... hDNA was first detected physically as DNA of intermediate density ('"heavy-light'") produced by recombination between DNA containing heavy isotopes (of C and N) and that containing ordinary (light) isotopes (176). Bromodeoxyuridine-containing DNA has also been used as a source of "heavy"' DNA, but excision-repair activities on this DNA can produce confusion. ...
... Hershey and Chase showed that 2% of phage particles contain homologous loci from both phage in the cross (Hershey and Chase 1951), and in 1954, Leventhal offered the partial replica or copy choice hypothesis to explain the recombinant phage (Levinthal 1954). However, Meselson and Weigle described breakage and reunion as the method of recombination seven years later (Meselson and Weigle 1961) Lysogeny, the ability of some phage to remain latent for several generations, was observed in 1921 by two groups (Bordet andCiuca 1921, Gildemeister 1921). Lysogenic strains of bacteria were shown to harbor and maintain non-infectious phage, prophage, and it was shown that the prophage could be induced to a vegetative state by external factors in 1951 Gutmann 1950, Lwoff et al. 1950). ...
Lysogeny is a defining feature of temperate bacteriophages. Temperate bacteriophages are able to establish lysogeny by integrating into the host chromosome or extrachromosomally, as a plasmid-like prophage in the host cytoplasm. In either case, host factors are involved in both the establishment and maintenance of lysogeny. Mycobacteriophage L5 forms an integrated prophage through the action of a phage-encoded tyrosine recombinase. L5 integrase (Int) binds to the phage attachment site, attP, and the bacterial attachment site, attB. Int binds attP bivalently by binding to the core, where strand exchange occurs, and to arm-type binding sites that flank the core. A host-encoded DNA binding protein, mIHF, is required for recombination and binds between the core and arm-type binding sites of attP. mIHF is thought to catalyze the bending of attP required for bivalent binding between the core and arm-type binding sites. attP core consists of a seven base pair overlap region flanked on either side by imperfect inverted repeats that make up the recombinase binding elements or RBEs. The RBEs were shown to be essential for core binding by creating attPs with mutations in the RBEs. When both of the RBEs are mutated Int can neither perform recombination, nor create specific complexes that involve core binding. When the right side RBE is wild type with mutant left side RBE, recombination can occur, and complexes involving core binding are observed. However, Int is unable to catalyze recombination when the left side RBE is wild type and the right is mutated, indicating that contact must be made with attP on the right side of core for a stable Int/attP core complex to be formed. The middle domain connects the two outer domains, but its function in recombination is not well understood. To determine the function of the middle domain, mutations were made at conserved residues in the middle domain of Int, and these mutants were characterized. These mutants are able to catalyze recombination, but do not form the recombinagenic complexes observed by wild type Int. Increasing the mIHF concentration in these reactions augments the recombination efficiency and stabilizes recombinagenic complexes that involve Int/core binding.
The ribosomes repeatedly undergo subunit exchange, by dissociation into their subunits and reformation from a pool of free subunits that continuously recycle through ribosomes. This chapter describes an in vitro assay to study the mechanism of ribosomal subunit exchange and its role in protein synthesis. The assay is capable of measuring a single cycle of ribosome dissociation and reformation from subunits. The ribosomal subunit exchange can be demonstrated in vivo by centrifugal analysis of the density distribution of ribosomes and ribosomal subunits following transfer of a culture uniformly labeled with heavy isotopes to a medium containing only light isotopes. Hybrid ribosomes originating in vivo display essentially the same patterns. These particles may be separated into two species by density-gradient equilibrium centrifugation, if they are not resolved in sedimentation velocity analysis. A sensitive method of measuring small differences in the extent of ribosomal subunit exchange is to label two heavy cell cultures with different radioisotopes.
Mit der Bestätigung der Chromosomentheorie war die Frage nach der zellulären Grundlage der Vererbung beantwortet. Die Aufklärung ihrer molekularen Grundlage hatte jedoch noch nahezu ein halbes Jahrhundert zu warten. Zugang zur Entdeckung der chemischen Verbindung, die die Erbanlagen enthält, erhielt man durch die Beobachtung, daß es möglich ist, erbliche Eigenschaften durch Infektion von Mäusen mit abgetöteten Erregern zu übertragen. Eine solche Übertragung von Erbinformation wird als Transformation bezeichnet. Die chemische Analyse der transformierenden Substanz ließ erkennen, daß es sich um Desoxyribonukleinsäure (DNA) handelt.
The effects of extracellular UV-irradiation on the replication of DNA were tested with phage T2. Cells of E. coli B/1 were multiply infected with UV-irradiated T2. The kinetics of P32-incorporation into phage DNA were significantly different from the unirradiated control. With unirradiated phages the time curve is linear during the second half of the latent period after as short nonlinear increase. With UV-irradiated phages however the amount of DNA increases exponentially during the whole latent period. Such kinetics might be expected from semiconservative DNA-replication if templates were limiting. The reported findings suggest that other regulatory mechanisms normally limiting DNA-synthesis are inactivated by UV. The kinetics determined by semiconservative replication could then be clearly observed with irradiated phages. The nature of the normally regulating mechanisms is discussed.
In these groups of plants meiosis occurs in the formation of the spores, microspores and megaspores, if both types exist. However, much more detailed information is available concerning the divisions of microsporocytes because of the abundance of cells available for study and the facility with which preparations of microsporocytes are made. The essential features of meiosis are quite uniform throughout the group when the process goes to completion. The aberrations that have evolved usually either alter the timing and sequence of events or allow the process to proceed without one or more of its characteristic features. Very few anomalous new features have been introduced as has occurred in the insects and related forms, for example.
The bacteriophage f1 is a small, single-stranded DNA-containing phage. The DNA is in a ring form containing about 6000 nucleotides. The phage particle itself is filamentous, does not kill its host cells, and is continuously extruded from the cell surface while the cells continue to grow (Marvin and Hohn, 1969).
As discussed repeatedly during this symposium, recombination between DNA molecules proceeds in several steps (see Fig. 1): (A) Breakage or nicking of parental molecules (Meselson and Weigle, 1961), (B) formation of “joint” intermediates (Anraku and Tomizawa, 1965), some of them branched (Broker and Lehman, 1971; Benbow et al., this symposium), (C) conversion of “joint” into covalently linked “recombinant” molecules, which may still contain mismatched base pairs in the heteroduplex regions (Tomizawa, 1967; Kozinski et al., 1967), (D) repair of mismatches in heteroduplex regions (Spatz and Trautner, 1970; Fox, Meselson, this symposium), and (E) replication of “recombinant” DNA molecules, which resolves unrepaired mismatches in heteroduplex regions. The temporal sequence of some of these steps is not well defined; i.e., breakage may precede or follow the formation of “joint” molecules and repair may precede or follow the conversion of “joint” into “recombinant” molecules.
This chapter focuses on empirical tests of evolutionary theory using viruses, specifically bacteriophage or phage, beginning by reviewing phage biology and the phage life cycle, and then describing laboratory procedures for cultivating phage and assaying fitness. It also reviews the theoretical models that have been used to describe phage ecology and evolution, in order to emphasize the advantages of using phage experimental systems to test theoretical predictions. The chapter then discusses recent phage experiments that have investigated the genetic basis and molecular mechanisms underlying adaptation. Finally, it emphasizes that the insights gained from experimental evolution studies with phage have important implications not only for phage ecology and evolution, but also for predator–prey and host–parasite interactions in general.
λ Recombination and Recombineering, Page 1 of 2
Abstract
The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.
DNA ligases refer to enzymes that catalyze the formation of a phosphodiester linkage between DNA chains. DNA ligases are essential reagents in studies on nucleic acid structure and metabolism. Their value derives from the specificity of the reaction and their ability to join polynucleotide chains covalently. In combination with polynucleotide kinase end-group labeling, DNA ligase can also be used to identify 3′-and 5′-end groups at single-strand interruptions by nearest neighbor analysis. DNA ligase was first identified in extracts of uninfected and T-phage- infected E. coli in 1967. This chapter focuses on the DNA ligases of E. coli and phage T4-induced enzymes. It considers studies on ligases from other sources, which supplement, or differ from those obtained with the E. coli and T4 enzymes. The chapter discusses the purification and physical properties of the ligases and outlines the properties and substrate specificities of the reactions catalyzed by the enzyme, including the intermediates in the reactions. It also highlights the in vivo roles of DNA ligases and describes the research applications of the enzyme..
Die Entdeckung der Bakterienviren oder Bakteriophagen durch Twort (1915) und d’Herelle (1917) führte sofort zur Veröffentlichung einer großen Zahl von Arbeiten über dieses Phänomen. Speziell stand im Vordergrund der Diskussion die Möglichkeit der Anwendung von Bakteriophagen in der Therapie von Infektionskrankheiten, Dieses Interesse an Phagen nahm jedoch bald wieder ab, indem sich herausstellte, daß sich die Hoffnungen, die man an eine solche Phagentherapie geknüpft hatte, nicht erfüllten. Mit den 30er und 40er Jahren trat ein neuer Aspekt der Phagenforschung in den Vordergrund: Forscher wie Burnet, Schlesinger und besonders Delbrück erkannten die Möglichkeit, mit Hilfe des Systems Bakteriophage—Bakterium allgemein biologische und virologische Probleme zu studieren. Wegen der im Vergleich zu anderen Viren leichten Handhabung dieses Systems wurden die Bakteriophagen zu „Modellviren“: die Erforschung ihrer Biologie lieferte Beiträge zum Verständnis des allgemeinen Virusproblems. Darüber hinausgehend sind die Bakteriophagen in den letzten Jahrzehnten zu einem der interessantesten Untersuchungsobjekte einer modernen „Molekularbiologie“ geworden. Grundsätzliche genetische und biologische Fragen wie die Organisation und Replikation genetischen Materials, die Mutation, die Rekombination genetischer Merkmale, der Zusammenhang zwischen genetischer Information und der durch sie gesteuerten Proteinsynthese wurden durch Experimente mit Bakteriophagen angegangen.
Mit der gedanklichen Aufteilung des Erbguts in einzelne Gene begründete Mendel 1865 die Wissenschaft der Genetik. (Die Bezeichnung „Genetik“ wurde 1906 durch Bateson, der Ausdruck „Gen“ 1909 durch Johannsen eingeführt.) Gene waren bei sexueller Fortpflanzung neu kombinierbar, d. h. sie verhielten sich als Einheiten bei der Rekombination. Jedes Gen steuerte die Ausbildung eines bestimmten Merkmals und war somit zugleich die Einheit einer Funktion. Später erkannte man das Vorkommen von Mutationen. Gene konnten in verschiedenen Zuständen vorliegen und wurden so auch zu Einheiten der Mutation.
The article highlights Meselson's unique position in science and society as an innovative scientist involved in several ‘crucial experiments’ in molecular biology; a policy analyst in the domain of biological and chemical weapons for US administrations in the 1960s and 1970s; and a public intellectual shaping the public opinion against the use of such weapons in both war and peace times. Meselson's role in the ‘Meselson–Stahl experiment’ (hereafter the ‘M–S experiment’) which established the semiconservative mode of deoxyribonucleic acid (DNA) replication for which both Meselson and Stahl remain best known, is highlighted.
Genetic material possesses the capacity for self-duplication. The total amount of genetic information as well as its order within the genome remains the same from nuclear generation to nuclear generation. Each daughter genome arising from a replication generally consists of an exact copy of the original genetic information. With each mitotic division such copies are transmitted to the daughter cells. The identity of the genetic information is thus assured for each cell of a multicellular organism. Nevertheless, such an inflexible transmission of hereditary material would prevent evolution. This problem is overcome by two fundamental properties of the genetic material, namely recombination and mutation.
An endonuclease activity observed in extracts of T4 phageinfected Escherichia coli B cells has been purified about 250-fold. The enzyme, "T4 endonuclease II," appears to be an "early" enzyme induced after T4 phage infection. T4 endonuclease II introduces predominantly single strand breaks into native λ DNA in vitro although at high concentrations of enzyme some double strand breakage of the DNA occurs. The single strand breaks produced by T4 endonuclease II contain 3'-hydroxyl and 5'-phosphate termini. While all four deoxynucleotides are found at the 5'-phosphate terminus, T4 endonuclease II produces breaks preferentially adjacent to deoxyguanosine and deoxycytidine residues. The enzyme shows at least a 10-fold greater activity on native DNA than on denatured DNA. The enzyme makes limited numbers of breaks in λ DNA and the average size of the limit product sedimented in alkaline sucrose gradients is about 1000 nucleotides long. This enzyme has no effect on T4 DNA, whether glucosylated or nonglucosylated, and may be involved in the breakdown of host cell DNA which occurs after T4 phage infection.
Studies on the degradation of DNA, alkylated with methylmethanesulfonate, by endonuclease II of Escherichia coli are presented. With a DNA gel assay, some of the kinetics of degradation have been examined. With extensively alkylated DNA, only a limited number of double-strand breaks were made. Activity was maximum over a pH range from 8.0 to 9.0. No absolute requirement for added divalent metals was observed. The rate of the reaction was stimulated by added Mg²⁺ or Mn²⁺, but was not inhibited by 8-hydroxyquinoline or several other chelating agents. An exception was ethylenediaminetetraacetic acid. Sulfhydryl reagents and high ionic strength inhibited the enzyme while transfer RNA and caffeine did not. An approximate sedimentation coefficient was 3.6. With lightly alkylated DNA, the enzyme made predominantly single-stranded breaks, and the ratio of single-to double-strand breaks was approximately 3.7:1. It is concluded that the enzyme can hydrolyze a phosphodiester bond near an alkylated base in a native DNA molecule which has no single-strand breaks in this region. With more extensively alkylated DNA, viscosity experiments, which measure double-strand breaks, give an n value of approximately 1.
The activity of endonuclease II of E. coli on several species of native nonalkylated DNA has also been studied. The enzyme makes a limited number of single-strand breaks, with T4 DNA, approximately 3 to 4 per single-strand. Several factors which affect the reaction of the purified enzyme with alkylated DNA also affect the reaction with nonalkylated DNA. These are no absolute metal requirement, normal enzyme activity with 8-hydroxyquinoline but enzyme inhibition by EDTA, and no inhibition by tRNA. There was no induction of an enzyme with properties of endonuclease II after infection with T4.
Enzyme activity was present in a series of mutants deficient in the ultraviolet repair process, in recombination, in DNA synthesis at elevated temperatures, and in host cell restriction as well as in a strain sensitive to methylmethanesulfonate.
Overcoming cellular senescence that is induced by telomere shortening is critical in tumorigenesis. A majority of cancers achieve telomere maintenance through telomerase expression. However, a subset of cancers takes an alternate route for elongating telomeres: recombination-based alternative lengthening of telomeres (ALT). Current evidence suggests that break-induced replication (BIR), independent of RAD51, underlies ALT telomere synthesis. However, RAD51-dependent homologous recombination is required for homology search and inter-chromosomal telomere recombination in human ALT cancer cell maintenance. Accumulating evidence suggests that the breakdown of stalled replication forks, the replication stress, induces BIR at telomeres. Nevertheless, ALT research is still in its early stage and a comprehensive view is still unclear. Here, we review the current findings regarding the genesis of ALT, how this recombinant pathway is chosen, the epigenetic regulation of telomeres in ALT, and perspectives for clinical applications with the hope that this overview will generate new questions.
(Da in diesem Kapitel der erst in Kapitel 8 diskutierte Zusammenhang zwischen Gen und Enzym vorausgesetzt wird, mag es bei der ersten Lektüre des Buches für den Anfänger zweckmäßig sein, Kapitel 7 zunächst zu überspringen und erst nach Studium von Kapiteln 8 und 9 hierher zurückzukehren.)
Mit der gedanklichen Aufteilung des Erbguts in einzelne Gene begründete Mendel 1865 die Wissenschaft der Genetik. (Die Bezeichnung „Genetik“ wurde 1906 durch Bateson, der Ausdruck „Gen“ 1909 durch Johannsen eingeführt.) Gene waren bei sexueller Fortpflanzung neu kombinierbar, d. h. sie verhielten sich als Einheiten bei der Rekombination. Jedes Gen steuerte die Ausbildung eines bestimmten Merkmals und war somit zugleich die Einheit einer Funktion. Später erkannte man das Vorkommen von Mutationen. Gene konnten in verschiedenen Zuständen vorliegen und wurden so auch zu Einheiten der Mutation.
Viruses are the simplest biological structures possessing an autonomous genetic system, usually consisting of one macromole-cule of RNA or DNA, which controls the development and reproduction of the virion inside the host cell. In the simplest cases, virus RNA undergoes the same fate in the host’s cells as the messenger RNA which is synthesized in cell nuclei. For some period of time this virus RNA is fully adapted to conditions in the cell, using all the residual metabolic system of the cytoplasm or nucleus for synthesis of virus proteins. In some cases virus DNA becomes an element built into the host’s genetic system. Nevertheless, viruses can be regarded as autonomous genetic systems actively programming their development and reproduction.
The transmission of genetic information from cell generation to cell generation necessitates a duplication of the hereditary material prior to each cell division. Such self-duplication, which occurs according to a predetermined pattern, consists of the copying of the hereditary determiners of the cell and is called replication.
This article highlights Stahl's role as a leading expert on the central biological process of ‘genetic recombination’ (Stahl, 1987, 1988) during the last six decades as his main research interest, as well as his collaborations in deoxyribonucleic acid (DNA) replication, mutagenesis and genetic mapping. Starting with Stahl's PhD thesis (Stahl, 1956) on the genetic recombination of phage T4, and continuing with Stahl's seminal collaboration on the ‘Meselson–Stahl experiment’ which established how DNA replicates, a discovery for which both Meselson and Stahl remain best known, the article grounds Stahl's various contributions to molecular biology in his institutional legacies at the University of Rochester (PhD 1955), Caltech in the mid- and late 1950s and the University of Oregon at Eugene since 1959. The article further highlights Stahl's role as a foundation member of its Institute of Molecular Biology, his long-term collaboration with his late spouse Mary Morgan Stahl (1934–1996), his mentorship of two generations of students and his current collaboration, as a professor emeritus, with his former PhD student and partner Henriette (Jette) Foss. Stahl's contributions to the history of molecular biology, as both author and editor, are also mentioned.
Any explanation of the facts of heredity in terms of molecular structure must eventually account for self-duplication, for specificity, and for variation and modification of the genetic material. The helical structure of DNA has certain implications, some or all of which may prove to be important to its biological function: they are the subject of this and the following chapters.
DNA-Moleküle sind nicht völlig stabil. Sie unterliegen chemischen oder photochemischen Schädigungen und Fehlern, die bei der DNA-Replikation eingeführt werden und in fehlgepaarten Basenpaaren resultieren. Mechanistische Studien in der Arbeitsgruppe von Paul Modrich haben gezeigt, wie Replikationsfehler durch stranggerichtete Fehlpaarungsreparatur in E. coli und im Menschen korrigiert werden.
DNA molecules are not completely stable, they are subject to chemical or photochemical damage and errors that occur during DNA replication resulting in mismatched base pairs. Through mechanistic studies Paul Modrich showed how replication errors are corrected by strand-directed mismatch repair in Escherichia coli and human cells.
Genetische Rekombination ist neben Mutation und Selektion einer der wesentlichen Faktoren der Evolution. Die Neuzusammenstellung von Erbgut ist daher ein biologisches Grundphänomen, an dessen Aufklärung seit der Wiederentdeckung der Mendelschen Versuche gearbeitet wird. Trotz dieser Bemühungen sind die Molekularprozesse des Geschehens noch unverstanden. Wegen der Bedeutung der Rekombination soll dennoch versucht werden, die verschiedenen Teilprobleme zu beleuchten.
Two-factor crosses were performed between am mutants of φX174 in which one or both of the single-stranded parental bacteriophages were labeled with heavy isotopes. Recombinants were formed that contained DNA from each of the two parental phages and were composed almost entirely of parental RF DNA. A small amount of the parental-strand DNA (less than 700 nucleotides on the average) was removed and replaced during the formation of a recombinant molecule.
Bei der Reduplikation biologischer Systeme muß von Generation zu Generation ein gewisser Grundbestand an Information, der in den Erbanlagen niedergelegt ist, weitergegeben werden. Dies setzt vor jeder Zellteilung eine Kopierung der genetischen Information voraus. Man kann heute mit Sicherheit annehmen, daß in autonomen Systemen die DNS der einzige Träger genetischer Information ist. Es gibt zwar noch andere Makromoleküle in der Zelle, die eine Information weiterleiten können, wie z. B. die RNS oder die Proteine, aber nur die DNS gewährleistet durch den Mechanismus der identischen Reduplikation eine konstante Informationsübermittlung.
Das genetische Material besitzt die Fähigkeit zur identischen Verdoppelung. Hierbei bleibt nicht nur die Summe aller Einzelinformationen erhalten, sondern auch ihre Ordnung innerhalb des Genoms. Jedes durch Autoduplikation entstandene Tochtergenom enthält im allgemeinen eine genaue Kopie der ursprünglichen genetischen Information. Bei jeder mitotischen Teilung werden solche Kopien an die Tochterzellen weitergegeben. Dadurch ist die Identität des genetischen Materials für jede Zelle eines vielzelligen Organismus sichergestellt. Jedoch würde eine derartig starre Weitergabe des Erbgutes eine Evolution verhindern. Dieser Nachteil wird durch zwei Grundmerkmale des genetischen Materials ausgeglichen, nämlich durch Rekombination und Mutation.
Die Weitergabe der genetischen Information von Zellgeneration zu Zellgeneration erfordert vor jeder Zellteilung eine identische Verdoppelung der erbtragenden Substanz. Diese Autoduplikation erfolgt nach vorgegebenem Muster. Sie besteht also in einer Kopierung der schon in der Zelle vorhandenen Erbanlagen und wird als Replikation bezeichnet.
Ihrer Funktion als Informationsträger entsprechend ist DNA im Prinzip stoffwechselstabil. Dies bedeutet, daß jeder DNA-Einzelstrang über ungezählte Generationen hinweg — theoretisch für alle Ewigkeit — in seiner physischen Identität erhalten bleiben kann. Wir haben jedoch (in §§ 4/2 und 6/11) gesehen, daß vielerlei Faktoren wie UV oder bestimmte Chemikalien die Erbsubstanz auf kritische Weise schädigen können. Es ist nun schon seit vielen Jahren bekannt, daß besonders Bakterienzellen Mechanismen entwickelt haben, um durch Strahlung oder chemisch induzierte DNA-Schäden wieder zu beheben. Hierbei geht es nicht um Letalmutationen, sondern um chemische Veränderungen, die sich als „letale Blockierung“ (vgl. §4/2) störend auf die basengepaarte Doppelstruktur der DNA und deren Replikation auswirken. Als solche wurden gefunden:
1
Pyrimidin-Dimere. Der größte Teil des UV-Schadens ist auf die Entstehung von Pyrimidin-Dimeren (zumeist Thymin-Thymin-Dimere) zurückzuführen. Diese bilden sich durch eine photochemische Reaktion, bei der zwei auf demselben Strang unmittelbar benachbarte Pyrimidine, z. B. Thymin und Thymin, oder Thymin und Cytosin, durch zwei zusätzliche kovalente Bindungen miteinander verkoppelt werden, wodurch sie, aus ihrer normalen Lage gezerrt, die Paarungsfähigkeit verlieren, d. h. keine komplementäre Base finden.
DNA joining enzymes (ligases) are enzymes that catalyze the synthesis of phosphodiester bonds in duplex DNA, coupled to the cleavage of the pyrophosphate bond of adenosine triphosphate (ATP). This chapter focuses on the E. coli DNA ligase because it is the only ligase presently available in physically homogeneous form; it is also the most thoroughly studied. The subjects discussed include (1) isolation and physical properties of the E. coli DNA ligase, (2) chemical mechanism of the reaction that it catalyzes, and (3) its role in vivo. DNA joining activities have been measured by a variety of assay methods: the change in sedimentation coefficient after covalent closure of hydrogen-bonded λ-DNA circles, differential adsorption to hydroxylapatite after denaturation of hydrogen-bonded λ-DNA dimers, conversion of internally located 5′-32P-labeled phosphoryl groups to a form resistant to alkaline phosphatase, linkage of one polynucleotide chain to a second one attached to cellulose, and restoration of transforming activity to DNA previously treated with pancreatic DNase. Assays that measure the first step in the ligase-catalyzed reaction have also been described; the formation of the enoyme-adenylate intermediate from DPN has been used for the E. coli enzyme, and the T4-induced ligase has been assayed by an ATP- pyrophosphate exchange reaction.
1.1. All the known markers in the temperate phage λ are linked to each other.2.2. Mating between pairs of intracellular particles occurs at random with respect to partner but is rather rare (average number of rounds of mating 0.5, assuming two reciprocal recombinants are formed simultaneously).3.3. Double crossing over in a single mating event occurs frequently in λ.4.4. There appears to be little or no interference between two crossovers which occur in the same mating event.5.5. No correlation between reciprocal recombinants in single bursts is found.
1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria.
2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth.
3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactivation of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane.
4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny.
5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially unchanged membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur.
6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactivity. The progeny of phage particles labeled with radioactive phosphorus contain 30 per cent or more of the parental phosphorus.
7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the interaction between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this interaction are remarkably stable. The nature of the interaction is otherwise unknown.
8. The sulfur-containing protein of resting phage particles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented.
Mutations (h+ → h) to extended host range occur in variants of the temperate coliphage lambda. The h mutants are adsorbed by a mutant bacterial host which does not adsorb h+ phage. An h locus has been genetically mapped. Considerable phenotypic mixing, corresponding to nearly random association of h phenotype with genotype, has been observed among the progeny phage after mixed infections by h and h phage. No phenotypic mixing has been found for strong virulence in lambda.
Forty independently occurring clear plaque-forming mutants of λ have been isolated. Among them three different phenotypes can be recognized. Crosses between mutants indicate that all of the mutant loci are closely linked to one another, occupying a segment of the λ linkage group which is about of the total known genetic length. More detailed mapping reveals that the mutant loci fall into three adjacent regions, one for each phenotype.Mixed infection with a pair of phenotypically different mutants, each of which lysogenizes very poorly or not at all, produces the high frequency of lysogenization characteristic of infection with wild type. This phenomenon is called cooperation. The bacteria surviving such a mixed infection are lysogenic either for one or both of the infecting types of phages. Two non-allelic clear mutations having the same phenotypic expression show a cis-trans position effect in lysogenization.The kinetics of cooperation suggest the existence of an intermediate state in lysogenization.
This communication presents a new method for the study of the molecular weight and partial specific volume of macromolecules, with some illustrations based on results with deoxyribonucleic acid (DNA) and several viruses. The method involves observation of the equilibrium distribution of macromolecular material in a density gradient itself at equilibrium. The density gradient is established by the sedimentation of a low-molecular-weight solute in a solution subject to a constant centrifugal field.
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See Doermann, A. H., and M. B. Hill, Genetics, 38, 79 (1953).
- R D Wagner
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12 Strain "Y-2 original" from Wagner, R. D., Univ. Texas Publ. No. 4445, 104 (1944).
13 Kaiser, A. D., Virology, 3, 42 (1957).
9 We are grateful to Academician A. N. Nesmeyanov for suggesting Soyuzchimexport, Moscow, as a source of supply for high-purity C13. 10 Isomet Corporation
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Weigle, J., M. Meselson and K. Paigen, J. Molec. Biol., 1, 379 (19591.
9 We are grateful to Academician A. N. Nesmeyanov for suggesting Soyuzchimexport, Moscow,
as a source of supply for high-purity C13.
10 Isomet Corporation, Palisades Park, New Jersey.
11 Davern, C., doctoral dissertation, California Institute of Technology (1959).
- See Kaiser
See Kaiser, A. D., Virology, 1, 424 (1955).
- R D Wagner
- Univ
Strain "Y-2 original" from Wagner, R. D., Univ. Texas Publ. No. 4445, 104 (1944).
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Kellenberger, G., M. L. Zichichi, and J. Weigle, Nature, 187, 161 (1960).
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Appleyard, R. K., J. F. McGregor, and K. M. Baird, Virology, 2, 565 (1956).
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Kellenberger, G., M. L. Zichichi, and J. Weigle, these PROCEEDINGS, 47, 869 (1961).