Showing papers in "Basic life sciences in 1982"
TL;DR: With a limited supply of arable land supporting an ever-increasing human population, the threat of crop loss to agricultural pests becomes continually more acute, and pesticides have become an essential component of modern agriculture.
Abstract: With a limited supply of arable land supporting an ever-increasing human population, the threat of crop loss to agricultural pests becomes continually more acute. Thus pesticides have become an essential component of modern agriculture. As competing organisms evolve resistance to commonly used agents, new and more effective poisons and repellants must constantly be developed. The fundamental problem in pesticide development is to produce chemicals that act specifically against certain organisms without adversely affecting others. Because of the similarities in the structural, metabolic and genetic components of all life forms, absolute species specificity is frequently difficult to attain. Furthermore, such toxic chemicals improperly used may engender biological effects beyond those for which they were originally manufactured.
87 citations
TL;DR: An important area being studied is that of strain selection and improvement for maximum productivity in the fermentation process.
Abstract: Recent interest in ethanol as a potential fuel or fuel supplement has stimulated research into various aspects of the fermentation process. Techniques such as continuous fermentation, vacuum distillation and methods for cell recycle have been investigated (6,10,11), but another important area being studied is that of strain selection and improvement for maximum productivity.
40 citations
TL;DR: Streptococcus mutans is thought to play an important role in the etiology of dental caries in humans and studies with mutants indicate that S. mutans’ ability to synthesize water-insoluble glucans from sucrose is closely linked to its pathogenicity.
Abstract: The genus Streptococcus contains a large number of species able to cause infection in humans and in animals. Our research efforts have revolved around the study of streptococci that normally reside in the human oral cavity. In this regard, Streptococcus mutans is thought to play an important role in the etiology of dental caries (tooth decay) in humans (5). Its virulence has been well established in animal model systems (see 5) and studies with mutants indicate that S. mutans’ ability to synthesize water-insoluble glucans from sucrose is closely linked to its pathogenicity.
37 citations
TL;DR: Alcohol dehydrogenase catalyzes the inter-conversion of an alcohol and an aldehyde with NAD+ as a cofactor and in many organisms there is a distinctive tissue specificity in the distribution of different ADH isozymes.
Abstract: Alcohol dehydrogenase (E.C. 1.1.1.1. ADH) catalyzes the inter-conversion of an alcohol and an aldehyde with NAD+ as a cofactor. In most higher organisms that have been studied, several different isozymes of ADH are present. The main function of these isozymes is presumed to be catabolic, to degrade various alcohols or sterols. This presumption is based primarily on the substrate preferences of the various isozymes and the absence of a fermentative pathway for alcohol production during glycolysis. In many organisms there is a distinctive tissue specificity in the distribution of different ADH isozymes.
32 citations
TL;DR: Ethylating agents are more oxygen-specific than the analogous methylating agent and, in a substantial number of cases, also more mutagenic at lower levels of treatment.
Abstract: Simple directly acting alkylating agents can be classified in terms of their mutagenic efficiency and their chemical reactivity. The most mutagenic are the N-nitroso compounds and these have a preference for reacting with nucleic acid oxygens in vitro and in vivo. In contrast, the alkyl sulfates are generally poor mutagens and react almost exclusively with base nitrogens. Other classes of alkylating agents also show correlations between oxygen reaction and mutagenicity. Ethylating agents are more oxygen-specific than the analogous methylating agent and, in a substantial number of cases, also more mutagenic at lower levels of treatment.
31 citations
TL;DR: The development of this procedure will be described here with the hope that some of the methods will be applicable to the development of such systems for other filamentous fungi.
Abstract: In comparison to yeast (15), the development of recombinant DNA technology for filamentous fungi is in its early stages. Recombinant DNA technology is dependent on two different though related matters: an efficient transformation system for the organism and an appropriate vector. In addition, transformation in any organism involves two processes: a system which permits the uptake of DNA into the cell and the subsequent integration of the DNA into a chromosome or its maintenance as a self-replicating entity in the recipient strain. An efficient transformation system for Neurospora crassa has been developed utilizing a cloned gene from Neurospora (4,27). The development of this procedure will be described here with the hope that some of the methods will be applicable to the development of such systems for other filamentous fungi.
30 citations
TL;DR: Pesticides are the most controversial for their known or potential genotoxicity (mutagenicity-carcinogenicity-teratogenicity) and will comprise the subject matter of this paper.
Abstract: Of the some 1500 chemicals used in agriculture (99), pesticides are the most controversial for their known or potential genotoxicity (mutagenicity-carcinogenicity-teratogenicity) and will comprise the subject matter of this paper. Pesticides are a large diverse group of compounds which include fungicides, herbicides, insecticides, fumigants, growth regulators and inhibitors, acaricides, rodenticides, soil nematicides, seed sterilants, soil conditioners, and chemosterilants. In addition, there are emulsifiers and solvents which are combined with pesticides and which are potential mutagens in their own right (25).
30 citations
TL;DR: The evidence is strongest for the association between farming and risk of leukemia, however, the specific leukemogenic agent or agents have yet to be identified and the urgent need for similar epidemiologic studies in the U.S.
Abstract: Farmers are rather self-sufficient and routinely perform tasks normally associated with other occupations such as machine repair, carpentry, welding, equipment operating, pesticide application, and livestock handling. They may come in contact with a wide spectrum of chemical, physical, and biologic agents in the performance of these tasks. Of particular concern are pesticides; zoonotic viruses, microbes, and fungi; solvents, fuels and oils; dusts; metal fumes; and mycotoxins. The subset of exposures experienced by individuals would vary, however, according to the specific type of farming operation.
28 citations
TL;DR: The DNA methyltransferase acting on O6-methylguanine appears to be primarily responsible for the adaptive response to the mutagenic effect of simple alkylating agents.
Abstract: Several repair enzymes are induced in Escherichia coli as a consequence of exposure to methylating agents such as N-methyl-N’- nitro-N-nitrosoguanidine. These include at least two different glycosylase functions, which act specifically on alkylated DNA by catalyzing the release of 3-methyladenine and 7-methy1guanine in free form. The apparently most important repair function induced during this adaptive response to alkylating agents, however, reduces the mutagenic activity of these agents by allowing improved repair of O6- methylguanine (Cairns, 1980; Schendel and Robins, 1978). This reaction can be faithfully duplicated in vitro employing cell-free extracts from adapted E. coli. The active factor has been shown to be an induced protein of molecular weight about 17,000, which catalyzes the transfer of a methyl group from the O6-position of an alkylated DNA guanine residue to one of its own cysteine residues. The protein acts in an analogous fashion on ethylated DNA by removing the ethyl group from O6-ethylguanine. The reaction is associated with suicide inactivation of the protein, since it appears to lack the capacity to release the blocking alkyl group. There is no detectable change in molecular weight of the protein accompanying methylation, so the inactivation probably depends on the modification of a reactive sulfhydryl group. S-Methylcysteine has been identified in the methylated protein by amino acid analysis and by conversion of this residue to its sulfone derivative by hydrogen peroxide treatment. S-Methylcysteine has not been found previously in enzymatically methylated proteins. Moreover, the reaction represents the only known case of protein methylation that is not dependent on S- adenosylmethionine as methyl donor. E. coli mutants unable to respond to alkylating agents by adaptation (isolated by P. Jeggo) also lack the methyltransferase, while mutants that express the adaptive response in a constitutive fashion (isolated by B. Sedgwick) also produce the methyltransferase constitutively. Thus, the DNA methyltransferase acting on O6-methylguanine appears to be primarily responsible for the adaptive response to the mutagenic effect of simple alkylating agents.
26 citations
TL;DR: This chapter explores the validity of the assumption that almost every pest-control chemical is a “potential mutagen” or "potential carcinogen" and presents an environmental view which modifies but does not entirely overcome it.
Abstract: The terms “pesticide” and “mutagen” seem continually to be linked in the public mind. According to news accounts, almost every pest-control chemical is a “potential mutagen” or “potential carcinogen.” In this chapter, I would like to explore the validity of this assumption and present an environmental view which modifies but does not entirely overcome it.
25 citations
TL;DR: Japanese immigrants to the United States show a decrease in the incidence of stomach cancer with an increase in that of intestinal cancer, presumably because of change in their life style.
Abstract: The incidences of cancers in various organs of the body differ in different countries (14,100). For instance, in Japan stomach cancer is the predominant cancer of the digestive tract, but in most Western countries intestinal cancer is predominant. Japanese immigrants to the United States show a decrease in the incidence of stomach cancer with an increase in that of intestinal cancer, presumably because of change in their life style (34).
TL;DR: Results suggest that beta-NF inhibits AFB1 carcinogenesis at least in part by altering MFO activities such that detoxication is enhanced and initial DNA damage by AFB1 is reduced.
Abstract: Trout were fed a range of dietary components which altered their carcinogenic response to aflatoxin B1 (AFB1). Dietary protein at levels substantially exceeding nutritional requirements were synergistic with AFB1. Cyclopropene fatty acids (CPFA) were carcinogenic when fed alone at 20 or 55 ppm, and synergistic when fed with AFB1 In contrast, several flavonoid and indole compounds, especially β-naphthoflavone (β-NF) and indole-3-carbinol, Inhibited the carcinogenic response when fed prior to and along with AFB1.
TL;DR: The error rate for DNA replication in Escherichia coli has been estimated as one error per 108−1011 base pairs replicated, considerably higher than that predicted on the basis of free-energy calculations or that achieved by DNA polymerases in vitro.
Abstract: The error rate for DNA replication in Escherichia coli has been estimated as one error per 108−1011 base pairs replicated. This level of accuracy is considerably higher than that predicted on the basis of free-energy calculations or that achieved by DNA polymerases in vitro. The existence of an excision repair mechanism acting upon mismatched base pairs could account, at least in part, for the accuracy achieved in vivo. Such a repair mechanism has been postulated to account for a number of genetic observations related to gene conversion and high negative interference phenomena. Furthermore, the increased spontaneous mutability of Pnemococcus hex - and E. coli uvrE - mutants that appear to be deficient in the repair of some mismatched base pairs indicates the involvement of mismatch repair in the suppression of spontaneous mutation rates, The existence of a mismatch repair system capable of efficiently correcting mispaired bases implies that a strand discrimination system must exist which allows the discrimination between the “incorrect” newly synthesized daughter strand and the “correct original parental strand.
TL;DR: The Streptomyces present a practical model for genetic manipulation and must have severely constrained codon usage patterns which should maximize the G+C content in the third position of codons.
Abstract: Antibiotics are economically-important chemicals produced by a variety of species of prokaryotic and eukaryotic microorganisms. Species of the prokaryotic genus, Streptomyces, produce about 70 percent of all known antibiotics (19). Therefore, the Streptomyces present a practical model for genetic manipulation. Streptomyces are also fundamentally interesting for at least two reasons. First, they undergo a differentiation cycle of spore germination, mycelial growth, aerial mycelium formation and sporulation (17). In general, the relationships between the differentiation cycles and antibiotic production are only poorly understood. Second, the Streptomyces contain circular genomes (17,19) with guanine plus cytosin (G+C) contents of 69 to 73 percent (49). This G+C content approaches the genetic code upper limit (50), beyond which the amino acid content of proteins would have to deviate substantially from the bacterial norm. Since the latter is highly unlikely (47), the Streptomyces must have severely constrained codon usage patterns which should maximize the G+C content in the third position of codons. It is not known if there are additional constraints in noncoding sequences which, for instance, might affect promoter sequences.
TL;DR: Genetic analysis and biochemical experiments indicate that the LexA gene product is the direct repressor of these genes and that proteolytic cleavage of the lexA protein by the recA protease is required for their induction.
Abstract: We have used the operon fusion vector Mud(Ap, lac) to generate a set of fusions within the Escherichia coli chromosome in which β- galactosidase (the product of the lacZ gene carried by the phage) is induced in response to DNA damaging agents such as UV, mitomycin C, 4-nitroquinoline-l-oxide, and N-methyl-N’-nitro-N-nitrosoguani- dine. This induction is not seen in recA - or lexA - cells. We have identified two members of this set of inducible genes as being uvrA and uvrB; the products of these two genes are required for excision repair of pyrimidine dimers and other lesions. In addition we have recently isolated a Mud(Ap, lac) insertion in the umuC gene. The product of this gene is specifically required for most chemical mutagenesis in E. coli and for inducible (Weigle) reactivation of UV- irradiated bacteriophage λ. Expression of β -galactosidase in the umuC::Mud(Ap, lac) fusion strain was induced by UV and a variety of agents in a recA + lexA +-dependent fashion. In all, the expression of ten E. coli genes is now known to be induced by DNA damage. Genetic analysis and biochemical experiments indicate that the lexA gene product is the direct repressor of these genes and that proteolytic cleavage of the lexA protein by the recA protease is required for their induction.
TL;DR: The isolation of host mutants of Escherichia coli K12 which exhibit increased frequencies of precise excision of transposon Tn10 are reported, supporting the idea that precise and nearly precise excison occur by closely related pathways which are distinct from those pathways which promote the third type of excision event.
Abstract: We have described three illegitimate recombination events associated with, but not promoted by, transposon Tn10: precise excision, nearly precise excision, and precise excision of a nearly precise excision remnant. All three are structurally analogous: excision occurs between two short direct repeat sequences, removing all intervening material plus one copy of the direct repeat. In each case, the direct repeats border a larger inverted repeat. We report here the isolation of host mutants of Escherichia coli K12 which exhibit increased frequencies of precise excision of Tn10. Nineteen of the 39 mutants have been mapped to five distinct loci on the E. coli genetic map and have been designated texA through texE (for Tn10 excision). Mapping and genetic characterization indicate that each tex gene corresponds to a previously identified gene involved in cellular DNA metabolism: recB and/or recC, uvrD, mutH, mutS, and dam. The role of these various DNA repair and recombination genes in an illegitimate recombination process such as Tn10 excision will be discussed. In addition to an increase in precise excision frequency, all 39 tex mutants display an increased frequency for nearly precise excision. However, none of the mutants are increased for the third excision event, precise excision of a nearly precise excision remnant, supporting the idea that precise and nearly precise excision occur by closely related pathways which are distinct from those pathways which promote the third type of excision event.
TL;DR: The following account of the status of current knowledge of the molecular biology of methanogens and suggestions for genetic approaches to be used in their study are, in part, derived from the very open and constructive discussions held at these two meetings.
Abstract: There have been two meetings*, convened during the past year, specifically to determine how microbial genetics could be productively applied to obligately anaerobic microorganisms and to methanogenic species in particular. One of us (JNR) attended both of these meetings. The following account of the status of current knowledge of the molecular biology of methanogens and suggestions for genetic approaches to be used in their study are, in part, derived from the very open and constructive discussions held at these two meetings.
TL;DR: The atypical citrate-utilizing ability to two strains of E. coli has been shown to be plasmid-encoded and the C. freundii viaB locus appears to encode the Vi antigen as well as the genetic "switch" mechanism controlling reversible Vi antigen expression.
Abstract: Genetic and biochemical studies of the metabolic and virulence properties of enterobacteria have been conducted, both for basic scientific and industrial reasons, for more than 40 years. Despite this long time span, only limited genetic characterization of enteric organisms other than Escherichia coli K-12 has been accomplished. Though several reasons can be offered for this lack of information, a major problem often encountered is the difficulty in genetically manipulating these other enteric organisms, e.g., Salmonella typhimurium, Salmonella typhi, Citrobacter freundii, wild-type E. coli, or Shigella species. In addition to facing DNA restriction barriers in many of these organisms, Hfr strains can not be easily constructed by classical procedures. Nevertheless, about 20 years ago my colleagues and I began intergeneric genetic transfer experiments with different enterobacteria to characterize their genetic constitution (2.3,8,13). Initially, classical genetic methods were used (i.e., F, F′, and Hfr strains), but these techniques presented many problems and resultant events usually occurred at a low frequency. Despite the difficulties, these studies led to the initial descriptions of conjugative plasmids coding for lactose and sucrose utilization in Salmonella (7,18,26,28) and involved the first mapping of chromosomally located antigenic determinants of Salmonella typhi, the causative agent of typhoid fever (11,13,14).
TL;DR: Egypt is a semi-arid country where the roughly 2.5 million acres of arable land lie in the Nile River delta and valley, and on this narrow strip, almost all of Egypt's 42 million people live and work.
Abstract: Egypt is a semi-arid country where the roughly 2.5 million acres of arable land lie in the Nile River delta and valley. On this narrow strip, almost all of Egypt1s 42 million people live and work. The Nile water is used for irrigation as well as for industrial and domestic purposes. The river is also used for the disposal of agricultural waste water. Industrial wastes may also be flowing into the river and its tributaries.
TL;DR: The mitochondrial DNA (mtDNA) is a dispensable genome in the facultative aerobe Saccharomyces cerevisiae, Since cell viability is maintained even if mtDNA is nonfunctional, mitochondrial mutagenesis can be studied.
Abstract: The mitochondrial DNA (mtDNA) is a dispensable genome in the facultative aerobe Saccharomyces cerevisiae, Since cell viability is maintained even if mtDNA is nonfunctional, mitochondrial mutagenesis can be studied. Marked dissimilarities exist between nuclear and mtDNA with respect to base composition, structure, multiplicity, and heterogeneity of molecules; differences in mutagenic processes are consequently expected. Mitochondrial mutants belong to two classes that both exhibit non-Mendelian inheritance; the rho − (or “petite” mutation) results from massive deletions with a preferential loss of a specific segment, accompanied by repetitions of retained sequences; the other type is due to point mutations or small deletions and includes antibiotic resistant mutants and mutants defective in the synthesis of one or more components of the mitochondrial membrane complex (mit−) or in the mitochondrial protein machinery (syn−). The rho− mutation is extremely frequent even spontaneously (from 1 to a few percent), whereas mitochondrial point mutations are relatively rare even with induced mutagenesis. Nuclear mutations due to molecular modifications similar to those seen in rho− are rather infrequent. The genetic control of mitochondrial mutagenesis requires gene products involved either (1) in both nuclear and mitochondrial mutagenesis, the genes being located in the nucleus; or (2) specifically in mitochondrial mutagenic processes, the genes being located either in the nuclear or in the mitochondrial genomes.
TL;DR: UV mutagenesis in yeast depends on the function of the RAD6 locus, a gene that is also responsible for a substantial fraction of wild-type resistance, suggesting that this eukaryote may possess a misrepair mechanism analogous to that proposed for Escherichia coli.
Abstract: UV mutagenesis in yeast depends on the function of the RAD6 locus, a gene that is also responsible for a substantial fraction of wild-type resistance, suggesting that this eukaryote may possess a misrepair mechanism analogous to that proposed for Escherichia coli. The molecular mechanism responsible for RAD6 repair or recovery is not yet known, but it is different from either excision or recombination-dependent repair, processes carried out by the other two main repair pathways in yeast. RAD6-dependent mutagenesis has been found to have the following characteristics.
TL;DR: This conclusion may help solve the C-value paradox, namely, that higher organisms appear to have much more DNA than is required for the synthesis and control of gene products, and that there is enormous variation of DNA content between different taxonomic groups or even between quite closely related species of higher organisms.
Abstract: A strong case has recently been made for the existence of selfish DNA in higher organisms (Doolittle and Sapienza, 1980; Orgel and Crick, 1980). Given that genetic elements exist which have the means of reproducing themselves by transposition to new locations, they would be expected to gradually accumulate in the genome, especially in diploid organisms. The essence of the argument is that each insertion of a nonfunctional DNA sequence, which only very slightly increases the size of the genome, would have a neglible phenotypic effect on the individual and therefore would not be selected against. For the amount of DNA to increase, all that is required is that the probability of a DNA sequence being added to the genome is greater than the probability of it being eliminated by natural selection or by random deletion. There will also be positive selection for genetic elements which increase by mutation their efficiency of transposition. Not only will genomes increase in size, but also most of the DNA will become “junk,” with no function for the organism. This conclusion may help solve the C-value paradox, namely, that higher organisms appear to have much more DNA than is required for the synthesis and control of gene products, and that there is enormous variation of DNA content between different taxonomic groups or even between quite closely related species of higher organisms.
TL;DR: Though the data published show that the fusion technique can be successfully applied for genetic analysis even in systems having no natural way to do it, further studies are needed to find the conditions where it works in predictable ways.
Abstract: Induced fusion of protoplasts is a relatively new way of genetic manipulation in microbial systems. The individual steps of the manipulation are shown in the scheme of Figure 1. The rigid cell wall of bacteria, fungi, yeast, and algae (as well as of plant cells) can be removed without influencing the viability and integrity of the cell. The wall-less microorganism is surrounded by a plasma membrane, in hypertonic media it is spherical in shape, and is called a protoplast. The protoplasts, under appropriate conditions, do resynthesize cell wall material and revert to the original form with characteristic morphology.
TL;DR: Most laymen and many scientists think only of industrial pollution and harmful chemicals when discussing roles for halogenated organic molecules in nature, but this paper hopes to persuade you that Mother Nature does like some halogenation compounds and indeed, has reserved halogensated compounds for some very special functions and purposes.
Abstract: I note that the title of this Conference indicates GENETIC ENGINEERING OF MICROORGANISMS in large print and in small print, “for chemicals”. In this paper, you will have to be satisfied with the “for Chemicals” part of that title because we have not yet arrived at the genetic engineering phase. We soon hope to enter into genetic engineering especially with respect to the production of one of our halogenating enzymes and I will refer briefly to that at the end of this manuscript. I am sure you have seen the television commercials where Mother Nature, accompanied by flashes of lightning and loud noises says “Mother Nature likes this” or “Mother Nature doesn’t like that”. It was with that thought in mind that I arrived at a title for this paper. Most laymen and many scientists think only of industrial pollution and harmful chemicals when discussing roles for halogenated organic molecules in nature. However, I hope to persuade you that Mother Nature does like some halogenated compounds and indeed, has reserved halogenated compounds for some very special functions and purposes.
TL;DR: In this article, procedures for assessing the carcinogenic potential of agricultural and other chemicals are discussed, considering both the classical long-term in vivo carcinogen bioassay in rodent or other species and the more recently developed shortterm in vitro tests based on genetic alterations in bacterial and other test systems.
Abstract: Pesticides and other agricultural chemicals are now widely used throughout the world as a means of improving crop yields in order to meet the increasing demands being placed upon the global food supply. In Canada, the use of such chemicals is controlled through government regulations established jointly by the Department of Agriculture and the Department of National Health & Welfare. Such regulations require a detailed evaluation of the toxicological characteristics of the chemical prior to its being cleared for use. In this paper, procedures for assessing the carcinogenic potential of agricultural and other chemicals are discussed. Consideration is given to both the classical long-term in vivo carcinogen bioassay in rodent or other species and the more recently developed short-term in vitro tests based on genetic alterations in bacterial and other test systems.
TL;DR: The methodology for obtaining mutants and for determining the amino acid substitutions responsible for these changes is presented, and several new mutants have been shown to be due to the substitution of an arginine for a tryptophan at position 54.
Abstract: Selection of petite strains of yeast (that is, strains unable to respire aerobically) on media containing allyl alcohol will result in enrichment for mutants at the ADC1 locus. This locus codes for the constitutive alcohol dehydrogenase, ADH-I, which is primarily responsible for the production of ethanol in yeast. The mutant enzymes are functional, and confer resistance to allyl alcohol on the cell by shifting the NAD-NADH balance in the direction of NADH. These mutants exhibit altered Km’s for cofactor, substrate, or both, and often have altered Vmax’s. In this paper, the methodology for obtaining these mutants and for determining the amino acid substitutions responsible for these changes is presented. Several new mutants have been at least approximately localized, and one, DB-AA3-N15, has been shown to be due to the substitution of an arginine for a tryptophan at position 54. This substitution would be expected, by analogy with the known tertiary structure of the horse liver alcohol dehydrogenase, to decrease the hydrophobic environment of the active site pocket. The substitution has a pronounced effect on the Km for ethanol, but far less on that for acetaldehyde.
TL;DR: The characterization of mutant polymerases is extended to include the role of T4 DNA polymerase in frame fidelity, and this work concludes that T4 polymerase plays a central role in replication fidelity.
Abstract: Mutant T4 DNA polymerases which alter mutation rates in vivo have been used to approach questions of replication fidelity Most studies have characterized “mutator” or “antimutator” polymerases by their influence upon base-pair substitution mutation, particularly transitions We are extending the characterization of mutant polymerases to include the role of T4 DNA polymerase in frame fidelity
TL;DR: Haloaromatic pathways illustrate the same basic principles as those for non-halogenated substrates: the need to match throats and stomachs, and the importance of proper pathway regulation to ensure substrate catabolism.
Abstract: Hydrocarbons have many different structures, but these microbial substrates have two general characteristics which influence the nature of oxidation pathways. (1) Hydrocarbons are usually hydrophobic molecules, and oxidizing activities are consequently located in cellular membranes. (2) Hydrocarbons are composed of several basic structural units (such as aromatic rings and aliphatic chains). In order to break down these composite substrates, oxidation pathways are organized into segments that are conveniently labeled “throats” and “stomachs”. Throats contain the initial activities which convert a specific hydrocarbon substrate into one of a few common intermediates, such as fatty acids or catechols. Stomachs contain the subsequent activities which transform the intermediates into the organic acid substrates of central metabolic pathways. Typical stomachs include β-oxidation and catechol ring fission pathways. Detailed genetic analysis has been carried out for a few hydrocarbon oxidation pathways, including the plasmidborne alk and xyl systems for oxidation of n-alkanes and of toluene or xylenes. In both cases, there is duplication of plasmic-and chromosome-encoded activities at certain steps of the oxidation pathway. Induction specificities in both systems are often more important than enzyme specificities in determining growth phenotypes on different substrates. Oxidation of halogenated aromatic substrates, such as chlorobenzoic acid or the pesticide 24D, has been the subject of less detailed genetic study, but haloaromatic pathways illustrate the same basic principles as those for non-halogenated substrates: (i) the need to match throats and stomachs, and (ii) the importance of proper pathway regulation to ensure substrate catabolism. The use of broad host-range cloning vectors means that recombinant DNA methods are available for use with hydrocarbon-oxidizing gram-negative bacteria. However, detailed consideration of the possible rate-limiting steps in hydrocarbon oxidation indicates that cloning and overproduction of particular enzymes may often not be successful in strain improvement for specific biodegradations or bioconversions. A particular problem can be the toxicity of membrane hydrocarbon-oxidizing proteins when they are produced at very high levels.
TL;DR: Most spontaneous polar mutations in Escherichia coli appear to the insertions, which is the most common insertion; however, certain DNA rearrangements are exemplified by DNA fusion and DNA dissociation that occur when replication-transposition functions of Mu are induced.
Abstract: It can be argued that all organisms exhibit two levels of DNA rearrangements. At a low level they may occur sporadically in cells, perhaps largely because of spontaneous activity of transposable genetic elements. A high level may be induced in special circumstances if functions that cause rearrangements are hyperactive. As an example of low level genetic rearrangements, we have studied the occurrence of spontaneous polar mutations in the early regions of prophage Mu. We isolated 49 independent prophage mutants, which are defective in replication and expression of late genes; 44 were in the B region and 5 were in the A region. In the B region, 68% were IS1 insertions, 9% were IS5 insertions and 9% were IS2 insertions; 14% showed no insertion. In the A region, all 5 were IS5 insertions. Thus most spontaneous polar mutations in Escherichia coli appear to be insertions. IS1 is the most common insertion; however, certain DNA regions may show preference for a specific element. High level DNA rearrangements are exemplified by DNA fusion and DNA dissociation that occur when replication-transposition functions of Mu are induced.
TL;DR: This paper will deal with plasmids, a key molecular ingredient for the genetic manipulation of microorganisms using recombinant DNA techniques.
Abstract: The title of this conference in itself suggests the merging of several traditional disciplines of study (chemistry, biochemistry, microbiology, and genetics) with recent and dramatic developments in molecular genetics. The revolutionary breakthroughs of recombinant DNA research are providing unique opportunities for the genetic manipulation of microorganisms. One of the major objectives of this conference is to assemble scientists of diverse expertise to focus collectively on the problem of constructing microorganisms with new or greatly enhanced capacities to produce chemical substances of agricultural, industrial, and medical importance. For this purpose, it is necessary to consider the most effective means of merging our understanding of the physiological, biochemical, and ecological properties of microorganisms with the powerful technological developments that have occurred in molecular genetics. This paper will deal with plasmids, a key molecular ingredient for the genetic manipulation of microorganisms using recombinant DNA techniques.