[Problems in the clinical application of human-recombinant cytokines. Production of human recombinant cytokines and application to genetic recombination].
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Animals↗
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Results for “Recombination, Genetic”
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Genetic recombination of human immunodeficiency virus.
We investigated genetic recombination of the human immunodeficiency virus (HIV) in a tissue culture system. A clonal cell line expressing a single integrated HIV provirus with a termination codon affecting pol gene expression was transfected with different defective mutants derived from an infectious molecular clone of HIV. Replication-competent viral particles were recovered, passaged, and plaque purified. Restriction analyses of the proviral DNA corresponding to several of these viruses indicated that their emergence was the result of genetic recombination.
Cells, Cultured↗
Further details of a hypothesis for the initiation of genetic recombination from recognition sites.
Consideration of the initiation of genetic recombination from fixed sites recognised by an initiation complex, has provided more details of the envisaged mechanism and implications of a recent hypothesis. It has been shown that the hypothesis allows for more than one recombinogenic event to result from a single binding of the recombination initiation complex to a recognition site in a DNA duplex. This capacity can explain data from fungal systems which are apparently inconsistent with the Meselson-Radding model of genetic recombination with respect to the positional relationship between tracts of hybrid DNA and sites of crossing-over. A mechanism for conversion, involving hybrid DNA formation, but without mismatch correction has also been proposed on the basis of this capacity. It is suggested that the hypothesis may apply generally to genetic recombination, in prokaryotes as well as eukaryotes.
Crossing Over, Genetic↗
Genetic recombination of bacterial plasmid DNA. Physical and genetic analysis of the products of plasmid recombination in Escherichia coli.
Derivatives of plasmid pBR322 DNA containing tet mutations were constructed by inserting XhoI linkers at various sites in the tetracycline resistance gene. Monomer plasmids containing either the tet-10 allele located at nucleotide position 23 or the tet-14 allele located at nucleotide position 1267 were used to construct a circular dimer containing one copy of each allele and a circular trimer containing one copy of the tet-10 allele and two copies of the tet-14 allele. Genetic recombination of these plasmid DNAs to produce a functional tetracycline resistance gene could be detected as the production of tetracycline-resistant progeny during the growth of transformants or using a restriction mapping assay which detected the rearrangement of the mutant alleles. The structure of individual tetracycline-resistant recombination products was determined by restriction mapping. This analysis suggested that as many as 70% of the plasmid recombination events in Escherichia coli AB1157 could have involved gene conversion events. The formation of these recombination products was most easily predicted by a model involving figure 8 recombination intermediates and the formation of symmetric regions of heteroduplex. Recombination in JC10287 delta(srlR-recA)304 occurred at 5% of the wild-type frequency and appeared to occur by a similar mechanism. Recombination in JC9604 recA56 recB21 recC22 sbcA23 occurred at 20 times the wild-type frequency and appeared to involve multiple independent recombination events.
DNA Restriction Enzymes↗
Genetic recombination in the dinoflagellate Crypthecodinium cohnii.
Genetic recombination in dinoflagellates has been detected with the use of chemically induced carotene-deficient mutants of Crypthecodinium cohnii.
Animals↗
Genetic recombination in bacteriophage phi chi 174.
Genetic recombination in bacteriophage phiX174 usually takes place early in the infection process and involves two parental replicative form (double-stranded) DNA molecules. The host recA protein is required; none of the nine known phiX174 cistron products is essential. The products of a single recombination event are nonreciprocal and asymmetric. Typically, only one of the parental genotypes and one recombinant genotype are recovered from a single cell. An alternative, less efficient recombination mechanism which requires an active phiX174 cistron A protein is observed in the absence of the host recA gene product.
Alleles↗
[Gene rIIB from bacteriophage T4 in genetic recombination].
The effect of the rIIB gene on genetic recombination in bacteriophage T4 was studied. Relationships between recombination frequency and the physical distance were determined in three series of isomarker two-factor crosses between rII mutants. In the first series of intergenic crosses (rIIa x rIIb), the rII gene function was restored owing to complementation. In the second series of crosses, identical to the first one, the rIIB gene function was suppressed, because the rIIa parent carried an additional amberlike mutation in the rIIB gene. The recombinants were scored by plating lysates on the amber-suppressor Escherichia coli strain, on which an amberlike mutation was not expressed phenotypically. In the third series, all crosses were intragenic (rIIb x rIIb). In two series of crosses in the absence of the rIIB function, the relationships between recombination frequency and the physical distance were identical, whereas enhanced recombination frequencies were observed in the rIIB+ background. The magnitude of the rIIB-related effect depended on distance, reaching the maximum in the region located 100 to 200 bp from the beginning of the rIIB gene. The possible role of the rIIB protein in genetic recombination is discussed.
Bacteriophage T4↗
A general model for genetic recombination.
A general model is proposed for genetic recombination. Its essential new feature is the hypothesis that recombination is initiated by a single-strand (or asymmetric) transfer, which may, after isomerization, become a two-strand (or symmetric) exchange. The likelihood of this transition from asymmetric to symmetric strand exchange determines certain characteristic features of recombination in any particular organism.
Chromatids↗
Genetic recombination of poliovirus in a cell-free system.
Genetic recombination of plus-strand RNA viruses is an important process for promoting genetic variation. By using genetically marked poliovirus RNAs, we have demonstrated that genetic recombination can occur in a cell-free system that generates infective virus from added poliovirus RNA. Recombinant polioviruses were isolated, and the region of crossing over was roughly mapped. Recombinants could be isolated even under conditions where the yield of viruses from one of the parental RNAs was depressed to levels comparable to or less than the yield of recombinant viruses, an observation suggesting that only one of the recombining RNAs needs to be replication-competent. The generation of poliovirus recombinants in a cell-free system offers new possibilities for studying recombination and evolution of RNA viruses.
Biological Evolution↗
Conversion of US3-encoded protein kinase gene from pseudorabies virus in a diploid gene located within inverted repeats by genetic recombination between the viral genome isomers.
The pseudorabies virus (PRV) genome consists of two components, long (U(L)) and short (U(S)) regions. The U(S) region is the only one capable of inverting itself relative to the U(L) region during productive infection, generating two equimolecular isomeric forms of viral DNA. Here we describe a recombinant virus (gIp2) generated by genetic recombination between pseudorabies viral isomers. This recombination event was observed in the parental virus gIS8, which was obtained by insertion of the alpha4-TK herpes simplex virus type 1 (HSV1) gene. The growth of gIS8 virus in the presence of 5-bromodeoxyuridine (BrdU) yielded gIp2. This was generated by nonhomologous recombination either between the two viral genomic isomers of gIS8, P and I(U/S), or between the same P isomer using nonhomologous and homologous recombination, with loss of the HSV1 sequences and duplication of the PRV US3-encoded protein kinase gene. Virus gIp2 is negative for TK, gI, gE, 11K and 28K and shows an in vitro replication capacity in neuronal cells approximately 22 times lower than that of parental virus gIS8, and similar to that of the Bartha vaccine virus strain in monkey kidney and human neuronal cells.
Animals↗
Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination.
Superhelical [3-H]DNA (replicative form I, RFI) of bacteriophage phiX174 slowly but spontaneously took up 32-P-labeled homologous single-stranded fragments at 4 degrees. Uptake was accelerated by heating to 75 degrees. RFI did not take up single-stranded fragments derived from DNA of Escherichia coli or from separated strands of phage lambda. Uptake was inhibited by low concentrations of ethidium bromide. Relaxed circular phiX174 DNA did not take up homologous fragments. Per molecule of RFI, the complexes contained as much as 90 nucleotide residues of homologous fragment. The 32-P-lebeled fragments were largely resistant to digestion by exonuclease I, and were not displaced by heating complexes at 60 degrees for 1 min in 16 mM or 100 mM NaCl. Under comparable conditions of temperature and salt all of the fragments were displaced from complexes in which at least one phosphodiester bond was cleaved by pancreatic DNase, but a significant fraction of the fragments was retained in complexes that were relaxed by digestion with S1 nuclease. These observations are interpreted to mean that S1 nuclease digested the plus (viral) strand of the recipient RF at the site of uptake in some instances. Transfection of E. coli by heterozygous complexes produced recombinant progeny, thereby showing that genetic information can be transferred from the fragment of plus strand to progeny plus strands. We propose that both uptake of a third strand by superhelical DNA and the action of nucleases on the resulting complex may simulate early steps in genetic recombination.
Centrifugation, Density Gradient↗
Genetic recombination among temperature-sensitive mutnats of Rous sarcoma virus.
Genetic recombination of RSV has been studied, using ts mutations in both initiation and maintenance of transformation as markers. The progeny of a single cycle of mixed infection appears to contain no recombinants, but yields heterozygous particles or viral clumps. On subsequent cycles of infection some of these heterozygotes/clumps persist, but they also segregate recombinant viruses. Some of the markers in these recombinants show evidence of linkage and thus probably recombine by intramolecular exchanges. Studies of td ts mutants (the class most frequently isolated from stock virus) show that recombination between them occurs at a level sufficient to explain cooperative transformation by these viruses. Furthermors, this genetic recombination is probably a necessary prerequisite for cooperative transformation since complementation between the mutants is absent or inefficient. The simplest explanation for this apparent lack of complementation is that the td ts mutants are all derived by lesions in the same cistron.
Avian Sarcoma Viruses↗
Biochemical analysis of genetic recombination in eukaryotes.
Recent studies concerning molecular mechanisms of genetic recombination in eukaryotes are reviewed. Since many of these studies have focused on the testable predictions arising from the hybrid DNA theory of genetic recombination, this theory is summarised. Experiments to determine the time of meiotic crossing-over and the structure of the synaptonemal complex which facilitates meiotic crossing-over are described. Investigations of DNA nicking and repair events implicated in recombination are discussed. Properties of proteins which may facilitate hybrid DNA formation, and biochemical evidence for hybrid DNA formation are presented. Finally, a nuclease which has been implicated in gene conversion is described.
Animals↗
Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage Lambda.
In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage mu Red recombination systems on the rescue of the O plus gene from the prophage by a superinfecting O minus phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants than does the Rec system, and its action requires DNA replication. The presence of UV lesions in the mu DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.
Coliphages↗
Modeling interference in genetic recombination.
In analyzing genetic linkage data it is common to assume that the locations of crossovers along a chromosome follow a Poisson process, whereas it has long been known that this assumption does not fit the data. In many organisms it appears that the presence of a crossover inhibits the formation of another nearby, a phenomenon known as "interference." We discuss several point process models for recombination that incorporate position interference but assume no chromatid interference. Using stochastic simulation, we are able to fit the models to a multilocus Drosophila dataset by the method of maximum likelihood. We find that some biologically inspired point process models incorporating one or two additional parameters provide a dramatically better fit to the data than the usual "no-interference" Poisson model.
Animals↗
A primary linkage map of the porcine genome reveals a low rate of genetic recombination.
A comprehensive genetic linkage map of the porcine genome has been developed by typing 128 genetic markers in a cross between the European Wild Boar and a domestic breed (Large White). The marker set includes 68 polymerase chain reaction-formatted microsatellites, 60 anchored reference markers informative for comparative mapping and 47 markers which have been physically assigned by in situ hybridization. Novel multipoint assignments are provided for 54 of the markers. The map covers about 1800 cM, and the average spacing between markers is 11 cM. We used the map data to estimate the genome size in pigs, thereby addressing the total recombination distance in a third mammalian species. A sex-average genome length of 1873 +/- 139 cM was obtained by comparing the recombinational and physical distances in defined regions of the genome. This is strikingly different from the length of the human genome (3800-4000 cM) and is more similar to the mouse estimate (1600 cM). The recombination rate in females was significantly higher than in males.
Animals↗
Topography and kinetics of genetic recombination in Escherichia coli treated with psoralen and light.
Genetic exchanges appear to be involved in repair of cross-linked DNA. Kinetics for completion of repair and strand rejoining controlled by the recA(+) gene were examined in Escherichia coli treated with psoralen and light. The results suggest the following model for genetic recombination. After cross-linking treatment, cells in a population initiate repair in near synchrony. Removal of DNA cross-links, preparation of substrate for recombination, and initiation of the first recA-dependent event are completed in less than 1 min. Recombination events occur singly in each cell or chromosome, and require 2.3 +/- 0.4 min at 32 degrees for the recA(+)-dependent step. After completion of the first event, subsequent recombination events occur in a sequential or progressive fashion around the chromosome or in clusters which may consist of one or more domains of the folded chromosome. The time required to proceed to successive sites is either a constant, independent of the distance on the chromosome, or is quite small compared to 2.3 min. DNA substrate for recombination decays with approximate first-order kinetics and the rate is dependent on the number of unrepaired sites. Cell survival can be expressed as a competition between completion of all repair events and the simultaneous decay of chromosomes to forms not reparable by recombination.Equations relating kinetics for completion of repair, the size distribution of DNA molecules, and cell survival are derived for the above model, using as parameters only rate constants for recombination and decay of substrate, and number of events per chromosome. An excellent correlation is found between experimentally determined and theoretical values.
DNA Repair↗
Genetic recombination in Streptomyces griseus.
Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described.
Cephamycins↗