RESUMO
The Red recombination pathway of phage lambda is shown to target recombination to double-chain ends of DNA. A double-chain cut, delivered in vivo to only one of two parents participating in a lambda lytic cross by a type II restriction endonuclease, increases the proportion of crossing over in the interval containing the cut compared with other intervals. The stimulating effect of a cut is evident whether replication is inhibited or permitted. Cut stimulation can move away from the initial cut-site, presumably by double-chain degradation. Movement of the stimulating effect of a cut is dependent on the Escherichia coli gene recA when the cross is carried out under conditions that inhibit phage replication. When replication is permitted, all aspects of cut-stimulated recombination are independent of recA. Evidence is presented to show that the reaction that is stimulated by cutting is often non-reciprocal at the molecular level.
Assuntos
Bacteriófago lambda/genética , Recombinação Genética , Troca Genética , Replicação do DNA , DNA Viral , Modelos Genéticos , PlasmídeosRESUMO
Biopanning has been used extensively in conjunction with purified components, but there are also examples in which mixtures of targets have been investigated. This study introduces a methodological innovation, termed iterative panning and blocking (IPAB), to extend the range of specific interactions that can be probed in mixtures. Here this procedure is used to probe a mixture of high molecular mass components of human cord blood with phage-peptide display libraries. The initial panning recovered phage that bore the consensus motif Gly-Pro-Arg-Pro, a known fibrinogen-binding motif. These phage bound specifically to purified fibrinogen. A series of peptides containing the Gly-Pro-Arg-Pro motif efficiently blocked the binding of phage having the same motif, presumably by binding to their common target. A second round of panning was performed against the same target mixture in the presence of this blocking peptide. Phage recovered from this second panning exhibited a motif (Ser-His-Tyr) that was subsequently shown to bind specifically to complement component C1q. A second peptide containing this motif specifically blocked the interaction of the phage with C1q. A third round of panning performed in the presence of both the fibrinogen- and the C1q- blocking peptides yielded phage with a new peptide motif (Asn-Pro-Phe) that also bound specifically to C1q, apparently at a new site. The three motifs isolated through this iterative process were distinct in that each was blocked only by its corresponding peptide. This IPAB strategy can be applied to many high diversity selection procedures that target complex mixtures.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Biblioteca de Peptídeos , Peptídeos/antagonistas & inibidores , Peptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Ligação Competitiva , Clonagem Molecular , Complemento C1q/antagonistas & inibidores , Complemento C1q/metabolismo , Sequência Consenso/genética , Sangue Fetal/química , Fibrinogênio/antagonistas & inibidores , Fibrinogênio/metabolismo , Humanos , Ligantes , Peso Molecular , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Especificidade por SubstratoRESUMO
There is an apparent paradox between the reported requirements for lambda DNA packaging in vivo and in vitro. In vivo, DNA concatemers are required for packaging. On the other hand, in vitro, packaging extracts can encapsidate either linear or circular monomeric lambda DNA. Perhaps cellular nucleases restrict the in vivo ability of monomers to package by degrading a free double chain end present as an intermediate in the packaging reaction. Consistent with this hypothesis, enhanced packaging of monomers was found in an ExoV- host. No additional enhancement was noted in a host also mutant for sbcB and sbcC. We isolated a mutant phage for which in vivo packaging of monomeric lambda chromosomes is increased about 10(3)-fold. The responsible mutation (plm1 for packages lambda monomers) was mapped to cro, sequenced, and found to cause a change from Ala29 to Ser in the alpha3 helix of Cro's DNA binding domain. Density transfer experiments showed that packaging of both plm1 and wild-type lambda was aided by allowing some DNA synthesis. However, the packaged chromosomes had not themselves undergone a full round of replication and therefore were not part of a canonical concatemer made by replication. Other tests showed that packaged phage had not been part of concatemers made by recombination or by annealing at cos. Our results with wild-type lambda also favor models in which two cos sites are needed for packaging, but these sites need not be in cis. In lambda plm1, replication intermediates may serve as substrates for encapsidation.
Assuntos
Bacteriófago lambda/fisiologia , DNA Viral/genética , Proteínas de Ligação a DNA , Montagem de Vírus , Cosmídeos , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Mutação , Recombinases Rec A/genética , Proteínas Repressoras/metabolismo , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
Chimeras of RNA and DNA have distinctive physical and biological properties. Chimeric oligonucleotides that contained one, two or three ribonucleotides whose phosphodiester backbone was covalently continuous with DNA were synthesized. Site-directed mutagenesis was used to assess genetic information transfer from the ribonucleotide positions. Transfer was scored by the formation or reversion of an ochre site that also corresponded to a restriction cleavage site. This allowed physical as well as genetic assay of mutational events. Bases attached to the ribonucleotides were able to accurately direct the synthesis of progeny DNA. The results suggest that in vivo DNA polymerases utilize a "running start" on a DNA backbone to continue across a covalent backbone junction into a region of ribonucleotides and then back again onto a normal DNA backbone. The phenomenon is designated short-patch reverse transcription (SPRT) by analogy to short-patch mismatch correction and reverse transcription as the term is generally used. The possibility is considered that SPRT contributes to an unrecognized pathway of mutagenesis.
Assuntos
DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , Transcrição Gênica , Sequência de Bases , Quimera , Primers do DNA , DNA Bacteriano/genética , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , Mapeamento por RestriçãoRESUMO
We examined linkage relationships for RecBC-mediated recombination in lytic cycle crosses of lambda phages bearing two cohesive end sites (cos) oriented in the same direction. The relationships obtained imply that a given recombinant tends to be packaged from the cos site that is the nearer one to the right of the exchange. In view of the previously established coupling of entry of a recombinase at a cos cut and initiation of DNA packaging by that cos cut, these results imply that the recombinase (presumably the RecBC gene product) enters lambda's chromosome at the right end.
Assuntos
Bacteriófago lambda/genética , DNA Viral/metabolismo , Proteínas de Escherichia coli , Exodesoxirribonucleases/metabolismo , Bacteriófago lambda/efeitos dos fármacos , Enzimas de Restrição do DNA , Escherichia coli/genética , Exodesoxirribonuclease V , Fenótipo , Especificidade da Espécie , Transdução Genética , Trimetoprima/farmacologiaRESUMO
The double-strand-break repair (DSBR) model was formulated to account for various aspects of yeast mitotic and meiotic recombination. In this study three features of the DSBR model are tested for Red-mediated recombination between phage lambda and lambda dv, a plasmid that is perfectly homologous to about 10% of lambda. The results support the applicability of the DSBR model to lambda's Red system: (1) Creating a double-strand-break (DSB) within the region of homology shared by phage and plasmid increases their genetic interaction by about 20-fold. A DSB outside the region of shared homology has no such effect. (2) Both patches, i.e., simple marker rescue, and splices, i.e., co-integration of the phage and plasmid, are stimulated by a DSB in the region of shared homology. (3) Co-integrants harbor a duplication of the region of shared homology. Among co-integrants that were formed by the creation of a DSB, there is a preferential loss of whichever allele was in cis to a utilized cut site. The DSBR model as originally formulated involves the isomerization and cleavage of Holliday junctions to resolve the canonical intermediate. We propose as an alternative mechanism that a topoisomerase can resolve the canonical DSBR intermediate.
Assuntos
Bacteriófago lambda/genética , Reparo do DNA , Escherichia coli/genética , Plasmídeos , Recombinação Genética , Modelos Genéticos , Homologia de Sequência do Ácido NucleicoRESUMO
The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric RNA fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal RNA fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.
Assuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 8 , Leucemia Mieloide/genética , Translocação Genética , Acetiltransferases/genética , Doença Aguda , Adolescente , Adulto , Idoso , Proteína de Ligação a CREB , Diferenciação Celular , Quimera , Feminino , Histona Acetiltransferases , Humanos , Hibridização in Situ Fluorescente , Íntrons , Leucemia Mieloide/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Neoplasia Residual/genética , Neoplasia Residual/patologia , Proteínas Nucleares/genética , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genéticaRESUMO
Two peptide motifs that bind to C1q have been identified from phage displayed libraries. A first panning cycle recovered phage that displayed a [N/S]PFxL motif. A synthetic peptide with that motif blocked those phage from binding to C1q. A second panning cycle was conducted with the [N/S]PFxL motif peptide present, leading to recovery of phage displaying a different motif, SHY. The two motifs are specific for C1q and are competed by DNA and the cognate synthetic peptide but not by immunoglobulins. Phage displayed peptide sequences containing the [N/S]PFxL have significant sequence similarity to a region of complement component C4, suggesting a possible site of interaction between C4, or one of its processed forms, and C1q. The SHY motif peptide induces C4 consumption in a hemolytic assay, suggesting that it activates C1 independent of immune complexes. This peptide may activate C1 by a mechanism similar to the beta-amyloid peptides found in Alzheimer's disease.
Assuntos
Proteínas de Transporte , Ativação do Complemento/imunologia , Complemento C1q/metabolismo , Complemento C4/química , Peptídeos/química , Proteínas/química , Animais , Bacteriófagos , Sítios de Ligação , Ligação Competitiva , Bases de Dados Factuais , Cobaias , Humanos , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Proteínas/genética , Proteínas/imunologia , Proteínas/metabolismo , Análise de Sequência de Proteína/métodos , Homologia de Sequência de AminoácidosRESUMO
Screening phage-displayed peptide libraries (biopanning) is an important technique for acquiring peptide ligands and for mapping peptide epitopes recognized by antibodies (Ab). In biological samples, other materials, not only contaminants but also natural constituents, often interfere with biopanning. Capture methods use anchoring Abs that reduce the need for purification of the intended panning target from a crude sample. This capture method is analogous to sandwich ELISA. However, when the target molecule concentration is low in the initial mixture, the panning of a captured target often yields epitopes that bind to the capture antibody rather than the target of interest. We have developed a methodology that utilizes specific blocking reagents of the capture Ab to extend the sensitivity and applicability of the capture approach to phage panning. A flowchart is presented to enable the worker to begin panning with the simplest approach and then to employ sandwich capture and specific blocking reagents as necessary.
Assuntos
Proteínas Sanguíneas/análise , Ensaio de Imunoadsorção Enzimática/métodos , Hibridomas/química , Biblioteca de Peptídeos , Animais , Especificidade de Anticorpos , Proteínas Sanguíneas/imunologia , Epitopos/análise , Epitopos/imunologia , Hibridomas/imunologia , Imunoglobulina M , Sensibilidade e EspecificidadeRESUMO
Handwerger and colleagues demonstrated that a particular clinical isolate of Enterococcus faecium, designated GUC, and here redesignated as GUCR, can conjugatively transfer vancomycin resistance. The vancomycin resistance is encoded by a chromosomally born linked set of genes in the donor, designated the vanA cluster, to the chromosome of an E. faecalis recipient, JH2-2. Here it is reported that an earlier isolate of E. faecium from the same patient who later harbored the vancomycin-resistant E. faecium GUCR lacks the vanA gene cluster but is otherwise similar (by SmaI chromosomal fingerprint and metabolic fingerprinting) to the vancomycin-resistant GUCR. Therefore, "GUCS" is a strong suspect as the base strain for the clinical acquisition of the vanA cluster present in GUCR. Thirteen laboratory-generated vanA transconjugants derived from conjugation between GUCR and JH2-2 were subjected to further analysis, allowing a comparison between transfer in the laboratory and transfer that occurred in the clinical setting. Surprisingly, each JH2-2 transconjugant had a unique constellation of abilities to oxidize various members of a panel of potential carbon sources. This pattern was stable for each transconjugant, and it was not changed by growing the strains with or without vancomycin. Transconjugants had pulsed-field gel electrophoretic (PFGE) patterns largely consistent with that of JH2-2, the recipient in conjugation experiments. However, PFGE analysis showed that a large but variable amount of DNA, between 145 kb and 277 kb, was transferred into different transconjugants. The mechanism appears to be conjugative transposition in which new DNA is added to the pre-existing genome rather than substituting for a segment in the recipient. Mapping and hybridization studies of several transconjugants showed that each received similar, but not exactly the same, DNA fragment of at least 30 kb from the donor. Sequencing of 16S ribosomal genes was used to confirm that the recipient and donor strains used in transconjugation experiments were different species. Sequence analysis was also used to consider the possibility that rRNA operons might be mobilized in conjugation, but no evidence for the transfer of rDNA operons was found. An apparent insertion sequence in E. faecium almost identical to IS 1485 and 57% sequence identity to IS 199 of Streptococcus mutans was found in the region of DNA transferred. The results imply new consequences of conjugative transfer and interspecies recombination.
Assuntos
Antibacterianos/farmacologia , Enterococcus faecium/genética , Recombinação Genética , Vancomicina/farmacologia , Sequência de Bases , Conjugação Genética , DNA Bacteriano , DNA Ribossômico , Resistência Microbiana a Medicamentos/genética , Eletroforese em Gel de Campo Pulsado , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fenótipo , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The activity of neurones was recorded from the supplementary motor area (SMA) of monkeys while they were performing a discrete, arbitrary wrist movement. The cells responded similarly whether there was a triggering stimulus at the time of the movement or not. This experiment indicates that SMA neurones are active both in relation to externally triggered and internally initiated (voluntary) actions.
Assuntos
Atividade Motora/fisiologia , Córtex Motor/fisiologia , Neurônios/fisiologia , Punho/fisiologia , Animais , Eletromiografia , Macaca mulatta , Córtex Motor/anatomia & histologiaRESUMO
Sex and recombination generate variation via processes that depend on an underlying complementarity between participants. Sex between DNA segments depends on their sequences having enough in common. Viewed in this way, sex does not depend on genes that originate in separate cells. Sex in the single genome uses many of the same mechanisms as intergenomic sex but has not been properly appreciated as a source of variation or as a selectable process. Mutation is the generation of new sequences rather than the novel grouping of pre-existing alleles. Mechanisms of mutation that depend on pre-existing sequence similarities in the haploid genome are a source of variation with significant and special characteristics.
RESUMO
Evolution and development are both lineage processes but are often conceptualized as occurring by different and mutually exclusive mechanisms. It is conventionally asserted that evolution occurs via the random generation of diversity and the subsequent survival of those that pass selection. On the other hand, development is too often presented as proceeding via the unfolding of a deterministic program encoded in the DNA sequence. In biology, universal generalizations are rare and dogmas are often wrong for particular cases. Deterministic mechanisms contribute some of the new DNA sequences that subsequently become substrates for natural selection. Conversely, stochastic and selective mechanisms are intrinsic to development, and also to maintenance of the immune, and possibly, nervous systems. Cancer appears to be another process that straddles distinctions between evolutionary and developmental modes of hereditary change and stabilization. DNA sequence changes are an essential feature of many cancers, but there are also aspects of the disease similar to developmental lineage gone awry. The literature suggests that the cellular changes that give rise to cancer occur by mechanisms commonly associated with both evolutionary and developmental lineage pathways.
Assuntos
Evolução Biológica , Biologia do Desenvolvimento , Animais , Transformação Celular Neoplásica , Sistema Imunitário/fisiologiaRESUMO
Homologous recombination can provide a basis for the construction of an ordered set of overlapping clones. The principle is to make two libraries, each in a vector that has a different selectable marker flanking the insert site. Recombination between the flanking markers, leading to a selectable phenotype, can only occur as the consequence of crossing over between inserts. The two libraries are crossed in a matrix, allowing the construction of an ordered set. The logic, akin to S. Benzer's (1961, Genetics 47:403-415) for the arrangement of deletion and point mutations, has a graph theoretic formulation, which helps to cope with the complex and noisy data inherent in the physical mapping of genomes rich in repeated sequences. The minimum length of identity required for homologous recombination is called the MEPS (minimum efficient processing segment) and is a property of each recombination pathway. The amount and the type of sequence similarity required for two sequences to recombine is different from that implied by either the conservation of restriction sites or by most procedures of hybridization.
Assuntos
Recombinação Genética , Deleção Cromossômica , Cromossomos Fúngicos , Biblioteca Gênica , Genoma Humano , Humanos , Mutação , FenótipoRESUMO
Wild-type Escherichia coli are resistant to genetic transformation by purified linear DNA, probably in part because of exonuclease activity. We demonstrate that E. coli containing a recD mutation could be easily transformed by linearized plasmids containing a selectable marker. The marker was transferred to the chromosome by homologous recombination, whereas plasmid markers not in the region of homology were lost.