Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.     

Analysis of fidelity mechanisms with eukaryotic DNA replication and repair proteins

We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (alpha, beta, delta, gamma), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells.     

Top-down and bottom-up regulation of herbivores: Spodoptera frugiperda turns tables on endophyte-mediated plant defence and virulence of an entomopathogenic nematode

Abstract.  1. The fungus Neotyphodium lolii forms a symbiotic relationship with its grass host Lolium perenne (perennial ryegrass). The fungus benefits from access to plant nutrients and photosynthate, whereas the plant benefits from acquired chemical defence against herbivory.
2. This study examined the potential for endophyte-mediated plant defences to influence interactions between fall armyworm Spodoptera frugiperda , and the entomopathogenic nematode Steinernema carpocapsae and clarified biological mechanisms underlying the observations made.
3. In laboratory and greenhouse experiments, S. frugiperda larvae were fed endophytic or non-endophytic L. perenne then exposed to S. carpocapsae or injected with the nematodes' symbiotic bacteria Xenorhabdus nematophila .
4. In all instances, S. frugiperda larvae fed endophyte-infected grass suffered significantly lower mortality than those fed non-endophytic plants. Although larvae fed endophyte-infected grass often had significantly lower biomass than those fed uninfected grass, these differences did not account for altered susceptibility to S. carpocapsae .
5. Endophyte-mediated reductions in herbivore susceptibility to the nematode pathogen represent a herbivore adaptation that effectively turns the tables on both plant and natural enemy by reducing the virulence of the nematodes' symbiotic bacteria while expanding the temporal window of herbivory.     

A small deletion in the Duchenne/Becker muscular dystrophy locus —a functionally important region?

Summary A DNA deletion in a patient with Becker muscular dystrophy (BMD) has been delineated by restriction endonuclease mapping. The deletion is unusually small, removing six kilobases (kb) of DNA distal to pERT 87-1 (DXS164). This region has previously been shown to contain an exon of a candidate gene which, when defective, causes Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy. Removal of this exon and surrounding DNA is apparently sufficient, in this case, to cause a BMD phenotype. The occurrence of this deletion in DXS164 would appear to confirm that this region is part of the BMD locus. Many DMD patients have deletions in and around this region, adding further evidence for the allelic nature of the two disorders. This fortuitous deletion may identify a functionally important domain of the protein product in terms of the severity of phenotype manifested.     

Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy

Summary Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.     

The beta form is the dominant interleukin 1 released by murine peritoneal macrophages

Using highly specific polyclonal antisera raised against recombinant murine IL-1 alpha and beta, we performed solid-phase immunoabsorption studies on supernates of resident and adjuvant-elicited CBA/J mouse peritoneal macrophages. Antibody specificity was established by reciprocal absorption studies and Western blot analysis. Supernates obtained from macrophages cultured for 18 hr in the presence of 1 microgram/ml lipopolysaccharide (LPS) were subjected to immunoabsorption. Approximately 78-90% of the released bioactive material was IL-1 and about 80% of this could be attributed to IL-1 beta. Analogous to that reported for human monocytes, these data suggest that IL-1 beta is the predominant released form of IL-1.     

Duchenne muscular dystrophy: deficiency of dystrophin at the muscle cell surface

Dystrophin is the altered gene product in Duchenne muscular dystrophy (DMD). We used polyclonal antibodies against dystrophin to immunohistochemically localize the protein in human muscle. In normal individuals and in patients with myopathies other than DMD, dystrophin was localized to the sarcolemma of the fibers. The protein was absent or markedly deficient in DMD. The sarcolemmal localization of dystrophin is consistent with other evidence that there are structural and functional abnormalities of muscle surface membranes in DMD.     

Arachidonic acid metabolites and their circadian rhythm in patients with allergic bronchial asthma

The aim of this study was to investigate circadian variation in concentrations of arachidonic acid (AA) metabolites in relation to the circadian pattern in bronchial patency. Blood samples were obtained at 4-hr intervals from 2000 of 1 day until 1400 of the next from 12 diurnally active asthmatic and six diurnally active non-asthmatic patients. Bloods were analyzed for the prostanoids thromboxane A2 (measured as stable metabolite 6-keto-PGF1a), PGE2 and PGF2a. Airways patency was assessed by self-measurement of peak expiratory flow (PEF). In asthmatics, circadian variation was detected in PEF as well as PGE2 and TXB2. The circadian trough of the PEF rhythm closely coincided with the circadian peak of the PGE2 and TXB2 rhythms. In the controls, the PEF was not circadian rhythmic. Of the AA metabolites only 6-keto-PGF1a exhibited 24-hr bioperiodicity in the controls. The controls exhibited a significantly higher circadian mean of PEF (P less than 0.001), while the asthmatics had a lower 24-hr average PGE2 but greater mean TXB2/PGE2 ratio. The obstructive effect caused by the overall 24-hr deficiency of PGE2 in asthmatics is possibly amplified by the increased of TXB2 during the early morning hours. This dissociation of the temporal patterns in TXB2 and PGE2 levels over the 24 hr is discussed as a characteristic finding for asthmatics.