Evidence for plasmid like DNA in a filamentous fungus, the ascomycete Podospora anserina

Summary The existece of plasmid like DNA was demonstrated in senescent mycelia of Podospora anserina (strain s) by biophysical and electronmicroscopic methods. According to their contour length of about 1.4 and 2.7 m respectively the molecular weight for the monomer is in the range of 3·10⁶.This work was supported by the Deutsche Forschungsgemeinschaft. The sojourn of P.A.L. for six months in Bochum was made possible by a Senior U.S. Scientist Award of the Alexander von Humboldt Stiftung (Bonn).     

Multiphasic activation of smooth muscle adenylate cyclase by pretreatment with guanyl-5′-yl imidodiphosphate (Gpp(NH)p) suggests multiple enzyme populations

The activation of uterine smooth muscle adenylate cyclase was studied by pretreating the particulate form of the enzyme with the GTP analog guanyl-5′-yl imidodiphosphate (Gpp(NH)p). Pretreatment with Gpp(NH)p left the enzyme in an irreversibly activated state which survived subsequent washing in guanyl nucleotide-free buffer. Activation under these conditions was multiphasic with rapid and slow components. At 23 °C slow activation proceeded at about $\text{1}\text{10}\text{th}$ the rate of rapid activation. The onset of the slow phase took longer at lower temperatures. Routine adenylate cyclase assay conditions (conversion of [³²P]ATP to cyclic [³²P]AMP) carried out without pretreatment probably characterized the rapidly activated component. The simplest kinetic model suggests not only the generally accepted two-step association reaction, but also implies the existence of more than one enzyme form, each of which is characterized by a separate activation rate. The complex kinetics of activation might be explained by a heterogeneous mixture of unassociated and preassociated nucleotide binding and catalytic subunits.     

Comparison of yeast mitochondrial Phe-tRNA synthetase subunits to their cytoplasmic counterparts: Isolation and determination of amino acid compositions

The α and β subunits of yeast mitochondrial Phe-tRNA synthetase are separated and isolated by means of chromatography on DEAE-cellulose, after enzyme alkylation with iodoacetate. The comparison of amino acid compositions of yeast mitochondrial and cytoplasmic native Phe-tRNA synthetases and their components shows significant differences. Results indicate that the two enzymes are coded for by different nuclear genes.     

Fusion of newly formed phagosomes with endosomes in intact cells and in a cell-free system

Phagosomes are membrane-bound vesicles, formed by the receptor-mediated internalization of particulate ligands, which exchange soluble and membrane proteins with other endocytic compartments as a part of their maturation process. This exchange of material is undoubtedly mediated by fusion of phagosomes with other membrane-bound compartments of the endocytic pathway. By using a particulate probe (fixed Staphylococcus aureus coated with mouse anti-dinitrophenol monoclonal antibody) localized in phagosomes and a soluble probe (dinitrophenol-derivitized beta-glucuronidase) internalized by receptor-mediated endocytosis, we have studied phagosome-endosome and phagosome-lysosome fusion in intact cells and in a cell-free system. Vesicle fusion was assessed by measuring beta-glucuronidase activity associated with S. aureus particles after lysis of the membranes. In intact macrophages, newly formed phagosomes fused with early endosomes and with lysosomes. Fusion with lysosomes was observed to commence after a short lag period of about 5 min. In broken-cell preparations, phagosomes were able to fuse with early endosomes. It was not possible to reconstitute phagosome-lysosome fusion in vitro. In vitro phagosome-endosome fusion required energy and cytosolic- and membrane-associated proteins. A nonhydrolyzable analog of GTP stimulated fusion at low cytosol concentrations and inhibited fusion at high cytosol concentrations. These observations indicate that the mechanisms mediating phagosome-endosome fusion are similar to those described for endosome-endosome fusion. Our results suggest that exchange of material with endosomes is an important step in the process of phagosome maturation.     

Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum.

An in vitro endosome fusion assay using Dictyostelium discoideum is described. The method requires endocytosis of anti-dinitrophenol (DNP) IgG or DNP-derivitized beta-glucuronidase into two sets of cells. After homogenizing the cells, the vesicles were mixed, and fusion was measured by quantitating immune complex formation between DNP-beta-glucuronidase and anti-DNP IgG. Fusion was dependent upon ATP, temperature, pH, ionic strength, and cytosol and sensitive to detergent, dilution, trypsin, N-ethylmaleimide, and guanosine 5'-3-O-(thio)triphosphate. Although weak bases, ionophores, hadacidin, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and caffeine inhibit endocytosis in vivo, these reagents had no affect on in vitro endosome fusion. Comparison of Dictyostelium with mammalian cells showed differences in the temperature, pH, and salt requirements for fusion, possibly reflecting differences in the life-styles of various cell types. Like mammalian cells, Dictyostelium required GTP-binding protein(s) and an N-ethylmaleimide-sensitive factor for endosome fusion. Thus, the mechanism driving endosome fusion may have been conserved throughout evolution. Electron microscopic studies confirmed in vitro endosome fusion and revealed endosomes were being engulfed by other endosomes, resulting in formation of multivesicular elements (i.e. autophagic vesicles). This system may be useful for characterizing mutations, evolution, and developmental regulation along the endocytic pathway.     

Break-Join Recombination in Phage λ

In phage lambda, when DNA replication is blocked, recombination mediated by the Red pathway occurs only near the double-chain break site, cos, that defines the termini of the virion chromosome. The recombinants initiated by cos contain newly synthesized DNA near cos, in amount corresponding to a few percent of the length of lambda. A restriction enzyme cut delivered to one parent far from cos results in elevated recombination near the restriction site. Recombinants induced by this cut have a similarly small amount of DNA synthesis in these replication-blocked crosses. When restriction cuts are introduced in the presence of normal amounts of all of the DNA replication enzymes, many of the resulting recombinants still enjoy, at most, a small amount of DNA synthesis associated with the exchange event. Thus, these experiments fail to support the previously considered possibility that Red-mediated recombination in lambda proceeds largely through a break-copy pathway.     

Further Tests of a Recombination Model in Which χ Removes the Recd Subunit from the Recbcd Enzyme of Escherichia Coli

When one of two infecting lambda phage types in a replication-blocked cross is chi + and DNA packaging is divorced from the RecBCD-chi interaction, complementary chi-stimulated recombinants are recovered equally in mass lysates only if the chi + parent is in excess in the infecting parental mixture. Otherwise, the chi 0 recombinant is recovered in excess. This observation implies that, along with the chi 0 chromosome, two chi + parent chromosomes are involved in the formation of each chi + recombinant. The trimolecular nature of chi +-stimulated recombination is manifest in recombination between lambda and a plasmid. When lambda recombines with a plasmid via the RecBCD pathway, the resulting chromosome has an enhanced probability of undergoing lambda x lambda recombination in the interval into which the plasmid was incorporated. These two observations support a model in which DNA is degraded by Exo V from cos, the sequence that determines the end of packaged lambda DNA and acts as point of entry for RecBCD enzyme, to chi, the DNA sequence that stimulates the RecBCD enzyme to effect recombination. The model supposes that chi acts by ejecting the RecD subunit from the RecBCD enzyme with two consequences. (1) ExoV activity is blocked leaving a highly recombinagenic, frayed duplex end near chi, and (2) as the enzyme stripped of the RecD subunit travels beyond chi it is competent to catalyze reciprocal recombination.     

Identification of glycoinositol phospholipid linked and truncated forms of the scrapie prion protein

Analysis of carboxy-terminal peptides derived from endoproteinase Lys-C digests of the scrapie isoform of the hamster prion protein revealed that the majority of the molecules are glycoinositol phospholipid linked through ethanolamine attached at serin-231. However, approximately 15% of PrPSc had a carboxy-terminal peptide that ends at glycine-228. It is intriguing that this glycine is part of the PrP sequence Gly-Arg-Arg, which is an established target sequence for the proteolysis and release of bioactive peptides from larger precursors. The mechanism of formation, as well as the role of the truncated carboxy terminus in the dissemination and neuropathology of scrapie, remains to be determined.