Fluorescence method for measuring the kinetics of fusion between biological membranes

An assay is presented that allows continuous and sensitive monitoring of membrane fusion in both artificial and biological membrane systems. The method relies upon the relief of fluorescence self-quenching of octadecyl Rhodamine B chloride. When the probe is incorporated into a lipid bilayer at concentrations up to 9 mol% with respect to total lipid, the efficiency of self-quenching is proportional to its surface density. Upon fusion between membranes labeled with the probe and nonlabeled membranes, the decrease in surface density of the fluorophore results in a concomitant, proportional increase in fluorescence intensity, allowing kinetic and quantitative measurements of the fusion process. The kinetics of fusion between phospholipid vesicles monitored with this assay were found to be the same as those determined with a fusion assay based on resonance energy transfer [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099]. Octadecyl Rhodamine B chloride can be readily inserted into native biological membranes by addition of an ethanolic solution of the probe. Evidence is presented showing that the dilution of the fluorophore, occurring when octadecyl Rhodamine containing influenza virus is mixed with phospholipid vesicles at pH 5.0, but not pH 7.4, resulted from virus-vesicle fusion and was not related to processes other than fusion. Furthermore, by use of this method, the kinetics of fusion between Sendai virus and erythrocyte ghosts and virus-induced fusion of ghosts were readily revealed. Dilution of the probe was not observed upon prior treatment of fluorescently labeled Sendai virus with trypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular organisation of the genes involved in the production of F7(2) fimbriae, causing mannose-resistant haemagglutination, of a uropathogenic Escherichia coli 06:K2:H1:F7 strain

Summary The genes responsible for the formation of the F7₂ fimbriae of the uropathogenic E. coli strain AD110 (O6:K2:H1:F7) have been cloned on the recombinant plasmid pPIL110-35 (Van Die et al. 1983). The F7₂ fimbriae, like the F7₁ fimbriae of AD110, are responsible for mannose resistant haemagglutination (MRHA).The molecular organisation of the genes of pPIL110-35 involved in the expression of MRHA was studied by: (a) analysis of transposon and Tn5 insertion mutants. Mutations that cause an MRHA-deficient phenotype were located in discrete groups within an 11.5 kb restriction fragment of pPIL110-35, separated by insertion mutations that do not inactivate MRHA. (b) complementation experiments. Restriction fragments of pPIL110-35 subcloned in the vector pBR322 were tested for their ability to complement transposon insertion mutations in the corresponding regions of pPIL110-35. Five complementation groups were distinguished.Five genes (designated A-E) involved in the expression of MRHA can be distinguished by these results. The products of these genes were analysed in minicells. The results indicate that gene B codes for a 75 K dalton protein, gene C for a 23 K dalton protein and gene E for a 36 K dalton protein. No product of gene D was observed. Gene A probably codes for the 17 K dalton subunit polypeptide of the F7₂ fimbriae, as will be discussed.     

Ultrastructural localization of zinc in rat exocrine pancreas by autoradiography

Summary ⁶⁵Zn was infused at a constant rate for 10 days into a rat. Glutaraldehyde fixed, Epon-araldite embedded ultrathin sections of pancreatic tissue were coated with Ilford L4 emulsion and at 211 days exposure were developed. Silver grains were found over the zymogen granules and over the rough endoplasmic reticulum of exocrine cells. Islet tissue was not observed in these studies. The failure of other zinc localization methods to demonstrate zinc in acinar tissue is discussed as are some of the pitfalls of the autoradiographic method and suggestions for future improvement.Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, Madison. Supported in part by USPHS Research Grant AM-05606 from the Nat. Institute of Arthritis and Metabolic Diseases.Supported by an NIH post-doctoral fellowship.     

The evolution of male-female dimorphism: Older than sex?

This contribution considers the evolution of a dimorphism with respect to cell fusion characteristics in a population of primitive cells. These cells reproduce exclusively asexually. The evolution towards asymmetric fusion behaviour of cells is driven by selection promoting horizontal transfer of an endosymbiontic replicator. It is concluded that evolution of asymmetric cell fusion in this scenario is more likely than evolution of sexual differentiation in a sexually reproducing population. Pre-existing dimorphism with respect to cell fusion may thus have been the basis for the establishment of sexual differentiation at the level of gamete fusion, and this in turn is fundamental to the evolution of two different sexes, male and female.     

Fusion of enveloped viruses with cells and liposomes

The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion.Influenza virus andSendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case ofSendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a “fresh” batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via “inactive” sites. Details of the low pH inactivation of fusion capacity ofinfluenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA₂, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes.     

Analysis of Fusarium causing dermal toxicosis in marram grass planters

In the European coastal dunes, marram grass (Ammophila arenaria) is planted in order to control sand erosion. In the years 1986 to 1991, workers on the Wadden islands in the Netherlands planting marram grass showed lesions of skin and mucous membranes, suggesting a toxic reaction. Fusarium culmorum dominated the mycoflora of those marram grass culms that were used for planting. This plant material had been cut and stored for more than one week in the open. The Fusarium toxin deoxynivalenol (DON) was detected in the suspect marram grass culms. Isolated F. culmorum strains were able to produce DON in vitro in liquid culture as well as in experimentally inoculated wheat heads. Pathogenicity tests, toxin test as well as RAPD analysis showed that the F. culmorum strains were not specialized for marram grass but may form part of the West-European F. culmorum population infecting cereals and grasses. Storage on old sand-dunes with plant debris may have led to the high occurrence of F. culmorum and contamination with DON. Marram grass culms should be obtained from young plantings on dunes on the seaward slopes and cut culms should not be stored.     

Meiotic Recombination Initiated by a Double-Strand Break in Rad50Δ Yeast Cells Otherwise Unable to Initiate Meiotic Recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad(+) strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad(+) and in rad50Δ cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50Δ cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50Δ diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination.     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5′ ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.