Characterization of pseudomonads isolated from diseased fleece.

A total of 59 Pseudomonas isolates was obtained from 11 samples of diseased fleece taken from live sheep. All but four of the isolates could be assigned to one of nine Pseudomonas species, of which P. aeruginosa, P. alcaligenes, P. mendocina , and P. putida were the most common. P. aeruginosa was found in four of the fleece samples and, when present, appeared to predominate. Although several of the isolates of P. aeruginosa lacked the ability to produce pyocyanin (and some produced neither pyocyanin nor fluorescein), nearly all produced several virulence factors. Of the other pseudomonads, many produced proteinase, esterase, and catalase, several were able to grow at 42 degrees C and reduce nitrate, and some also produced lipase and hemolysin and, like P. aeruginosa, might serve to initiate (or sustain) the dermatitis frequently associated with fleece rot in sheep.     

Accumulation of Plastoquinone A during Low Temperature Growth of Winter Rye

Chloroplasts isolated from rye (Secale cereale L. cv Puma) grown at 5°C (RH) accumulated 260% more plastoquinone A (PQA) per plastid than chloroplasts isolated from rye grown at 20°C (RNH). The number of plastoglobuli increased by 270% in RH chloroplasts compared with RNH plastids. When RH plastids were lysed and washed, the number of plastoglobuli associated with thylakoid membranes decreased significantly, yet the PQA levels remained high. Room temperature fluorescence induction indicated that (a) there is no change in the size of the PQA pool immediately available for photochemistry in RNH and RH thylakoids and (b) there is a pool of oxidized PQA present in RNH and RH thylakoids which is not available for photochemistry. The accumulated PQA in RH thylakoids may reflect an increased nonphotochemical function such as regulation of thylakoid protein phosphorylation or protection against photoinhibition.     

Tissue fixation by osmium tetroxide. A possible role for proteins.

The osmiophilia, under the conditions of normal tissue fixation, of the histidine, lysine, tryptophan, cysteine and methionine side chain of proteins is suggested by in vitro studies on blocked amino acids representative of such protein side chains, and the chemical nature of the reaction products elucidated. The chemical feasibility of inter- or intramolecular cross-linking of protein by OsO4 at these and other sites is demonstrated, as in the cross-linking of protein with unsaturated lipids such as methyl oleate, methyl linoleate and linolenate, and cholesteryl acetate. The relevance of these results to the process of tissue fixation by OsO4 is discussed.     

Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins

We have used low shear viscometry and electron microscopy to study the interaction between pure actin filaments and microtubules. Mixtures of microtubules having microtubule-associated proteins (MAPs) with actin filament have very high viscosities compared with the viscosities of the separate components. MAPs themselves also cause a large increase in the viscosity of actin filaments. In contrast, mixtures of actin filaments with tubulin polymers lacking MAPs have low viscosities, close to the sum of the viscosities of the separate components. Our interpretation of these observations is that there is an interaction between actin filaments and microtubules which requires MAPs. This interaction is inhibited by ATP and some related compounds. Electron micrographs of thin sections through mixtures of actin and microtubules show numerous close associations between the two polymers which may be responsible for their high viscosity.     

Drosophila topoisomerase II-DNA interactions are affected by DNA structure.

The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions.     

HMf, a histone-related protein from the hyperthermophilic archaeon Methanothermus fervidus, binds preferentially to DNA containing phased tracts of adenines.

HMf, a histone-related protein from Methanothermus fervidus, was found to bind preferentially to a DNA that is intrinsically bent as a result of the presence of phased oligo(dA) tracts. The intergenic regions in M. fervidus DNA are A+T rich and frequently contain oligo(dA) tracts, some of which may have the size and phasing required to create a net bending in one direction. The binding of HMf to bent DNA could play a direct role in gene expression and stabilization of the genome of this organism.     

Early stages in RecA protein-catalyzed pairing. Analysis of coaggregate formation and non-homologous DNA contacts.

RecA protein will catalyze the in vitro pairing of homologous DNA molecules. To further explore the events involved in the search for homology, we have applied a nitrocellulose filter binding assay to follow pairing, and a sedimentation assay to follow the generation of aggregates (termed coaggregates) formed between RecA-complexed single-stranded (ss) DNA and double stranded (ds) DNA. Electron microscopy (EM) was used to visualize the structures involved. RecA protein promoted the pairing of circular M13 ssDNA and linear M13mp7 dsDNA efficiently in the absence of coaggregates. Indeed, pairing of homologous ss- and dsDNAs involved coaggregate formation only if the dsDNA was circular. For DNAs containing only a few hundred base-pairs of homology, for example pUC7 dsDNA and M13mp7 ssDNA, pairing and joint formation was observed if the dsDNA was superhelical but not if it was topologically relaxed or linear with the homology internal to an end of the dsDNA. The effect of non-covalently attached heterologous dsDNA on the RecA-promoted joining of M13 ssDNA and linear M13mp7 dsDNA (with non-M13 sequences at both ends) was found to depend on the topology and concentration of the heterologous DNA. A tenfold excess of superhelical pBR322 DNA strongly inhibited pairing. However, addition of relaxed or linear pBR322 DNA to the pairing reaction had little effect. As seen by EM, superhelical pBR322 DNA inhibited joint formation by excluding the homologous dsDNA form the coaggregates. EM also revealed heterologous DNA interactions presumably involved in the search for homology. Here the use of EM has provided a direct visualization of the form and architecture of coaggregates revealing a dense interweaving of presynaptic filaments and dsDNA.