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J L Carrascosa

Publications and source records attributed to J L Carrascosa.

At least 19 recordsLinked to original sources
PubMed1992

DNA conformational change induced by the bacteriophage phi 29 connector.

Translocation of viral DNA inwards and outwards of the capsid of double-stranded DNA bacteriophages occurs through the connector, a key viral structure that is known to interact with DNA. It is shown here that phage phi 29 connector binds both linear and circular double-stranded DNA. However, DNA-mediated protection of phi 29 connectors against Staphylococcus aureus endoprotease V8 digestion suggests that binding to linear DNA is more stable than to circular DNA. Endoprotease V8-protection assays also suggest that the length of the linear DNA required to produce a stable phi 29 connector-DNA interaction is, at least, twice longer than the phi 29 connector channel. This result is confirmed by experiments of phi 29 connector-protection of DNA against DNase I digestion. Furthermore, DNA circularization assays indicate that phi 29 connectors restrain negative supercoiling when bound to linear DNA. This DNA conformational change is not observed upon binding to circular DNA and it could reflect the existence of some left-handed DNA coiling or DNA untwisting inside of the phi 29 connector channel.

Bacillus Phages↗
PubMed1992

Streptomyces lividans possesses a GroEL-like chaperonin.

Streptomyces lividans grown at 45 degrees C produces a GroEL-like chaperonin. This protein is specifically synthesized in bacterial cell cultures upon heat shock induction. It has a similar size (62 kDa) to the GroEL-like proteins from Escherichia coli and Bacillus subtilus and shows immunological cross-reaction with serum raised against GroEL from E. coli. The S. lividans 62-kDa protein assembles into oligomers around 20S that show a morphology consistent with a barrel showing six-fold and seven-fold symmetries as previously described in E. coli and B. subtilis.

Bacterial Proteins↗
PubMed1992

Role of the amino-terminal domain of bacteriophage phi 29 connector in DNA binding and packaging.

The connector of bacteriophage phi 29 is required for prohead assembly, binds DNA, and drives DNA packaging into viral proheads. Limited proteolysis of the connector protein with endoproteinase Glu-C from Staphylococcus aureus V8 and chymotrypsin showed that a domain of the NH2-terminal region is involved in DNA binding and in the subsequent packaging into preformed proheads, but not in prohead assembly. Mutants in specific amino acids of the NH2-terminal domain, obtained by directed mutagenesis techniques, showed that the Ala1-Arg2-Lys3-Arg4 region of the connector is absolutely necessary for DNA packaging into the proheads as well as for efficient DNA binding.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of T3 connector purified from overexpressing bacteria.

The bacteriophage T3 connector has been purified from overexpressed protein in Escherichia coli, harboring a plasmid containing the gene encoding p8 protein. The connector, which is composed of 12 copies of p8, has been crystallized in two-dimensional sheets and studied by electron microscopy from negatively stained specimens. A two-dimensional Fourier filtering and averaging procedure was performed with crystalline specimens. In addition, single particle averaging techniques were used with other preparations. The average images obtained from these two approaches gave similar results. A three-dimensional reconstruction from two-dimensional crystals of T3 connectors was obtained by collecting several sets of tilted views and using standard Fourier procedures. The resolution of the three-dimensional map was 1.65 nm. The reconstructed connector shows two main domains: a wider one with 12 small units in the periphery and with an external diameter of 14.9 nm, and a smaller one with 8.5 nm diameter. The height of the reconstructed connector has been determined to be around 8.5 nm. The reconstruction clearly shows an internal open channel running along the longitudinal axis of the particle and having an average diameter of 3.7 nm.

Capsid↗
PubMed1991

Binding studies and localization of Escherichia coli lipopolysaccharide in cultured hepatocytes by an immunocolloidal-gold technique.

In this study, the uptake and localization of an Escherichia coli lipopolysaccharide, and the temperature effects on these processes, were studied in rat cultured hepatocytes using a binding assay and an immunocolloidal gold technique. The lipopolysaccharide was found to bind to the cell membrane and microvilli after short incubation times, at both 4 degrees C and 37 degrees C. This was followed by a dispersed localization into the cytoplasm, reaching mitochondria. The uptake was found not to be receptor-mediated. A decrease of temperature, delays, but does not prevent, the lipopolysaccharide internalization.

Animals↗
PubMed1991

Characterization of a versatile in vitro DNA-packaging system based on hybrid lambda/phi 29 proheads.

We have studied the assembly of bacteriophage lambda head proteins on the phage phi 29 connector to produce in vitro chimeric proheads, whose ability to package different types of DNA depends on the physical integrity of the phi 29 connector. Terminal protein-free phi 29 as well as nonviral DNAs have been shown to be efficiently packaged by this hybrid system. An RNA, that can be provided by any of the extracts used in the complementation mixture, was required for DNA packaging, both by the hybrid system as well as by the homologous lambda system. The DNA-packaging activity of RNase-treated proheads can be restored by adding a mixture of ribosomal RNAs. There is also a requirement for a minimal length of DNA to be stably packaged. The packaging protein p16 of phi 29 can replace the lambda terminase complex in the in vitro packaging system, both with the chimeric as well as genuine lambda proheads.

Bacteriophage lambda↗
PubMed1991

Electron microscopy study of GroEL chaperonin: different views of the aggregate appear as a function of cell growth temperature.

We have studied two members of the family of morphogenetic factors or chaperonins, the GroEL-like factors from Escherichia coli and Bacillus subtilis, in order to determine the possible structural basis of their related function in promoting the correct and efficient assembly of biological oligomers. The main objective of this work has been to study by transmission electron microscopy the possible changes that these factors may undergo when subjected to a number of different conditions such as changes in temperature in vivo and in pH in vitro. We applied both rotational and multivariate statistical analyses of single particles to images of GroEl-like aggregates from the two bacteria. The most striking result is the finding of two distinct "front views" of these aggregates, from both E. coli and B. subtilis. One view, which has not been described earlier, shows a sixfold symmetry and is most abundant at growing temperatures below 37 degrees C. After heat shock, a view showing seven morphological units becomes dominant. On the basis of our analysis it is clear that GroEL-like morphogenetic factors from two unrelated bacteria such as E. coli and B. subtilis present two distinct views: one sixfold and the other sevenfold. Their relative percentage of appearance is related to the temperature at which the cells were grown and also to the storage conditions (pH).

Bacillus subtilis↗
PubMed1991

Uptake and subcellular distribution of Escherichia coli lipopolysaccharide by isolated rat type II pneumocytes.

Treatment of isolated rat Type II pneumocytes with Escherichia coli lipopolysaccharide (LPS) induces a number of ultra-structural changes which become evident after 60 min of incubation. By using post-embedding immunolabeling methods and electron microscopy, we have followed the fate of LPS after different times of incubation. After an initial period of accumulation in the pneumocyte microvilli, the LPS molecules enter the cytoplasm, forming discrete patches which are dispersed in some areas. After longer incubation times, LPS localize in condensed chromatin-free areas inside the nuclei. LPS micelles were visualized after freeze-fracture and compared with the LPS-labeled membrane areas, showing that LPS micelles aggregate in particular membrane zones. The sugar-specific staining in microvilli areas, where Maclura pomifera agglutinin (MPA)-gold particles bind, indicates the presence of galactose derivatives in these membrane structures. Pre-treatment of pneumocytes with LPS inhibited the MPA-gold labeling, suggesting a relation between the MPA receptor and a possible LPS receptor. Finally, double immunolabeling experiments indicated an apparent LPS-tubulin association in some particular membrane regions, which could not be observed when LPS and actin were co-localized.

Animals↗
PubMed1990

Conformational changes in bacteriophage phi 29 connector prevents DNA-binding activity.

In vitro DNA packaging activity in a defined system derived from bacteriophage phi 29 depends upon the chemical integrity of the connector protein p10. Proteolytic cleavage of p10 rendered the proheads inactive for DNA packaging. A similar treatment on isolated connectors abolished the DNA-binding activity of the native p10, but the general shape and size of the connector was not changed as revealed by electron microscopy. Analytical ultracentrifugation showed that the proteolyzed connectors had a smaller sedimentation coefficient, while amino acid analysis after dialysis of the proteolyzed p10 confirmed the loss of 16 and 19 amino acids from the amino and carboxy termini, respectively. Low angle X-ray scattering revealed that proteolysis was followed by a small decrease in the radius of gyration and a reorganization of the distal domain of the cylindrical inner part of the connector. Characterization of the cleavage sites in the primary sequence allowed us to propose the location of the DNA-binding domain in the connector model.

Amino Acid Sequence↗
PubMed1990

Production of lambda-phi 29 phage chimeras.

Proheads of bacteriophage lambda which carry the connector of phage phi 29 instead of that of lambda have been produced in vitro. These hybrid proheads have a structure similar to that of normal lambda proheads. Furthermore, the chimeric proheads can package both lambda and phi 29 DNA. These data show that the connector domains involved in both head assembly and DNA packaging are functionally similar. The DNA-containing lambda-phi 29 proheads can be complemented in vitro with phi 29 tails to yield infective particles capable of DNA transfer.

Bacteriophage lambda↗
PubMed1990

Collagenous structures present in brain contain epitopes shared by collagen and microtubule-associated protein tau.

A novel type of collagenous fibers has been isolated from human brain and characterized by electron microscopy and optical diffraction. It was found that the morphology of the fibers is similar, but not identical, to that of skin collagen. Also, the collagenous fibers show some similarities with the paracrystals that could be assembled in vitro from purified microtubule-associated protein tau. Immunological analyses indicated the presence of epitopes in these collagenous fibers which react with antibodies against collagen and tau.

Alzheimer Disease↗
PubMed1988

Tau factor polymers are similar to paired helical filaments of Alzheimer's disease.

Tau factor, upon urea treatment, is able to polymerize in vitro. These polymers are composed of tau factor as shown by immunogold staining. The structure of tau polymers is very similar to that of paired helical filaments (PHFs) of Alzheimer's disease in their dimensions as well as in their periodicity. Metal shadowing of both polymers shows a similar twisting. Also, similar peptide maps were found for tau factor and a 33 kDa protein that is the main component of our PHF preparations.

Alzheimer Disease↗
PubMed1988

Bacteriophage T3 connector: three-dimensional structure and comparison with other viral head-tail connecting regions.

The bacteriophage T3 connector, which consists of 12 copies of protein gp8, has been studied by image processing of electron micrographs from negatively stained ordered aggregates. A three-dimensional reconstruction of T3 connectors was obtained by collection of tilted views and using the direct Fourier method, up to 2.3 nm resolution. The reconstructed unit cell contains two connectors whose main structural features are essentially identical, but facing in opposite directions. The T3 connector has a height of about 10.9 nm, with two clearly defined domains: a wider one 14.4 nm in diameter, with 12 morphological units in the periphery, and a narrower one, 9.7 nm in diameter. There is a channel clearly defined in the narrower domain that almost closes along the wider domain. Comparison of the three-dimensional structure obtained for the connector of phages T3 and phi 29, and that of the neck extracted from phage phi 29 particles, reveals striking similarities and significant differences. A model for a general connector to account for the common functions carried out by these viral assemblies is discussed together with the possible role of the channel for DNA translocation.

Computer Graphics↗
PubMed1988

Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging.

The gene 1 protein of Salmonella bacteriophage P22 is located at the DNA packaging vertex of the mature particle. The protein is incorporated into the procapsid shell during shell assembly and is required for DNA packaging. The unassembled precursor form of the gene 1 protein has been purified from cells infected with mutants blocked in procapsid assembly. The purified 90,000-dalton protein was dimeric or monomeric; upon storage in the cold it formed 20S cyclic dodecamers. Computer filtering of negatively stained electron micrographs revealed 12 arms and knobs projecting from a central ring, with a 30-A channel at the center. Similar dodecameric rings were released from disrupted procapsid shells. These results indicate that the gene 1 protein is organized as a cyclic dodecamer within the procapsid shell and serves as the portal through which P22 DNA is threaded during DNA packaging. The presence of a 12-fold ring located at a 5-fold portal vertex appears to be a conserved structural theme of the DNA packaging apparatus of double-stranded DNA phages.

Capsid↗
PubMed1987

Restoration of direct Fourier three-dimensional reconstructions of crystalline specimens by the method of convex projections.

We consider the problem of the three-dimensional (3-D) reconstruction of objects by the direct Fourier method (DFM) and their restoration by the method of projections on to convex sets (POCS). The main discussion is centered on the case of specimens arranged in a two-dimensional (2-D) crystal and imaged by transmission electron microscopy, although the conclusions could be extended to more general cases. We present results of the restoration of the 3-D reconstruction of a computer generated 2-D crystal under different conditions of data collection limitation. A preliminary application with a real biological specimen (the connector of bacteriophage phi 29) is also presented. These results indicate that POCS can be used practically, in certain cases, to restore 3-D reconstructions obtained by the DFM, giving grounds for the proposal of the study of a combined DFM + POCS (reconstruction + restoration) method for the determination of biological structures by electron microscopy and 3-D image processing.

Coliphages↗