Alpha 1-antitrypsin: apparent molecular weight heterogeneity shown by two-dimensional electrophoresis.

Two-dimensional (2-D) gel electrophoresis was used to examine charge and molecular weight variability of alpha 1-antitrypsin. Two-D electrophoresis resolved distinctive differences among individual phenotypes. Microheterogeneity of charge was seen for the different alleles that corresponded to the charge variability observed on isoelectric focusing gels. The molecular weights of the major components of each allele appeared to differ from each other by approximately 1,000, suggesting, that in addition to sialic acid, there may be differences in neutral sugar composition between the individual components. In comparison to the M allele components, the corresponding S and Z components had higher molecular weights. The MZ and MS phenotypes showed characteristic patterns of protein spot doublets. Computerized quantitation was used to separate and estimate the contribution of each component to the overall allele composition. The Z allele components contained about 15% of the total MZ quantity. The 2-D electrophoresis technique may offer a new approach for molecular structural studies of alpha 1-antitrypsin variants and similar glycoproteins.     

Detection and separation of heterogeneity in molecular complexes by statistical analysis of their two-dimensional projections

Progress in molecular structure determination by cryo electron microscopy and single particle analysis has led to improvements in the resolution achievable. However, in many cases the limiting factor is structural heterogeneity of the sample. To address this problem, we have developed a method based on statistical analysis of the two-dimensional images to detect and sort localised structural variations caused, for example, by variable occupancy of a ligand. Images are sorted by two consecutive stages of multivariate statistical analysis (MSA) to dissect out the two main sources of variation, namely out of plane orientation and local structural changes. Heterogeneity caused by local changes is detected by MSA that reveals significant peaks in the higher order eigenimages. The eigenimages revealing local peaks are used for automated classification. Evaluation of differences between classes allows discrimination of molecular images with and without ligand. This method is very rapid, independent of any initial three-dimensional model, and can detect even minor subpopulations in an image ensemble. A strategy for using this technique was developed on model data sets. Here, we demonstrate the successful application of this method to both model and real EM data on chaperonin-substrate and ribosome-ligand complexes.     

Conformational changes induced in the Saccharomyces cerevisiae GTPase-associated rRNA by ribosomal stalk components and a translocation inhibitor

The yeast ribosomal GTPase associated center is made of parts of the 26S rRNA domains II and VI, and a number of proteins including P0, P1α, P1β, P2α, P2β and L12. Mapping of the rRNA neighborhood of the proteins was performed by footprinting in ribosomes from yeast strains lacking different GTPase components. The absence of protein P0 dramatically increases the sensitivity of the defective ribosome to degradation hampering the RNA footprinting. In ribosomes lacking the P1/P2 complex, protection of a number of nucleotides is detected around positions 840, 880, 1100, 1220–1280 and 1350 in domain II as well as in several positions in the domain VI α-sarcin region. The protection pattern resembles the one reported for the interaction of elongation factors in bacterial systems. The results exclude a direct interaction of these proteins with the rRNA and are compatible with an increase in the ribosome affinity for EF-2 in the absence of the acidic P proteins. Interestingly, a sordarin derivative inhibitor of EF-2 causes an opposite effect, increasing the reactivity in positions protected by the absence of P1/P2. Similarly, a deficiency in protein L12 exposes nucleotides G1235, G1242, A1262, A1269, A1270 and A1272 to chemical modification, thus situating the protein binding site in the most conserved part of the 26S rRNA, equivalent to the bacterial protein L11 binding site.     

Fluorescence intensity multiple distributions analysis: concurrent determination of diffusion times and molecular brightness

Fluorescence correlation spectroscopy (FCS) has proven to be a powerful technique with single-molecule sensitivity. Recently, it has found a complement in the form of fluorescence intensity distribution analysis (FIDA). Here we introduce a fluorescence fluctuation method that combines the features of both techniques. It is based on the global analysis of a set of photon count number histograms, recorded with multiple widths of counting time intervals simultaneously. This fluorescence intensity multiple distributions analysis (FIMDA) distinguishes fluorescent species on the basis of both the specific molecular brightness and the translational diffusion time. The combined information, extracted from a single measurement, increases the readout effectively by one dimension and thus breaks the individual limits of FCS and FIDA. In this paper a theory is introduced that describes the dependence of photon count number distributions on diffusion coefficients. The theory is applied to a series of photon count number histograms corresponding to different widths of counting time intervals. Although the ability of the method to determine specific brightness values, diffusion times, and concentrations from mixtures is demonstrated on simulated data, its experimental utilization is shown by the determination of the binding constant of a protein-ligand interaction exemplifying its broad applicability in the life sciences.     

Different roles of P1 and P2 Saccharomyces cerevisiae ribosomal stalk proteins revealed by cross-linking

The stalk is an essential domain of the large ribosomal subunit formed by a complex of a set of very acidic proteins bound to a core rRNA binding component. While in prokaryotes there is only one type acidic protein, L7/12, two protein families are found in eukaryotes, phosphoproteins P1 and P2, which presumably have different roles. To search for differences zero-length cross-linking by S-S bridge formation was applied using Saccharomyces cerevisiae mutant P1 and P2 proteins carrying single cysteine residues at various positions. The results show a more exposed location of the N-terminal domain of the P2 proteins, which in contrast to P1, can be found as dimers when the Cys is introduced in this domain. Similarly, the Cys containing C-terminal domain of mutant P2 proteins shows a notable capacity to form cross-links with other proteins, which is considerably lower in the P1 type. On the other hand, mutation at the conserved C-domain of protein P0, the eukaryotic stalk rRNA binding component, results in removal of about 14 terminal amino acids. Protein P2, but not P1, protects mutant P0 from this truncation. These results support a eukaryotic stalk structure in which P1 proteins are internally located with their C-terminals having a restricted reactivity while P2 proteins are more external and accessible to interact with other cellular components.     

An Eimeriid origin of isosporoid coccidia with Stieda bodies as shown by phylogenetic analysis of small subunit ribosomal RNA gene sequences

Morphological and life cycle features of the tissue cyst-forming coccidia have been difficult to interpret in devising taxonomic classifications for the various genera. In this study, we amplified the full small subunit rRNA gene sequence of Isospora robini McQuistion and Holmes, 1988, and the partial sequence of Isospora gryphoni Olsen, Gissing, Barta, and Middleton, 1998 by PCR. Both of these species vary from Isospora species of mammals in having Stieda bodies on the sporocysts. The sequences were cloned and sequenced and were incorporated into an alignment with other Isospora species lacking Stieda bodies as well as with other coccidia. Maximum parsimony analysis of these sequences produced a single most parsimonious tree that placed I. robini and I. gryphoni in a clade containing various other eimeriid species. The Isospora species lacking Stieda bodies were in the sarcocystid clade. Similar results were found by maximum likelihood analysis. These findings indicate that the genus Isospora as defined by several authors is polyphyletic. Taxonomic changes to the genus Isospora would have to incorporate the 2 major clades found by molecular phylogenetic analysis. Isospora species with Stieda bodies should be classified in the family Eimeriidae, whereas those without Stieda bodies should remain in the family Sarcocystidae.     

Nuclei of symbiotic arbuscular mycorrhizal fungi as revealed by in vivo two-photon microscopy

The present work reports the results obtained from in vivo studies on the distribution and behavior of nuclei of two arbuscular mycorrhizal (AM) fungi growing in symbiosis with tomato root organ cultures (AM monoxenic cultures). Upon staining with 4',6-diamidino-2-phenylindole and two-photon microscopy (2PM) observations, symbiotic thick runner hyphae appeared mostly opaque to 2PM and did not reveal nuclei within them; thin runner hyphae showed dimly stained nuclei along them, whereas nuclei were clearly visible within the branches of the so-called branched absorbing structures. When visible, nuclei appeared anchored laterally at regular intervals along the symbiotic AM extraradical hyphae. Other nuclei migrate through the hyphal central core; this migration occurs in pulses. Simultaneous observations on different areas of extraradical AM mycelium revealed the existence of lysed compartments along the fungal hyphae, containing nuclei remnants and/or chromatin masses. All these results give new insights in (i) the differential permeability of AM hyphae in the symbiotic versus the asymbiotic state; (ii) the behavior and distribution of nuclei along the symbiotic extraradical mycelium; (iii) the occurrence of ageing events within the AM fungal colony; and (iv) the existence of "healing" mechanisms aiming to restrict the damage induced by such ageing or lytic events. An AM fungal strategy for hyphal survival under adverse conditions is also suggested.     

Nuclei of symbiotic arbuscular mycorrhizal fungi as revealed by in vivo two-photon microscopy

Summary The present work reports the results obtained from in vivo studies on the distribution and behavior of nuclei of two arbuscular mycorrhizal (AM) fungi growing in symbiosis with tomato root organ cultures (AM monoxenic cultures). Upon staining with 4′,6-diamidino-2-phenylindole and two-photon microscopy (2PM) observations, symbiotic thick runner hyphae appeared mostly opaque to 2PM and did not reveal nuclei within them; thin runner hyphae showed dimly stained nuclei along them, whereas nuclei were clearly visible within the branches of the so-called branched absorbing structures. When visible, nuclei appeared anchored laterally at regular intervals along the symbiotic AM extraradical hyphae. Other nuclei migrate through the hyphal central core; this migration occurs in pulses. Simultaneous observations on different areas of extraradical AM mycelium revealed the existence of lysed compartments along the fungal hyphae, containing nuclei remnants and/or chromatin masses. All these results give new insights in (i) the differential permeability of AM hyphae in the symbiotic versus the asymbiotic state; (ii) the behavior and distribution of nuclei along the symbiotic extraradical mycelium; (iii) the occurrence of ageing events within the AM fungal colony; and (iv) the existence of “healing” mechanisms aiming to restrict the damage induced by such ageing or lytic events. An AM fungal strategy for hyphal survival under adverse conditions is also suggested.     

Vicia faba chloroplast DNA has only one set of ribosomal RNA genes as shown by partial denaturation mapping and R-loop analysis

Summary Broad-bean (Vicia faba) chloroplast DNA (cpDNA) was isolated and characterized. The intact DNA is circular and has a molecular weight of 79.8x 10⁶ dalton. Electron microscopic analysis of self-annealed intact single-strand circles show that it does not have a large double-stranded inverse repeat as seen in spinach chloroplast DNA. Only one ribosomal RNA gene (one set of 16S and 23S rRNA sequences) was found in preparations of R-loops between the Vicia rRNA and cpDNA circles. A restriction enzyme map for SalI and KpnI was derived by comparing the partial denaturation pattern of the fragments with the pattern of the intact circle. The map was confirmed by gel analysis. The ribosomal RNA gene was localized on the SalI fragment 3b by R-loop analysis. SalI fragment 1a although it contains a G-C rich region did not form R-loops with rRNA. Partial denaturation patterns of spinach cpDNA circles and BglI fragments were determined and from this the position of the fragments mapped. This confirmed the reliability of these methods for the arrangement of restriction enzyme fragments along circular molecules. The structures of the two cpDNAs were compared.     

Quantitative analysis of in vivo ribosomal events at UGA and UAG stop codons.

An in vivo translation assay system has been designed to measure, in one and the same assay, the three alternatives for a ribosome poised at a stop codon (termination, read-through and frameshift). A quantitative analysis of the competition has been done in the presence and absence of release factor (RF) mutants, nonsense suppressors and an upstream Shine-Dalgarno-like sequence. The ribosomal +1 frameshift product is measurable when the stop codon is decoded by wild-type or mutant RF (prf A1 or prf B2) and also in the presence of competing suppressor tRNAs. Frameshift frequency appears to be influenced by RF activity. The amount of frameshift product decreases in the presence of competing suppressor tRNAs, however, this decrease is not in proportion to the corresponding increase in the suppression product. Instead, there is an increase in the total amount of protein expressed from the gene, perhaps due to the purging of queued ribosomes. Mutated RFs reduce the total output of the reporter gene by reducing the amount of all three protein products. The nascent peptide has earlier been shown to influence the translation termination process by interacting with the RFs. At 42 degrees C in a temperature-sensitive RF mutant strain, protein measurements indicate that the nascent peptide seems to influence the binding efficiencies of the RFs.     

Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome

During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states. Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation. Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome. Isolated L12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment. The overall structure can be described as three ellipsoids joined by flexible linkers. No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution. In the (1)H-(15)N HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies. The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12. These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors.     

Dinoflagellates in evolution. A molecular phylogenetic analysis of large subunit ribosomal RNA

Summary The sequence of the large subunit ribosomal RNA (LsuRNA) gene of the dinoflagellateProrocentrum micans has been determined. The inferred rRNA sequence [3408 nucleotides (nt)] is presented in its most probable secondary structure based on compensatory mutations, energy, and conservation criteria. No introns have been found but a hidden break is present in the second variable domain, 690 nt from the 5 end, as judged by agarose gel electrophoresis and primer extension experiments.Prorocentrum micans LsuRNA length and G+C content are close to those of ciliates and yeast. The conserved portions of the molecule (1900 nt) have been aligned with corresponding sequences from various eukaryotes, including five protista, one metaphyta, and three metazoa. An extensive phylogenetic study was performed, comparing two phenetic methods (neighbor joining on difference matrix, and Fitch and Margoliash on Knuc values matrix) and one cladistic (parsimony). The three methods led to similar tree topologies, except for the emergence of yeast that groups with ciliates and dinoflagellates when phenetic methods are used, but emerges later in the most parsimonious tree. This discrepancy was checked by statistical analyses on reduced trees (limited to four species) inferred using parsimony and evolutionary parsimony methods. The data support the phenetic tree topologies and a close relationship between dinoflagellates, ciliates, and yeast.     

Phosphorylation of the yeast ribosomal stalk. Functional effects and enzymes involved in the process

The ribosomal stalk is directly involved in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes the acidic components correspond to the 12-kDa P1 and P2 proteins, and the RNA binding component is the P0 protein. All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure. Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins. Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37 degrees C. The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site. However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification. Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins. A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins. It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.     

Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1–3 ·m in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4–6 weeks in culture and progressively developed a broad territorial region up to 12 ·m wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly This revised version was published online in November 2006 with corrections to the Cover Date.