The acid-labile subunit of the serum insulin-like growth factor-binding protein complexes. Structural determination by molecular modeling and electron microscopy.

The acid-labile subunit (ALS) is a glycosylated 85-kDa member of the leucine-rich repeat (LRR) protein superfamily and circulates in ternary complexes with the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). These complexes are thought to regulate the serum IGFs by restricting IGF movement out of the circulation. However, little is known about how ALS binds to IGFBP-3 or -5, which link the IGFs to ALS. To investigate potential sites of interaction, the ALS structure has been modeled with the crystal structure of the LRR protein porcine ribonuclease inhibitor as a template. ALS is predicted to be a donut-shaped molecule with an internal diameter of 1.7 nm, an external diameter of 7.2 nm, and a thickness of 3.6 nm. These dimensions are supported by rotary shadowing electron microscopy of ALS. The internal face is lined with a substantial region of electronegative surface potential that could interact with the positively charged region on IGFBP-3 known to be involved in ALS binding. The model also predicts that three potential N-linked oligosaccharide sites within the LRR domain are clustered together, which may be important in light of recent studies showing ALS glycan involvement in complex formation with IGFBP-3.

Collagen-associated molecules in the cornea: localisation with monoclonal antibodies.

This paper describes the biochemical characteristics and immunostaining properties of four monoclonal antibodies (MAbs) raised to the extracellular matrix of bovine corneal endothelial cells in culture. All four antibodies labelled molecules in frozen sections of bovine cornea. MAb A15 labelled the collagen lamellae of the stroma. Immuno-electron microscopy located the label between rather than over the collagen fibrils. Antibody label was removed by collagenase treatment of the stroma. This antibody bound to several denatured collagen types in ELISAs and Western blots, the common epitope being located close to the collagenase binding site. A15 immunoprecipitated native polypeptides of 173 kDa and 150 kDa from the conditioned medium of corneal endothelial cells. Amino acid analysis of the 150 kDa molecule showed broad similarity to bovine type VI collagen although there was no immunological cross reactivity. The binding of this antibody in the corneal stroma may be to a collagen type VI-like molecule. MAb A70 bound to a collagenase sensitive molecule in corneal endothelial cell extracellular matrix in vitro, and to basement membranes in vivo. It showed staining typical of type IV collagen in frozen sections of cornea. MAbs A67 and A49 both labelled flexuous fibre-like structures in the cornea, which appeared to wrap around the fibrillar collagen lamellae. The molecule labelled by A67, of 86 kDa was sensitive to collagenase treatment of endothelial cell conditioned medium, yet totally resistant to collagenase treatment of the cornea, which completely removed the fibrillar collagen from the stroma. In the endothelial extracellular matrix, this antigen was resistant to all proteases tested and required severe denaturing conditions to remove antigenic activity. Amino acid analysis did not yield the high proportion of glycine typical of collagens, so if collagenous sequences occur they must be relatively short. The molecule labelled by A49 was a 51-kDa protein, of similar amino acid composition to antigen 67, and showed a similar distribution in frozen sections of bovine cornea. There was, however, no immunological cross reactivity between the two. Unlike antigen 67, antigen 49 was not sensitive to collagenase under any conditions and was very sensitive to protease treatment of the endothelial extracellular matrix. Immuno-electron microscopy showed labelling with both these antibodies between the collagen fibrils in the stroma. We postulate that these two molecules may be involved in stabilising the lamella structure of the corneal stroma.

Double immunogold labelling demonstrating expression of recombinant genes for production of an anti-fertility vaccine.

The multiple antibody technique for double immunogold labelling for the simultaneous localization of two antigens with negative staining was utilized to demonstrate the expression of recombinant genes in bacteria, with the primary antibodies being raised in different host species. For the production of a vaccine for immunological control of fertility, a multi-functional plasmid vector was introduced into the bacterium Pseudomonas aeruginosa containing the Dichelobacter nodosus fimbrial subunit gene with a grafted amino acid sequence of luteinizing hormone releasing hormone (LHRH) peptide. Fimbriae of this recombinant, when run on a SDS-polyacrylamide electrophoresis gel, gave a single broad band for LHRH peptide/D. nodosus subunit and were harvested to produce the anti-fertility vaccine which, when injected into mice, produced atrophy of the testes with absence of sperm, resulting in reversible castration. Double immunogold labelling of the recombinant P. aeruginosa bacteria demonstrated fimbriae with strong expression of the LHRH-peptide, expression of the D. nodosus subunit and absence of host fimbriae.

A study of electrophoretic mobility of DNA in agarose and polyacrylamide gels.

The aim of this paper is to clarify the mechanism of gel electrophoresis of DNA under constant-field conditions. We have conducted a large number of experiments on double-stranded DNA varying in length between approximately 10 and approximately 50,000 base-pairs, in both agarose and polyacrylamide gels ranging from 0.5% to 12% concentration, and with electric field strengths ranging from 0.5 to 8 V/cm. We have made (logarithmic) plots of velocity against length of DNA for all of the various test conditions. At the left-hand side of these plots, all of the empirical curves have a unique, standard shape. When the curves are normalized so that their left-hand parts coincide, a second feature emerges in that, while for any given test the curve follows the "master curve" up to a certain point, it then "breaks away" and becomes horizontal. We describe these two patterns of behaviour as "regions 1 and 2", respectively. We find simple yet comprehensive empirical formulae that fit the observations in the two regions of behaviour: these express the velocity in terms of length of DNA, electric field strength and gel concentration. We then construct two separate theories for the two regions of behaviour. The first theory involves the statistics of motion of an object through a random array of gel obstacles, with the instantaneous speed depending on the number of obstacles with which the object is currently in contact. The second theory is based on the mechanical hypothesis (for which there is other, independent support) that the DNA moves through the gel by piling up against a barrier, which eventually breaks or deforms under the resulting force, thereby allowing the DNA to move on to the next barrier. The statistical theory is an adaptation of existing work, while the mechanical one is new. We also describe experiments on the migration of repeated-sequence, curved DNA with length up to 1500 base-pairs, and we discuss its behaviour in terms of our two theories. Our studies by electron microscopy are consistent with the view that this repeated-sequence DNA adopts a superhelical configuration. Finally, we show that a very wide range of observations may be understood clearly by means of our two theoretical schemes.

Morphogenetic expression of Moraxella bovis fimbriae (pili) in Pseudomonas aeruginosa.

Type 4 fimbriae (pili) are found in a wide variety of gram-negative bacteria and are composed of small structural subunits which share significant sequence homology among different species, especially at their amino-terminal ends. Previous studies demonstrating morphogenetic expression of Bacteroides nodosus fimbriae from cloned subunit genes in Pseudomonas aeruginosa suggested that there is a common mechanism for type 4 fimbriae assembly and that the structural subunits are interchangeable (J. S. Mattick et al., J. Bacteriol. 169:33-41, 1987). Here we have examined the expression of Moraxella bovis fimbrial subunits in P. aeruginosa. M. bovis subunits were assembled into extracellular fimbriae in this host, in some cases as a homopolymer but in others as a mosaic with the indigenous subunit, indicating structural equivalence. This result contrasts with other studies in which recombinant P. aeruginosa expressing different subunits produced fimbriae composed almost exclusively of one subunit or the other (T. C. Elleman and J. E. Peterson, Mol. Microbiol. 1:377-380, 1987). Both observations can be explained by reversibility of subunit-subunit interactions at the site of assembly, with the forward equilibrium favoring chain extension between compatible subunits.

Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.

The ultrastructural morphology of native salivary gland chromosomes of Drosophila melanogaster: the band-interband question.

Native salivary gland chromosomes of Drosophila melanogaster, isolated without exposure to acid fixatives, have been examined in regions 1A-3B, 15A-17B, 19B-20D and 71E-73A and reveal improved aspects of preservation at the ultrastructural level. Three main points emerge: fine bands are well preserved allowing detection of some not recorded in maps made on classical acid-fixed preparations. Structures with the morphology of putative nascent ribonucleoprotein (RNP) particles are apparent in puffs, diffuse bands and virtually all interbands observed. At this level the morphology of native chromosomes is consistent with the hypothesis that all decondensed regions are members of a continuum of transcriptionally active structures. This notion is relevant to data obtained from other approaches to the band-interband question. (iii) Although the chromosomes have not been exposed to 45% acetic acid, at least some of the dark bands represented by the Bridges as doublets in their classical maps contain vacuoles which include putative RNP particles.

Isolation and characterization of Bacteroides nodosus fimbriae: structural subunit and basal protein antigens.

We examined the isolation of fimbriae from Bacteroides nodosus. It was found that the best preparations were obtained from the supernatant of washed cells cultured on solid medium, from which fimbriae could be recovered in high yield and purity by a simple one-step procedure. Analysis of such preparations by sodium dodecyl sulfate gel electrophoresis showed that greater than 98% of the protein consisted of fimbrial structural subunits whose molecular weight was ca. 17,000. These preparations also usually exhibited minor contamination with a polypeptide of ca. 80,000 molecular weight, as well as trace amounts of lipopolysaccharide. Attempts to release additional fimbriae by the traditional means of subjecting the bacterial cells to physical stress, such as shearing or heating, resulted primarily in an increase in the level of contamination, without significant gain in the yield of fimbriae. Removal of the 80,000-dalton component could not be achieved by any of a variety of techniques normally used in fimbriae purification, including isoelectric precipitation, MgCl2 precipitation, and CsCl gradient ultracentrifugation, implying a direct physical association with the fimbrial strand. Electron micrographs of fractions containing this protein show cap-shaped structures attached to the ends of what appeared to be fimbrial stubs. These observations suggest that the 80,000-dalton polypeptide may actually constitute the basal attachment site which anchors the fimbria to the outer membrane, analogous to a similar protein recently described in enterotoxigenic strains of Escherichia coli. In B. nodosus, this 80,000-dalton protein is a major surface antigen, and like the fimbrial subunit, exhibited variation in electrophoretic mobility between serotypically different isolates.

Nucleosome repeat structure is present in native salivary chromosomes of Drosophila melanogaster.

The regularly repeating periodic nucleosome organization is clearly resolved in the chromatin of the isolated salivary chromosomes of Drosophila melanogaster. A new microsurgical procedure of isolation in buffer A of Hewish and Burgoyne (1973, Biochem. Biophys. Res. Commun., 52:504-510) yielded native Drosophila salivary chromosomes. These chromosomes were then swollen and spread by a modified Miller procedure, stained or shadowed, and examined in the electron microscope. Individual nucleoprotein fibers were resolved with regularly repeated nucleosomes of approximately 10 nm diameter. Micrococcal nuclease digestion of isolated salivary nuclei gave a family of DNA fragments characteristic of nucleosomes for total chromatin, 5S gene, and simple satellite (rho = 1.688 g/cm3) sequences.

Ultrastructure of polytene chromosomes of Drosophila isolated by microdissection.

Drosophila polytene chromosomes prepared by a new micromanipulative procedure, which avoids acid squashing, have been examined at the ultrastructural level in the electron microscope. Puffs at 2B, 68C, 74EF, 75B and 85EF, have been examined in some detail, along with the chromocentre and various interbands. The ultrastructure of these chromosomes, which have never been exposed to acid protein denaturants, compares favourably with that of classical acid-fixed specimens. Ribonucleoprotein particles in puffs are seen to be organized in linear arrays and evidence is adduced for looped transcription units. Particles with characteristic sizes and morphologies are observed near the chromocentre, in puffs and in interbands. In interbands RNP particles and 'superbead'-like chromatin particles may be distinguished. Drosophila polytene chromosomes isolated by micro-manipulation should prove useful for the localization of native chromosomal proteins at an ultrastructural level.

An electron-microscope study of the lampbrush chromosomes of the newt Triturus cristatus.

The ultrastructure of lampbrush chromosomes has been examined in sections of end-embedded spread preparations, where the nuclear sap was dispersed prior to fixation, and in oocyte nuclei fixed entire, in 60-kV and i-MV electron microscopes. In spread preparations the axial chromomeres are seen to be organized as regularly spaced, unravelled skeins of DNP, each with a skein width of some 30 nm, though in some chromomeres there are regions where the DNP is much more densely packed. In both unravelled and dense regions, the 'ultimate' DNP fibres, wherever they can be identified, appear to be some 5 nm wide and thrown into loose coils. The unravelled state, although it clearly reflects an orderly packaging of the non-transcribing DNP, is an artifact of preparation; in sections of entire nuclei all chromomeres are seen to consist of DNP fibrils in the more densely packed state. The interchromeric fibril is single, and some 10 nm or less wide; it shows no sign of transcriptional activity. In sections of end-embedded preparations the RNPmatrix of most lateral loops, where transcription occurs, is seen to be made up of particles, each uniformly some 30 nm in diameter and strung together in linear array. These RNP particles are equally evident in sections of whole nuclei. In many loops the strings of particles are wound back on themselves to form regularly spaced, dense aggregates, each some 200-300 nm wide or wider; the larger aggregates can be resolved in the light micrpscope. The RNP particles are of the same dimensions throughout the lengths of individual lateral loops, and of substantially the same dimensions in loops of different gross morphologies. It is suggested that as each successive short length of RNA is transcribed from loop axis DNA, a protein associates with this RNA and winds it up to form a 'manageable' package, allowing transcription to proceed.