Heparin binding to monodisperse plasma fibronectin induces aggregation without large-scale changes in conformation in solution.

Plasma fibronectin was purified by gelatin affinity chromatography in the absence of urea and studied by photon correlation spectroscopy. Polydispersity in the observed translational diffusion coefficient (D20,w) was minimized by subsequent gel permeation fast protein liquid chromatography (FPLC) on Superose 6, which separated fibronectin monomers (D20,w = 2.15 +/- 0.03 x 10(-7) cm2 sec-1; polydispersity 5.2%) from aggregates. Addition of heparin to FPLC-purified fibronectin, at physiological pH, ionic strength and temperature, induced fibronectin aggregation, as shown by an increase of up to 60% in the static light-scattering intensity. Additional changes induced by heparin were an approximate 40% decrease in D20,w and an increase in polydispersity to 33%. After removal of aggregates by FPLC, the translational diffusion coefficient for fibronectin monomers was unaffected by the presence of heparin, in conditions where fluorescence polarization with fluoresceinamine-labelled heparin showed that 80% of the available heparin binding sites on fibronectin were occupied. Small differences in the circular dichroism spectrum of gelatin affinity-purified fibronectin were observed before and after removal of aggregates by gel permeation FPLC, and similar changes were seen when heparin was added to FLPC-purified fibronectin, without subsequent removal of aggregates. The results demonstrate the importance of minimizing polydispersity in the biophysical analysis of fibronectin in solution. We conclude that heparin binding to monomeric fibronectin occurs without large-scale changes in the conformation of the fibronectin molecule, although the possibility of more extended conformations in aggregated forms of fibronectin cannot be excluded.

Chick corneal development in vitro: diverse effects of pH on collagen assembly.

In vivo, the embryonic chick corneal epithelium lays down a stroma of collagen and proteoglycans whose fibrils are unusual as their diameter distribution peaks sharply about a mean of 20 nm. Such epithelia cultured on Nuclepore filters will also lay down a stroma containing 20 nm diameter fibrils, although there is only limited orthogonal organisation. We report here that collagen fibril morphology is critically dependent on the pH of the medium in which the corneal epithelium is cultured and that normal 20 nm diameter fibrils only assemble in a narrow band around neutral pH (approx. 6.9-7.4). At higher pH (7.6-8.1), fibrils in the distal region of the stroma more closely resemble those seen in non-corneal stroma as their diameters can be up to 200 nm even though fibrils near the basal lamina are only about 10 nm in diameter. At low pH (approx. 6.5), there are again wide fibrils, but with the hieroglyphic cross-sections typical of those seen in heritable disorders of N-terminal procollagen processing. Biochemical analysis by SDS-PAGE and fluorography confirms that N-terminal procollagen processing is deficient at this pH. At very low pH (approx. 5.8-6.2), there is little processing of procollagen and the stroma comprises filamentous material with the occasional banded structures typical of those formed by unprocessed procollagen at high concentration. Gel electrophoresis and peptide mapping showed that the collagens produced by the corneal epithelium of the primary stroma included types I, II and V and that total collagen production, as assessed by incorporation of [3H]proline, increased with pH, although the relative amounts of the different collagens produced remained essentially unchanged. While the biochemical data can account for the altered morphologies in the pH range 5.8 to 7.0, the sensitivity of fibril diameter to small changes around neutral pH remains unexplained, but points to the subtle, charge-based interactions necessary for the formation of 20 nm diameter fibrils in the developing cornea.

Procollagen binding to sphingomyelin.

The interactions of [3H]procollagen I with various phospholipids were studied by density gradient centrifugation. At physiological conditions of pH, ionic strength, and temperature, there was no evidence for procollagen binding to phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine liposomes. In contrast, procollagen I bound strongly to sphingomyelin liposomes in a reversible and saturable manner, with an apparent dissociation constant (Kd) of 2.6 nM. Binding occurred over a range of temperatures (4-37 degrees C) and was relatively unaffected by salt concentrations up to 1.2 M NaCl. Binding was observed in phosphate buffers, but not in the presence of high concentrations of Tris or Hepes. Bovine serum albumin had no effect on procollagen binding to sphingomyelin, and neither did unlabeled type I collagen, with or without the nonhelical telopeptides. Procollagen II and denatured procollagen I also bound to sphingomyelin. Procollagen binding to sphingomyelin at 35 degrees C was considerably reduced when small amounts of phosphatidylcholine were present, although binding was partially restored when the temperature was reduced below the corresponding phase transition temperature. Purified unlabeled procollagen COOH-terminal propeptides successfully competed for binding, and 125I-labeled COOH-terminal propeptides bound to sphingomyelin in the absence of procollagen. Weaker binding to sphingomyelin, mediated by the collagen triple helical region, was also observed; but this was dominated by the sphingomyelin-COOH-terminal propeptide interaction. The data suggest a novel mechanism for matrix vesicle-mediated biomineralization.

Decreased major urinary protein in male Bar Harbor 129 REJ dystrophic mice indicates a hormonal deficiency.

A significant decrease in major urinary protein (MUP) in adult male Bar Harbor 129REJ dystrophic mice correlated with a marked decrease in the amount of translatable MUPmRNA in the liver. Previous investigations have shown that MUP synthesis is under complex multihormonal regulation suggesting that the dystrophic mouse may have a hormonal deficiency.

Elevated plasma levels of haptoglobin in Duchenne muscular dystrophy:electrophoretic variants in patients with a severe form of the disease.

Analysis of plasma proteins of Duchenne muscular dystrophy (DMD) patients and normal age-matched boys using two dimensional gel electrophoresis and densitometry indicated a three-fold increase in the average haptoglobin level in DMD plasma. Two electrophoretic variants of haptoglobin in which the alpha 2 chain had more basic or more acidic spots were found in DMD patients with a rapidly progressing form of the disease. The basic variant was present in relatively low amount. Possible reasons for the elevated haptoglobin levels are discussed.

Myelin proteins and collagen in the spinal roots and sciatic nerves of muscular dystrophic mice.

Proteins of lumbosacral spinal roots and sciatic nerves of adult dystrophic mice (Bar Harbor 129REJ dydy) were analyzed by SDS gel electrophoresis to determine if identifiable proteins were affected. All peripheral nerve myelin proteins (P0 glycoprotein, P1 and Pr basic proteins, X protein and a high-molecular-weight protein) were decreased in the roots but not in the sciatic nerves. Central nervous system myelin proteins were not increased in either roots or sciatic nerves. Although dystrophic spinal roots and sciatic nerves contained less collagen, Type I, III and V collagens were present.

Sex chromosome associated satellite DNA: evolution and conservation.

Satellites visible in female but not in male DNA were isolated from the snakes Elaphe radiata (satellite IV, p = 1.708 g x cm-3) and Bungarus fasciatus (BK1 minor, p = 1.709 g x cm-3). The satellites cross hybridize. Hybridization of 3H labelled nick translated BK minor satellite DNA with the total male and female DNA and/or chromosomes in situ of different species of snakes revealed that its sequences are conserved throughout the snake group and are mainly concentrated on the W chromosomes. Snakes lacking sex chromosomes do possess related sequences but there is no sex difference and visible related satellites are absent. The following conclusions have been reached on the basis of these results. 1. The W chromosome associated satellite DNA is related to similar sequences scattered in the genome. 2. The origin and increment in the number of the W satellite DNA sequence on the W chromosome is assoicated with the heterochromatinization of the W. 3. Satellite sequences have become distributed along the length of the W and resulted in morphological differentiation of sex chromosomes. 4. Evolutionary conservation of W satellite DNA strongly suggests that functional constraints may have limited sequence divergence.

Primate repetitive DNAs: evidence for new satellite DNAs and similarities in non-satellite repetitive DNA sequence properties.

Repetitious DNA sequences have been isolated from a number of the primates in in both Suborders Anthropoidea and Prosimii by hydroxy-apatite chromatography at a Cot of 10. In addition to finding previously unreported possible AT-rich satellite DNAs in Orangutan, Gibbon, Rhesus and Slow Loris a clear similarity to human DNA was found in the nonsatellite repetitious DNA sequence properties of the primates in the Suborder Anthropoidea. This is based on the presence of the hydroxyapatitie isolated 1.703 and 1.714 g/cm3 DNA families in CsCl gradients in the analytical ultracentrifuge following renaturation and extensive DNA hyperpolymer network formation. Within the superfamily Hominoidea the amount of the 1.714 g/cm3 DNA family was greater than that of the 1.703 g/cm3 DNA family while the reverse situation was true within the Superfamily Cercopithecoidea. The orangutan 1.703 and 1.714 g/cm3 DNA families were shown to exhibit the same differential reassociation behavior demonstrated previously in human DNA (Marx et al., 1976a). These data are interpreted as preliminary evidence for a similar sequence organization in the Order Primates Suborder Anthropoidea.

Behaviour of sex chromosome associated satellite DNAs in somatic and germ cells in snakes.

Sex chromosome associated satellite DNAs is isolated from the snakes Elaphe radiata (sat III) (Singh et al., 1976) and Bungarus fasciatus (Elapidae) (minor satellite) are evolutionarily conserved throughout the suborder Ophidia. An autosome limited satellite DNA (B. fasciatus major satellite) is not similarly conserved. Both types of satellites have been studied by in situ hybridisation in various somatic tissues and germ cells where it has been observed that the W sex chromosome remains condensed in interphase nuclei. In growing oocytes however, the W chromosome satellite rich heterochromatin decondenses completely whilst the autosomal satellite rich regions remain condensed. Later, the cycle is reversed and the W chromosome condenses whilst the autosomal satellite regions decondense. In a primitive snake (Eryx johni johni) where the sex chromosomes are not differentiated and where there is no satellite DNA specific to them, these phenomena are absent. - The differential behaviour of autosomal and sex chromosome associated satellite DNAs is discussed in the light of gene regulation.

Conservation and chromosomal localization of DNA satellites in balenopterid whales.

DNA satellites were isolated from three balenopterid species, viz. the minke, sei, and fine whales. In each of them at least two DNA satellites were recognizable with buoyant densities in neutral CsCl of rho = 1.702/1.703 and rho = 1.710/1;711, respectively. cRNAs from each satellite group were used for filter and in situ hybridisations. Homo-and heterologous DNA-cRNA hybrids within each satellite group yielded virtually identical melting curve profiles showing conservation of at least a considerable part of the DNA satellite sequences. There was no evident sequence homology between the rho = 1.702/1.703 and the rho = 1.710/1;711 satellites by filter hybridisation.--The in situ hybridisation showed that in each species the rho = 1.702/1.703 satellite was located in centromeric-paracentromeric C-bands in a few pairs, whereas the rho = 1.710/1.711 satellite was located in terminal C-bands throughout the karyotypes.--The data on the whale DNA satellites indicate that the quantitative evolution of the sateliite DNA sequences preceded species divergence of the balenopterids and that the satellite sequences have remained relatively unaltered since the divergence took place. The function of satellite DNA is considered to imply the introduction of both chromosomal and genic polymorphisms and thus being of great importance in speciation, Based upon these concepts a model is postulated for the function of satellite DNA. According to this model at meiotic pairing euchromatinheterochromatin overlapping between homologous chromosomes is considered to be of a general occurrence. This overlapping is presumed to be accentuated by the size heteromorphism frequently observed between homologous heterochromatic segments (C-bands). In the region of such euchromatinheterochromatin overlapping, cross-over would be excluded. The overlapping is suggested to be rectified progresssively in the chromosome arms, leaving unaffected crossing-over distant to the euchromatin-heterochromatin junctions. The consequence of this will be that genes in the proximity of the junctions are collectively inherited and selected, whereas genes distant to the the heterochromatin will be independently assorted and selected.

Effect of different denaturing agents on the detectability of specific DNA sequences of various base compositions by in situ hybridisation.

In situ hybridisation of certain AT rich and GC rich satellite DNA complementary RNAs (cRNAs) to their homologous chromosomes at their respective optimal rate temperatures (TOPTS) after denaturation with various reagents (0.2 N HCl, 0.07 N NaOH, 90% formamide and heat) led to the following conclusions. -- Heat denaturation of chromosomal DNA in 0.1 X SSC at 100 degrees C gives significantly higher grain counts regardless of DNA base composition, HCl denaturation discriminates markedly against GC rich DNA. Chromosome morphology is best preserved after HCl and heat denaturation.

Satellite DNA and evolution of sex chromosomes.

The satellite DNA (SATELLITE III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (delta equals 1.700 g cm-3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.

The chromosomal localisation of satellite DNA in Ptyas mucosus (Ophidia, Colubridae).

Ptyas mucosus male DNA has a repetitious DNA satellite (p = 1.700 g cm-3) constituting 5% of the haploid genome. In situ hybridisation of radioactive complementary RNA (cRNA) has revealed that satellite sequences are located in the centromeric region of one pair of macrochromosomes and in the terminal region of 8 pairs of microchromosomes. These regions are constitutively heterochromatic as revealed by C-banding. The possibility of involvement of satellite rich microchromosomes in nucleolus organisation is discussed.

Influence of temperature on the detectibility and chromosomal distribution of specific DNA sequences by in situ hybridisation.

Hybridising certain AT-rich satellite complementary RNAs (cRNAs) to their homologous chromosomal DNA sequences at different temperatures of incubation results in a different dispersion of autoradiographic label throughout the karyotypes. The temperature at which most label, or cRNA-DNA hybrid formation, exists corresponds to the optimal rate temperature for the hybridisation of these same satellite cRNA-DNA hybrids as determined by RNA excess filter hybridisation. It is likely that the in situ hybridisation results can therefore be explained by the fact that there is a similar temperature-dependence on the rate of hybrid formation for both in situ and RNA excess hybridisation. This should have important implications for the designing of in situ hybridisation experiments in general.