Structural properties of barley nucleosomes

The structural properties of barley oligonucleosomes are investigated and compared to those of rat liver oligomers. Extraction of barley chromatin was performed using mild nuclease digestion of isolated nuclei leading to a low ionic strength soluble fraction. Oligonucleosomes were fractionated on sucrose gradients and characterized for DNA and histone content. Physico-chemical studies (sedimentation, circular dichroism and electric birefringence) showed that barley oligonucleosomes exhibit properties very close to those of the H1-depleted rat liver counterparts. Moreover, in situ, barley linker DNA was more sensitive to micrococcal nuclease digestion than that of rat liver. These results suggest that barley oligonucleosomes show a less compact structure than their rat liver counterparts and appear to be in contradiction with the very condensed organization of barley chromatin previously suggested.     

Distribution and conformation of crystalline nigeran in hyphal walls of Aspergillus niger and Aspergillus awamori.

Hyphal walls of Aspergillus awamori containing increased amount of the alpha-glucan, nigeran, became correspondingly more opaque when viewed in the electron microscope as shadowed preparations. However, increased polymer deposition was not accompanied by any significant change in wall thickness. The nigeran of both A. awamori and Aspergillus niger occurred in situ in a crystalline conformation identical to that of single crystals prepared with pure polysaccharide. Furthermore, this polymer was the dominant crystalline material in the hyphae whether or not they were enriched in nigeran. Enzymic digestion of nigeran in A. niger and A. awamori revealed that the bulk of the polymer was exposed to the cell's exterior. However, a certain fraction was accessible to enzymic attack only after the wall was treated with boiling water. A third portion, detectable only by x-ray diffraction, was associated with other components and could not be extracted, even with prolonged boiling. It was removed by hot, dilute alkali and was associated in the wall with another glucan fraction. Dry heating of A. niger walls altered their susceptibility to enzymic digestion of nigeran in situ. It is proposed that this treatment introduces interstices in the crystal surface that facilitate attack.     

A simplified procedure for the concentration,desalting, and thin-layer chromatography of mevalonic acid from reaction mixtures used to assay 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity

A procedure has been described for the quantitative isolation of [¹⁴C]-mevalonic acid from reaction mixtures used for the assay of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. It consists of absorbing the reaction mixtures on Whatman No. 4 filter-paper supports and concentrating the radioactive substrate and the product within a 2-mm² area of the paper by two-dimensional elution with nonpolar solvents. This procedure simultaneously results in desalting of the reaction mixture, thus facilitating an excellent thin-layer chromatographic separation of mevalonolactone uncontaminated by the radioactive substrate. Among other advantages of the method are (a) quantitative extraction of mevalonolactone, thus avoiding the necessity of using an internal standard; (b) possibility of simultaneous processing of a large number of samples with the elution being carried out overnight without frequent supervision; (c) simplicity of the technical operations involved; and (d) inexpensiveness of the materials needed for analysis.     

Low field electric birefringence study of monovalent cation-DNA interactions

The effects of monovalent cations on DNA have been studied using static and dynamic electric birefringence. Kerr's law is obeyed in a limited E range (<30 Vcm_?1) and the steady state birefringence values are close for the different cations. The birefringence kinetics have been analysed in terms of three relaxation times. On a semilogarithmice plot of Δn(t), the tail of the curve is linear over a wide range of time for Na⁺, K⁺, NH₄⁺ and Li⁺. Only for Cs⁺ solution is no linear part found and a much longer relaxation time is determined. This only contributes a small part of the total birefringence. With Cs⁺ this contribution is more field-dependent than for the other cations and we observe a larger molecular flexibility. On the other hand, with Li⁺ a greater stiffness of the DNA molecule appears. The electrical polarizabilities anisotropies decrease in the order: Cs⁺ >NH⁺₄ >K⁺ >Na⁺ >Li⁺. There are no significant differences in the optical anisotropy factors.     

3beta-Hydroxysteroid dehydrogenase activity in human fetal membranes.

A 3beta-hydroxysteroid dehydrogenase (3betaHSD) was demonstrated in term human fetal membranes (chorion and amnion) with both dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3beta-hydroxy-5-pregnen-20-one as substrates, and the subcellular distribution substrate and nucleotide specificity of the enzyme was studied. In both membranes the microsomal fraction (particles which sedimented at 105,000 g after 90 min) had the highest specific activity. The chorion was more active than the amnion but the enzyme in both tissues had similar substrate and nucleotide specificity. NAD was the preferred cofactor, and pregnenolone was a better substrate than dehydroepiandrosterone in the presence of NAD. However, with NADP as cofactor both steroids were equally good substrates. When the 3beta-hydroxysteroid dehydrogenase activity of chorion microsomes was compared with that of placental microsomes, the specific activities were found to be of the same order of magnitude, and the substrate, nucleotide specificity and steroid binding properties were almost identical.     

Sequences homologous to Tc(s) transposable elements ofCaenorhabditis elegans are widely distributed in the phylum nematoda

Summary To have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements fromCaenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found inCaenorhabditis and inTeratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and inBursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited toC. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.     

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

Summary We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145–167, 173–187, 197–226, 237–300, 311–329, and 367–387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K 12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding. The fact that the N-terminal third of the protein is highly conserved is in agreement with the idea that it is part of, or constitutes, the pore domain located within the transmembranous channel and that it is not accessible from the cell surface.     

Gene structure of mouse cathepsin B

The structure of a genomic DNA fragment encoding mouse cathepsin B was characterized. The genomic insert spans 15 kbp and contains 9 exons encoding the 339 amino acid residues of mouse preprocathepsin B. Intron break-points are not found at the junctions of the pre-peptide, pro-peptide and mature enzyme. Like other cysteine proteinase genes, the region around the cysteinyl active site is split by an intron, but in contrast with cathepsins L and H the intron break-point is located immediately after the active site.     

Protein turnover and proliferation. Turnover kinetics associated with the elevation of 3T3-cell acid-proteinase activity and cessation of net protein gain.

1. At least 95% of the total protein of A31-3T3 cell cultures undergoes turnover. 2. First-order exponential kinetics were used to provide a crude approximation of averaged protein synthesis, Ks, degradation, Kd, and net accumulation, Ka, as cells ceased growth at near-confluent density in unchanged Dulbecco's medium containing 10% serum. The values of the relationship Ka = Ks - Kd were : 5%/h = 6%/h - 1%/h in growing cells, and 0%/h = 3%/h - 3%/h in steady-state resting cells. 3. As determined by comparison of the progress of protein synthesis and net protein accumulation, the time course of increase in protein degradation coincided with the onset of an increase in lysosomal proteinase activity and decrease in thymidine incorporation after approx. 2 days of exponential growth. 4. After acute serum deprivation, rapid increases in protein degradation of less than 1%/h could be superimposed on the prevailing degradation rate in either growing or resting cells. The results indicate that two proteolytic mechanisms can be distinguished on the basis of the kinetics of their alterations. A slow mechanism changes in relation to proliferative status and lysosomal enzyme elevation. A prompt mechanism, previously described by others, changes before changes in cell-cycle distribution or lysosomal proteinase activity. 5. When the serum concentration of growing cultures was decreased to 1% or 0.25%, then cessation of growth was accompanied by a lower steady-state protein turnover rate of 2.0%/h or 1.5%/h respectively. When growth ceased under conditions of overcrowded cultures, or severe nutrient insufficiency, protein turnover did not attain a final steady state, but declined continually into the death of the culture.