The effect of various conditions of chromatin isolation on the nucleosomal structure of the isolated chromatin

Chromatin from calf thymus isolated under hypotonic conditions in the presence of various agents was investigated by methods of electron microscopy prior to and after EDTA treatment. It is shown that the presence of chelating agents and, especially, the application of considerable mechanical forces in the course of isolation may cause damage to the nucleosomal structure of the chromatin. Moreover, sufficiently great mechanical forces are liable to destroy the structure of the chromatin nucleosomal fibres even when they are packed in structures of a higher order of organization of the chromatin.     

The effect of template secondary structure on vaccinia DNA polymerase.

Vaccinia virus DNA polymerase will utilize a substrate consisting of phi X174 DNA primed with a strand of a unique restriction fragment, but the reaction is inefficient. Examination of the reaction products by alkaline agarose gel electrophoresis revealed a few discrete fragments, each corresponding to an extended primer strand. This result implies that specific barriers exist on the phi X174 template which impede, but do not completely halt, the progress of the enzyme. Only a few per cent of the template molecules were completely copied. Similar findings were reported by Sherman and Gefter using Escherichia coli DNA polymerase II and fd DNA (J. Mol. Biol. (1976) 103, 61-76). Several observations suggest that the barriers are regions of template secondary structure. Some barriers are more effective than others, and they increase in both effectiveness and number as the temperature is decreased. The same barriers are observed with T4 DNA polymerase, but none are detected with E. coli DNA polymerase I. Finally, the major barriers are located in regions of the phi X174 sequence known to contain hairpin structures of relatively high stability. The exact stopping point of one of the major barriers is within the duplex stem of a hairpin structure. These results show that DNA polymerases are a useful probe of the secondary structure of a single-stranded DNA.     

The effect of heparin on structural and functional properties of low density lipoproteins

Heparin binding to human low density lipoproteins (LDL) and the effect of heparin on the ability of LDL to bind to the LDL receptor has been investigated. Emphasis has been made on the physiological conditions of temperature, pH and the ionic strength. Intrinsic fluorescence spectroscopy of LDL has been applied to follow heparin binding. Fluorescence anisotropy has been measured to describe the changes in apoB and dansyl-heparin dynamics upon binding. Eu3+-labeled LDL binding to the intact LDL receptor has been monitored by time-resolved fluorescence spectroscopy technique. We have found that heparin binds to LDL under the physiological conditions, probably by Van der Waals interactions and hydrogen bonding. Temperature seems to be the most important factor influencing the interaction. Furthermore, the presence of heparin inhibits LDL binding to the intact LDL receptor that might have consequences on the cholesterol metabolism in vivo.     

The effect of chromatin structure on cisplatin damage in intact human cells

The influence of chromatin structure on cisplatin DNA damage was investigated in intact human cells. The epsilon-globin gene promoter was utilised as the target DNA sequence and the terminal transferase-dependent PCR technique was employed to examine adduct formation at base pair resolution. It was found that cisplatin preferentially damaged at runs of consecutive guanine bases in intact cells. By comparing the relative intensity of adduct formation in intact cells and in purified genomic DNA, it was possible to assess the influence of chromatin proteins on the extent of cisplatin DNA damage. Enhanced damage in intact cells was found at the CACC site where a member of the Sp1 family of proteins is thought to bind. It is postulated that protein binding at this site bends the DNA double-helix so that enhanced cisplatin binding occurs. The altered DNA binding of cisplatin in the presence of chromatin proteins could be important in the properties of cisplatin as an anti-tumour drug.     

Exploring the dermal “template effect” and its structure

Scar formation is the problem for clinic surgery. Recent studies showed that the scar formation was closely related to the dermal defect. Three-dimensional (3-d) structures of dermal tissues act as a template to modulate cell functions that are essential the regeneration of skin structure and function. The dermal tissue’s integrity and continuity is a prerequisite for repair to take place. Loss of the dermal tissue integrity and continuity due to trauma leads to a lack of the template effect, which may be one important mechanism that hinders the recovery of cell function, resulting in scar formation. These studies give us two questions: what is the three-dimensional (3-d) structure of the dermal tissue? How do the tissues form? Up to now, it is well known that the molecular structure of collagen, the micro-structure of microfibril, however, the mesoscopic structure of dermal tissues is still unclear. Our recently rudimentary studies showed the problem might be resolved by phase-contrast micro-tomography with synchrotron radiation, which is likely to open new avenues for further investigations on wound regeneration and skin tissue engineering.     

RNA chain elongation on a chromatin template.

The rate of RNA chain elongation has been measured with DNA and chromatin as template. RNA propagation on chromatin is about 50% of the rate found with DNA. Kinetic experiments demonstrate that the inhibition is not due to interference with the addition of the nucleoside triphosphates. Analysis of the dependence of propagation on the Tm of DNA shows that the chromatin proteins interfere with the translocation of the RNA polymerase along the DNA template.     

Phenobarbital stimulation of housefly chromatin template activity

The effect of phenobarbital on the chemical composition and template activity for Escherichia coli RNA polymerase of housefly chromatin is examined. Chromatin isolated from the resistant Fc housefly strain after administration of phenobarbital has a much higher template activity as compared to control chromatin. The effect is minimal in a susceptible housefly strain. Phenobarbital treatment alters the composition of the RNA polymerase product which results in an increase of the $\text{(A + U)}\text{(C + G)}$ ratio with chromatin from the Fc strain as template, while the $\text{(A + G}\text{)}\text{(}\text{C + U)}$ and $\text{(A + U}\text{)}\text{(}\text{C + G)}$ ratios are decreased when chromatin of the susceptible strain is used.A substantial portion of the intracellular phenobarbital is physically bound to nuclei and microsomes, but most of it is associated with cytosolic macromolecules. Less than 0.2% is covalently bound to cellular macromolecules. Phenobarbital apparently binds to a small mol. wt cytosolic macromolecule(s) as shown by sucrose density gradient centrifugation and Sephadex G-25 column chromatography.     

Features of the chromatin structure of erythrocytes depending on the properties of lysine-rich histones

A comparative analysis of chromatin from erythrocytes of frog, trout and hen has been performed in correlation with properties of the nucleosomal linker histones of H1 family. In the nucleosomes from frog erythrocytes the linker histone is represented by H1(0)-like variant with amino acid sequence highly homologous to that of the hen histone H5, however the arginine content in the proteins differs (3 mol% in the frog erythrocyte H1 and 12 mol% in the hen erythrocyte H5). On the other hand histone H5 from trout being significantly different in the primary structure from the hen histone H5 is at the same time rich in arginine (9 mol%). The nucleosomal repeat length, estimated by using agarose gel electrophoresis is 201, 213 and 213 b.p. in erythrocyte chromatin from frog, trout and hen, correspondingly. Chromatin packing density in fixed nuclei from erythrocytes of frog, trout and hen as determined using cytophotometric measurements is 0.144, 0.444 and 530 pg/mu 3, correspondingly. The data support the previously made suggestion that the increase in arginine content in nucleosomal linker proteins is connected with the increase of chromatin compaction in the nuclei and elongation of the linker in the nucleosome.