The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.     

In vitro replication of DNA containing either the SV40 or the polyoma origin

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).     

Wheat germ agglutinin binding sites on human urothelial cells of different grades of transformation

¹²⁵I-Wheat germ agglutinin (WGA) binding parameters of human urothelial cell lines of different grades of transformation (TGrll and TGrlll) were compared. The values of association constant (K_a) and the number of binding sites/cell for HCV29 (TGrll) cell line were about 3×10⁶M^–1 and over 4×10⁷, respectively. Two TGrlll cell lines, HCV29T and Hu549 revealed lower values for K_a, and considerably higher numbers of binding sites/cell (about 3×10⁸ and 2×10⁸, respectively). Binding of¹²⁵I-WGA to total cellular proteins resolved by SDS-PAGE and transferred to nitrocellulose showed multiple diffused bands in the range of 58–180 kDa. Some of these bands were characteristic for TGrll cells (124 kDa) or TGrlll cells (135 and 148 kDa).Abbreviations TGr transformation grade - WGA wheat germ agglutinin - sWGA succinylated wheat germ agglutinin - GlcNAc N-acetyl-d-glucosamine - BSA bovine serum albumin - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

MICROSPOROGENESIS AND TAPETAL DEVELOPMENT IN NORMAL AND MALE-STERILE CARROTS (DAUCUS CAROTA)

Zenkteler , Maciej . (U. Adam Mickiewicz, ul. Stalingradzka 14, Poznan, Poland.) Microsporogenesis and tapetal development in normal and male-sterile carrots (Daucus carota). Amer. Jour. Bot. 49(4): 341–348. Illus. 1962.—Meiosis and anther development proceed normally in fertile plants. Nine pairs of chromosomes are present at diakinesis and at metaphase I. The mature pollen grains possess 2 male gametes at the time of shedding; 80–92% of the pollen appears normal. A cross-shaped configuration at pachytene characteristic of a reciprocal translocation is present in the completely pollen-sterile plants indicating that one of the parents is homozygous for an interchange between 2 members of the chromosome complex. Chromosome bridges with fragments at anaphase I and anaphase II lead to aberrant chromosome distribution during meiosis. Complete microspore abortion is associated with a periplasmodium formation of the tapetum and anther wall deterioration.     

Increased Neural Cell Adhesion Molecule in the CSF of Patients with Mood Disorder

Abstract: Neural cell adhesion molecule (N-CAM) is involved in cell-cell interactions during synaptogenesis, morphogenesis, and plasticity of the nervous system. Disturbances in synaptic restructuring and neural plasticity may be related to the pathogenesis of several neuropsychiatric diseases, including mood disorders and schizophrenia. Disturbances in brain cellular function may alter concentrations of N-CAM in the CSF. Soluble human N-CAM proteins are detectable in the CSF but are minor constituents of serum. We have recently found an increase in N-CAM content in the CSF of patients with schizophrenia. Although the pathogenesis of both schizophrenia and mood disorders is unknown, ventriculomegaly, decreased temporal lobe volume, and subcortical structural abnormalities have been reported for both disorders. We have therefore measured N-CAM concentrations in the CSF of patients with mood disorder. There were significant increases in amounts of N-CAM immunoreactive proteins, primarily the 120-kDa band, in the CSF of psychiatric inpatients with bipolar mood disorder type I and recurrent unipolar major depression. There were no differences in bipolar mood disorder type II patients as compared with normals. There were no significant effects of medication treatment on N-CAM concentrations. It is possible that the 120-kDa N-CAM band present in the CSF is derived from CNS cells as a secreted soluble N-CAM isoform. Our results suggest the possibility of latent state-related disturbances in N-CAM cellular function, i.e., residue from a previous episode, or abnormal N-CAM turnover in the CNS of patients with mood disorder.     

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II ( PS II ) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl-washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl₂, (or urea + NaCl)-washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl₂, or (urea + NaCI)-treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.     

Notes on ecotone attributes and functions

We attempt to identify general properties of ecotones. Earlier attempts to do so encountered difficulties resulting from contradictory conceptions of ecotones. Thus, we begin with and center our discussion on a definition of ecotone. The definition is complex. It includes scaling, structural, and functional aspects. Based on this complex definition, we offer a brief review of what is an ecotone, what attributes it has, and how it influences other habitats of interest. We identify feedback as a possibly important but ignored function of ecotones. This discussion is presented in general terms which apply to a variety of ecological situations. We point out that results of an evaluation of ecotone attributes largely depends on the spatial and temporal scale at which ecotone is conceptualized and data are collected. We suggest that some of ecotone determinants scale naturally in a repeatable fashion among various aquatic systems. Finally, we point to the concentration of dynamic properties of ecotones as applied to land/water interface.     

Replication variants of the human inactive X chromosome

Replication variants of the inactive X chromosome were investigated in lymphocytes from six donors by means of terminal BrdU or thymidine incorporation. There were interindividual differences in the incidence of particular variants. In endoreduplicated and tetraploid cells both allocyclic X chromosomes showed the same replication sequence. The Xp22 band of the allocyclic X chromosome seemed to replicate later than the homologous material in some cells. Initiation time of DNA synthesis within the inactive X chromosome was found to be stable; termination time, however, varied greatly relative to the other chromosomes. Early completion of replication within the heterochromatic X chromosome could be demonstrated preferentially for the Xq25–27 terminal sequence, but other variants expressed the phenomenon also. A variable replication rate of the inactive X chromosome is believed to be responsible for its asynchronous, independent replication. The biological significance of the phenomenon is discussed with respect to cell differentiation.