Statistical mechanics of DNA topoisomers. The helical worm-like chain

Recent experimental data of Shore & Baldwin (1983b) and of Horowitz & Wang (1984) for the apparent twisting coefficient K, which determines the breadth of the Gaussian distribution of DNA topoisomers with different linking numbers N, show that the product of K and nbp (the number of base-pairs) is nearly a constant for nbp approximately greater than 2000, but that it increases sharply with decreasing nbp for nbp approximately less than 2000. The main purpose of the present paper is to explain theoretically such behavior of K as a function of nbp. Thus the statistical mechanics of DNA topoisomers in general is developed on the basis of a twisted worm-like chain, i.e. a special case of the helical worm-like chain. The previous treatments of the N-dependent ring-closure probability, i.e. the distribution of N, which are valid only for small chain length L, are extended to the range of larger L. The variance of N is then shown to be exactly the sum of those of the writhe Wr and the twist Tw. For small values of L, the distribution of Wr is not Gaussian, and its variance or moment (Wr2) increases rather steeply with increasing L. With these and known Monte Carlo results for freely jointed chains, an empirical interpolation formula for (Wr2) is also constructed to be valid for all values of L. It predicts that (Wr2)/L increases monotonically, with increasing L to its coil-limiting value. On the other hand, the distribution of N is actually Gaussian in the practical range of N for all values of L. The conditional distribution of Wr with N fixed is also evaluated. Finally, K is expressed in terms of the torsional constant C, the stiffness parameter lambda-1 (which is equal to the Kuhn segment length and twice the persistence length for this special case), and (Wr2). The derived equation predicts that nbpK decreases monotonically to its coil-limiting value with increasing nbp. This decrease arises from the fluctuation in Wr and its neglect leads to an underestimate of C by 7 to 10%, even for short DNA with nbp approximately equal to 200. From an analysis of the experimental data of the two groups, the estimates of C = 3.1 to 3.2 X 10(-19) erg cm and lambda-1 = 1000 to 1200 A are obtained.     

辣椒净光合速率Hayman双列杂交分析

用辣椒(Capsicum annuum L.)6个亲本,按(1/2)n(n-1)双列杂交法配制15个杂交组合,用Hayman双列杂交分析法估算不同开花结果时期净光合速率的遗传参数。阵列协方差(Wr)对阵列方差(Vr)的回归分析结果表明,辣椒开花结果前期、中期、后期净光合速率的遗传都不符合“加性-显性”模型。Wr Vr与亲本Yr间的相关分析表明含有更多高净光合速率,显性基因的亲本具有较大的Wr Vr值。遗传参数估算表明开花结果时期的净光合速率遗传是显性效应比加性效应更加重要,同时还存在显著上位性效应。狭义遗传力较小,开花结果中、后期杂种优势比前期明显。     

不同土壤水分条件下紫藤叶片生理参数的光响应

测定了不同土壤湿度下2年生紫藤叶片光合速率（P_n）、蒸腾速率（T_r）及水分利用效率（WUE）等生理参数的光响应过程,探讨了紫藤正常生长发育所需的土壤水分和光照条件.结果表明:紫藤叶片的P_n、T_r及WUE对土壤湿度和光照强度的变化具有明显的阈值响应.维持紫藤正常生长（同时具有较高P_n和WUE）的土壤湿度范围为:体积含水量（W_v）15.3%～26.5%、相对含水量（W_r）46.4%～80.3%,最佳土壤湿度约为W_v 23.3%、W_r 70.6%.紫藤叶片对光照环境的适应性较强,在光合有效辐射强度（PAR）为600～1 600 μmol·m^-2·s^-1时,P_n和WUE具有较高水平,饱和光强在PAR为800～1 000 μmol·m^-2·s^-1.紫藤叶片光合作用非气孔限制的发生与土壤湿度与光照强度密切相关,W_v为18.4%～26.5%、W_r为55.8%～80.3%时,光合作用主要受气孔限制,光照强度的影响较小;超出此范围后,其受光照强度的影响较大,出现由气孔限制转变为非气孔限制的PAR临界值.紫藤正常生长允许的最低土壤湿度约为W_v 11.9%、W_r 36.1%,允许最高PAR约为1 000 μmol·m^-2·s^-1,是紫藤叶片光合机构受到破坏的临界点.     

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.     

High frequency antigens of human erythrocyte membrane sialoglycoproteins, III. Studies on the EnaFR, Wrb and Wra antigens

The nature of the common erythrocyte antigens EnaFR and Wrb, that are both absent from En(a-) cells, and the rare Wra receptor, apparently encoded by an allele of Wrb, was investigated. Various modification, fractionation or cleavage products of erythrocyte membranes were used in hemagglutination inhibition assays. The EnaFR and Wrb antigens were shown to represent labile structures within the residues approx. 62-72 of the major (MN) sialoglycoprotein that require lipids, at least for complete expression of antigenic activity. During the course of these experiments, the arrangement of the MN glycoprotein's peptide chain with respect to the lipid bi-layer was also studied, using various proteinases. Furthermore, the MN glycoprotein was found to aggregate with the major membrane protein (band 3) in the presence of Triton X-100. The Wra antigen was shown to exhibit properties that differ considerably from those of the Wrb receptor. Analyses on the MN glycoprotein, isolated from the red cells of the only known Wra homozygote and two WraWrb individuals, did not reveal any amino-acid exchange within the residues 40-96 of the molecule. Therefore, the Wr locus that determines the presence or absence of the Wrb antigen on the MN glycoprotein might influence the post-translational modification of amino-acid residues, the structure of tightly bound lipids or the aggregation of the MN glycoprotein with a different protein such as band 3.     

Bleomycin-induced aggregation of presolubilized and nuclear chromatin from L1210 cells

These studies have identified a new activity of bleomycin (in addition to the well-known DNA cleavage): drug-induced chromatin aggregation. Bleomycin treatment of presolubilized chromatin from L1210 nuclei resulted in two types of effect as shown by sedimentation analysis of intact nucleoprotein. The first effect was a dose-dependent fragmentation of chromatin to mononucleosomes (12 S) and subnucleosomal fragments (under 5 S). The second effect was aggregation manifested by the generation of large chromatin particles (over 120 S) that sedimented faster than the original material (20-40 S). Bleomycin treatment of nuclei from L1210 cells resulted in a similar, almost bimodal, size distribution of drug-released chromatin fragments. Increasing levels of bleomycin produced a gradual enhancement of the amount of small fragments (under 5 S) accompanied by generation of large, aggregated particles. Aggregation caused by high drug concentrations significantly reduced the overall extent of chromatin solubilization and allowed only the release of the most degraded fragments from nuclei. The aggregation required intact nucleoprotein, since it was not detected after high-salt deproteinization of bleomycin-treated presolubilized or nuclear chromatin. The aggregation phenomenon reflects a novel activity of bleomycin which may contribute to the drug's antiproliferative properties.     

The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1] [2]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3] [4] [5] [6] [7]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) [8] [9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9] [10] [11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.     

Effect of insulin on the composition of chromatin phospholipid in rat liver cells

In vivo the action of insulin on rat liver chromatin phospholipid composition was investigated. It was shown that the hormone led to reliable increase (nearly 20%) of total phospholipid content. The same phenomenon was shown also in the fraction of active chromatin while the phospholipids content of non-active chromatin didn't changed. It was also shown that the content of three from six phospholipid fractions altered under the insulin action. The content of sphingomyelin and phosphatidilethanolamine increased and the quantity of phosphatidylinositol decreased under the hormone action. The decrease of monophosphoinositides content was accompanied by the reliable increase of triphosphoinositides amount. It was suggested that the fractions of chromatin polyphosphoinositides were redistributed under the action of insulin.     

Enniatin B synthesis by a Fusarium sambucinum Fuck culture

Three fungal strains belonging to the genus Fusarium Lk. ex. Fr. (F. sambucinum Fuck. 52377, F. avenaceum (Fr. Sacc.) 52311, F. gibbosum App. et. Wr. emend Bilai 52021) whcih form 800-1200 mg of enniatin B per litre during submerged cultivation have been selected. The morphology of F. sambucinum 52377 in the course of growth and production of enniatin B on the selected medium is described. The maximum accumulation of the product is found at the stationary growth phase. The active accumulation of fatty inclusions during this period suggests the participation of metabolism of fatty acids in the biosynthesis of enniatin B.     

Action of hydrocortisone on the lipid composition of active and inactive chromatin fractions in the rat liver

A composition of phospholipids and neutral lipids of rat liver chromatin and its active and inactive fractions has been investigated. It is shown that the hydrocortisone action results in marked increase in phospholipid/neutral lipid ratio of both chromatin and its active fraction. The changes in lipid content is clearly expressed in active chromatin fraction, the lipid content of inactive fraction is not changed. It is concluded that the increase of content in certain phospholipids and simultaneous decrease of neutral lipids in chromatin promotes the hormonal activation of genome.     

Chromatin-bound and free forms of poly(adenylic acid) polymerase in rat hepatic nuclei.

Isolated rat hepatic nuclei were shown to contain poly(A) polymerase in two distinct physiologically active forms. One form was associated with the chromatin fraction and was dependent on endogenous RNA, presumably mRNA. The other activity was localized in the nuclear sap in a 'free' form and was stimulated almost 30-40-fold by exogenously added poly(A). Isolated nucleoli were devoid of significant poly(A)-synthesizing activity.     

Formation of chromoneme-like fibrils induced in infusorian somatic nuclei by magnesium ions

A study was made of the effect of Mg2+ on higher-order chromatin structure in marconuclei (Ma) of infusoria Paramecium aurelia and Bursaria truncatella. In infusorian Ma, inactive chromatin is commonly packed in chromatin bodies sized 60-200 nm. When isolated chromatin or Ma were treated with Mg2+ (about 3 mM), chromatin bodies arranged into fibrils 100-300 nm in diameter, which resembled higher eukaryotic chromonemes. The formation dynamics of chromoneme-like fibrils was described. The results testified to the similarity of chromatin bodies to chromomeres of higher eukaryotes. Structurally intact central chromomere cores proved to be essential for the formation of chromoneme-like fibrils. Chromatin organization in infusoria was shown to follow the discrete-level model (nucleosomes-nucleomeres-chromomeres-chromonemes) assumed for higher eukaryotes.