植物大片段 DNA 的研究进展

植物大片段 DNA 的研究成为了基因组学研究的一个重要方面．对它的研究得益于容纳大片段 DNA 片段载体的发展．对构建植物大片段 DNA 的载体、植物大片段 DNA 的提取方法、植物大片段 DNA 的主要应用领域的最新进展进行了介绍．     

The kinase activity of SV40 large T antigen is mediated by a cellular kinase.

Large T antigen (large T) extracted from SV40-infected or transformed cells exhibits an in vitro protein kinase activity, whose origin and biological significance up to now had been obscure. We have addressed the questions of whether this activity is intrinsic to large T or arises by association with a cellular kinase, and, furthermore, whether this activity might play a biological role in vivo. Instead of analyzing large T from whole-cell lysates, where non-specific association of a cellular kinase(s) with large T might easily occur, we analyzed individual cellular subclasses of large T, isolated from their in vivo locations. In contrast to large T isolated from whole-cell lysates which was always kinase positive, none of the cellular subclasses of large T prepared by in situ fractionation of SV40-transformed mKSA cells exhibited detectable in vitro kinase activity. We could demonstrate that our fractionation conditions neither inactivated the large T-associated kinase activity nor dissociated it from large T when they were applied to kinase-positive large T isolated from whole-cell lysates. We conclude that large T does not contain an intrinsic kinase activity. This conclusion was further supported by our finding that it was possible to remove the large T-associated kinase activity from kinase-positive large T preparations and to reconstitute it by incubating the kinase-negative large T with cell lysates from various cell lines. Therefore, the simplest way of interpreting our results is that the in vitro kinase activity measured with large T preparations from whole-cell lysates is the result of an in vitro association of a cellular kinase(s) with large T during certain conditions of cell lysis.     

左侧大肠癌和右侧大肠癌蛋白质差异表达谱的建立及质谱分析

目的:初步探索左侧大肠癌和右侧大肠癌中蛋白质表达的差异,为左右侧大肠癌生物学特性上的差别提供功能基因组学上的依据。方法:以左右侧大肠癌组织为研究对象,提取组织总蛋白,依次进行二维凝胶电泳,凝胶图象分析,质谱及生物信息学分析。结果:成功建立了左侧大肠癌和右侧大肠癌的二维电泳图谱,进行质谱和生物信息学分析比较得到左侧大肠癌与右侧大肠癌的差异表达蛋白共有16个,其中左侧大肠癌中表达增加的蛋白有10个包括蛋白质二硫化异构酶A1,78 kDa葡萄糖调节蛋白,抑制素,热休克蛋白60,含硫氧还蛋白域的蛋白5,T-复合蛋白1ε亚单位,应急蛋白70,异柠檬酸脱氢酶,蛋白质二硫化异构酶A3,巨噬细胞加帽蛋白;左侧大肠癌表达降低的蛋白6个包括ATP合成酶β亚单位,延伸因子1-delta,热休克蛋白β1,载脂蛋白A-Ⅰ,转甲状腺素蛋白,热休克蛋白β6。结论:左侧大肠癌和右侧大肠癌中存在差异表达蛋白,这些差异表达蛋白可能是左右侧大肠癌生物学性质差异的分子遗传学基础。     

酸性同工铁蛋白、p53在大肠腺癌和大肠腺瘤组织中的表达及其意义的探讨

采用抗人胎盘酸性同工铁蛋白单克隆抗体、ｐ５３ 单克隆抗体和免疫组化方法,分别对３２ 例大肠腺癌、３０ 例大肠腺瘤和３０ 例非肿瘤性大肠粘膜组织进行检测。结果是酸性同工铁蛋白在５３．１％ 的大肠腺癌和１６．６７％ 的大肠腺瘤中为阳性表达, 二者阳性率比较存在显著性差异 （Ｐ＜ ０．０５）, 在非肿瘤性大肠粘膜组织中全部为阴性; ｐ５３ 在４３．８％ 的大肠腺癌和１３．３％ 的大肠腺瘤中为阳性表达; 在大肠腺癌组织中, 酸性同工铁蛋白与ｐ５３ 的表达符合率为７１．９％ （同为阳性者为３４．４％ , 同为阴性者为３７．５％ ）, 经统计学检验酸性同工铁蛋白的表达与ｐ５３ 的表达具有显著的相关性; 在３０ 例大肠腺瘤组织中, 酸性同工铁蛋白与ｐ５３ 同为阳性表达者３ 例, 该３ 例组织均可见非典型增生的病理变化。本研究结果表明大肠癌细胞内存在酸性铁蛋白抗原, 这些酸性铁蛋白若释放到血液中可能是造成患者血清铁蛋白水平升高的主要原因之一; 另外还提示对大肠腺瘤患者进行ｐ５３ 和酸性同工铁蛋白的检测可能作为判断其早期癌变的指标     

The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function.

Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action, showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T.     

Production of recombinant large proneurotensin/neuromedin N-derived peptides and characterization of their binding and biological activity.

Proneurotensin/neuromedin N (pro-NT/NN) is the common precursor of two biologically active related peptides, neuromedin N (NN) and neurotensin (NT). It undergoes a tissue-specific processing leading to the formation in some tissues and cancer cell lines of large peptides ending with the NT (large NT) or NN (large NN) sequence. In this study, we prepared and purified high amounts of recombinant large NT and large NN using the Drosophila S2 cell expression system. The binding and pharmacological properties of recombinant large peptides were characterized and compared to those of NT and NN using either COS cells transfected with the human subtype-1 NT receptor (hNTS1) or the human colon adenocarcinoma HT29 cell line that endogenously expresses hNTS1. Furthermore, the metabolic stability of the large peptides, when exposed to HT29 cells, was compared to that of NT and NN. Both large NT and large NN were able to bind to and activate hNTS1 with potencies that were approximately 10 times lower than that of their small counterpart. In addition, the large forms proved to be far less sensitive to degradation than the small peptides. Taken together, these data suggest that the large forms might represent endogenous, long-lasting activators of hNTS1 in a number of physiopathological situations.     

Mutational analysis of delta antigen: effect on assembly and replication of hepatitis delta virus.

Hepatitis delta virus requires a helper function from hepatitis B virus for packaging, release, and infection of hepatocytes. The assembly of large delta antigen (HDAg) is mediated by copackaging with the small surface antigen of hepatitis B virus (HBsAg), and the assembly of small HDAg requires interactions with large HDAg. To examine the molecular mechanisms by which small HBsAg, large HDAg, and small HDAg interact, we have established a virion assembly system in COS7 cells by cotransfecting plasmids encoding the small HBsAg, the small HDAg, and large HDAg mutants. Results indicate that sequences within the C-terminal 19-amino-acid domain flanking the Cxxx isoprenylation motif are important for the assembly of large HDAg. In addition, a large HDAg mutant bearing extra sequences separating the C-terminal 19-amino-acid domain from the common regions of the small and large HDAgs is capable, like the wild-type large HDAg, of copackaging with small HBsAg. The ability of assembly is also demonstrated for a large HDAg mutant from which nuclear localization signals have been removed. Furthermore, a cryptic signal within the N-terminal 50 amino acid residues other than the putative N-terminal coiled-coil structure and a subdomain between amino acid residues 50 and 65 of the large HDAg are important for the assembly of small HDAg as well as the trans-dominant negative regulation of large HDAg in hepatitis delta virus replication.     

Only a minor fraction of plasma membrane-associated large T antigen in simian virus 40-transformed mouse tumor cells (mKSA) is exposed on the cell surface.

The bulk of simian virus 40 (SV40) large T antigen in SV40-infected and -transformed cells localizes within the cell nucleus, while a minor fraction specifically associates with the plasma membrane (PM) and is exposed on the cell surface. PM-associated large T seems to span the lipid bilayer but, on the other hand, does not display typical features of a transmembrane protein. To further characterize the postulated transmembrane orientation of large T, we asked whether all large T molecules associated with the plasma membrane indeed are exposed on the cell surface. We compared the amount of cell surface-exposed large T, determined on living cells by a sensitive 3H-protein A-binding assay and by external immunoprecipitation, with that of total PM-associated large T extracted from isolated PM. We demonstrate that in mKSA cells (SV40-transformed BALB/c mouse fibroblasts), total PM-associated large T accounted for a substantial portion (ca. 2%) of total cellular large T. However, only 0.1 to 0.2% of it could be detected on the cell surface. Thus, only a minor fraction of PM-associated large T (less than 10%) is exposed on the surface of these cells. Interior PM-associated large T is stably associated with the plasma membrane, while the small fraction of surface-exposed large T is rapidly released from the cell surface.     

Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen.

Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T.     

各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding.

The effect of phosphorylation on the ability of simian virus 40 large T antigen to stimulate DNA synthesis in vitro was tested. Treatment of affinity-purified large T antigen with calf intestinal alkaline phosphatase resulted in the removal of 70 to 80% of the phosphate residues. Only serine-bound phosphate residues were affected. Phosphatase-treated large T antigen stimulated in vitro DNA synthesis fourfold over the untreated control. The stimulation was strongest at early times of DNA replication. At later times, DNA replication proceeded at equal rates with dephosphorylated and untreated large T antigen. The ATPase activity of large T antigen was not affected by phosphatase treatment. The origin-binding activity of large T antigen was tested over a wide range of large T antigen to DNA ratios, including DNA excess, and in the presence and absence of carrier DNA. Under no condition was an effect of dephosphorylation of large T antigen on its DNA-binding activity observed. These findings might indicate that phosphorylation at serine residues modulates the interaction of large T antigen with cellular factors. During DNA synthesis large T antigen was substantially rephosphorylated by kinases in the HeLa cell extract. As shown by two-dimensional peptide mapping, this phosphorylation occurred at all known in vivo sites. No phosphatase and protease activities were detectable in the HeLa cell extract.     

Identification and Precipitation of the Polyribosomes in Chlamydomonas reinhardi Involved in the Synthesis of the Large Subunit of d-ribulose-1,5-bisphosphate Carboxylase

The size classes of polyribosomes involved in the synthesis of ribulose-1,5-bisphosphate carboxylase large subunit were determined by binding radioiodinated specific antibodies to polyribosomal preparations from Chlamydomonas reinhardi. Antibodies specific to the denatured large subunit and to the native enzyme bound primarily to small polyribosomes (N = two to five ribosomes). The binding of antibodies to small polyribosomes was unexpected since the large subunit is a large polypeptide (molecular weight 55,000) coded for by a corresponding large mRNA (12-14S). Control experiments showed that this unexpected pattern of antibody binding was not a result of messenger RNA degradation, "run-off" of ribosomes from polyribosomes, or adventitious binding of the completed enzyme to a selected class of polyribosomes. In addition, polyribosomes bearing nascent large subunit chains have been immunoprecipitated from small polyribosome fractions. A large RNA species that can direct the synthesis of large subunit in vitro was extracted from small polyribosomes.     

Evidence for transmembrane orientation of acylated simian virus 40 large T antigen.

In mKSA cells (a simian virus 40-transformed BALB/c mouse tumor cell line), plasma membrane-associated large T antigen (large T) is found in two subfractions of the plasma membrane; a minor amount of large T is recovered from the Nonidet P-40 (NP-40)-soluble plasma membrane fraction, whereas the majority is tightly bound to a substructure of the plasma membrane, the plasma membrane lamina (PML). Only PML-associated large T is fatty acid acylated (U. Klockmann and W. Deppert, EMBO J. 2:1151-1157, 1983). We have analyzed whether these two forms of plasma membrane-associated large T might differ in features like cell surface expression or metabolic stability. In addition, we have asked whether one of the two large Ts might represent the hypothetic, large T-related protein T* (D. F. Mark and P. Berg, Cold Spring Harbor Symp. Quant. Biol. 44:55-62, 1979). We show that in mKSA cells grown in suspension culture, large T associated with the PML is also exposed on the cell surface. This form of large T, therefore, exhibits properties of a transmembrane protein. Large T in the NP-40-soluble plasma membrane fraction could not be labeled with radioiodine on the cell surface and, for this reason, does not seem to be oriented towards the cell surface. In contrast, when mKSA cells were grown on substratum (culture dish), we found that in these cells both NP-40-soluble large T as well as large T anchored in the PML could be cell surface iodinated. We also have analyzed the plasma membrane association of surface T antigen in mKSA cells grown in a mouse as ascites tumor. In tumor cells, only PML-bound large T is cell surface associated. We conclude that differences in extractibility of cell surface-associated large T most likely depend on cell shape and are not an artifact of cell culture. Both NP-40-soluble and PML-bound large Ts are associated with the plasma membrane in a metabolically stable fashion. Neither of the two large Ts represents T*.     

Morphological, cytochemical autoradiographic and quantitative studies of the cells in lymphocyte cultures with added tuberculin]

Lymphocyte culture experiments with purified tuberculin showed a transformation of small lymphocytes into medium sized, large and very large basophilic cells. During cell growth RNA synthesis and acid phosphatase activity increased. Some of the very large basophilic cells synthesized DNA. During the first and second day of culture experiment also medium sized and large weakly basophilic cells increased, which have a pale plasma and azurophilic granules. These cells did not synthesize DNA and showed only a low incorporation of 3H-uridine. The results of our experiments indicate a transformation of medium sized and large basophilic cells not synthesizing DNA into weakly basophilic (pale) cells showing azurophilic granules. In contrast to the lymphocyte culture experiments with tuberculin lymphocyte cultures without tuberculin showed no significant increase of medium sized, large and very large basophilic cells and of weakly basophilic cells.     

Free transfer of expanded parascapular, latissimus dorsi, and expander "capsule" flap for coverage of large lower-extremity soft-tissue defect

The coverage of large soft-tissue defects usually requires a large flap transfer, especially in a combination and expanded form. However, some large soft-tissue defects still cannot be covered by such flaps. In this article, we present a case of a civil war injury in a patient from Afghanistan who had severe trauma to the right knee, lower thigh, and upper leg and a marked soft-tissue defect. This large soft-tissue defect was covered with a large combined free flap of the expanded parascapular and latissimus dorsi muscle, including a large retrograde hinge flap of the tissue expander capsule and a complementary skin graft. The defect was covered completely, and the final result was excellent.     

The large genome constraint hypothesis: evolution, ecology and phenotype

BACKGROUND AND AIMS: If large genomes are truly saturated with unnecessary 'junk' DNA, it would seem natural that there would be costs associated ith accumulation and replication of this excess DNA. Here we examine the available evidence to support this hypothesis, which we term the 'large genome constraint'. We examine the large genome constraint at three scales: evolution, ecology, and the plant phenotype. SCOPE: In evolution, we tested the hypothesis that plant lineages with large genomes are diversifying more slowly. We found that genera with large genomes are less likely to be highly specious -- suggesting a large genome constraint on speciation. In ecology, we found that species with large genomes are under-represented in extreme environments -- again suggesting a large genome constraint for the distribution and abundance of species. Ultimately, if these ecological and evolutionary constraints are real, the genome size effect must be expressed in the phenotype and confer selective disadvantages. Therefore, in phenotype, we review data on the physiological correlates of genome size, and present new analyses involving maximum photosynthetic rate and specific leaf area. Most notably, we found that species with large genomes have reduced maximum photosynthetic rates - again suggesting a large genome constraint on plant performance. Finally, we discuss whether these phenotypic correlations may help explain why species with large genomes are trimmed from the evolutionary tree and have restricted ecological distributions. CONCLUSION: Our review tentatively supports the large genome constraint hypothesis.     

The potential for large carnivores to act as biodiversity surrogates in southern Africa

Biodiversity in southern Africa is globally extraordinary but threatened by human activities. Although there are considerable biodiversity conservation initiatives within the region, no one has yet assessed the potential use of large carnivores in such actions. Surrogate approaches have often been suggested as one such way of capitalizing on large carnivores. Here we review the suitability of the large carnivore guild (i.e., brown hyaena Hyaena hyaena, spotted hyaena Crocuta crocutta, cheetah Acinonyx jubatus, leopard Panthera pardus, lion Panthea leo and African wild dog Lycaon pictus) to act as surrogate species for biodiversity conservation in southern Africa. We suggest that the guild must be complete for the large carnivores to fully provide their role as ecological keystones. The potential for large carnivores to act as umbrella and indicator species seems limited. However, self-sustaining populations of large carnivores may be useful indicators of unfragmented landscapes. Moreover, diversity within the large carnivore guild may reflect overall biodiversity. Although the global appeal of the large African carnivores makes them important international flagships, we stress that international conservation funding must be linked to local communities for them to be important also locally. In summary, we suggest that the flagship value of these large carnivores should be used to promote biodiversity conservation in the region, and that the suggested relationship between large carnivore diversity and overall biodiversity is empirically tested. Finally we suggest that direct conservation activities should focus on enhancing the keystone values of large carnivores through complete guild conservation and restoration.     

Simian virus 40 small t antigen activates the carboxyl-terminal transforming p53-binding domain of large T antigen.

Expression of the simian virus 40 large T antigen (large T) in F111 rat fibroblasts generated only minimal transformants (e.g., F5 cells). Interestingly, F111-derived cells expressing only an amino-terminal fragment of large T spanning amino acids 1 to 147 (e.g., FR3 cells), revealed the same minimal transformed phenotype as F111 cells expressing full-length large T. This suggested that in F5 cells the transforming domain of large T contained within the C-terminal half of the large T molecule, and spanning the p53 binding domain, was not active. Progression to a more transformed phenotype by coexpression of small t antigen (small t) could be achieved in F5 cells but not in FR3 cells. Small-t-induced progression of F5 cells correlated with metabolic stabilization of p53 in complex with large T: whereas in F5 cells the half-life of p53 in complex with large T was only slightly elevated compared with that of (uncomplexed) p53 in parental F111 cells or that in FR3 cells, coexpression of small t in F5 cells led to metabolic stabilization and to high-level accumulation of p53 complexed to large T. In contrast, coexpression of small t had no effect on p53 stabilization or accumulation in FR3 cells. This finding strongly supports the assumption that the mere physical interaction of large T with p53, and thus p53 inactivation, in F5 cells expressing large T only does not reflect the main transforming activity of the C-terminal transforming domain of large T. In contrast, we assume that the transforming potential of this domain requires activation by a cellular function(s) which is mediated by small t and correlates with metabolic stabilization of p53.     

Evolutionary stability for large populations

We present a revision of Maynard Smith's evolutionary stability criteria for populations which are very large (though technically finite) and of unknown size. We call this the large population ESS, as distinct from Maynard Smith's infinite population ESS and Schaffer's finite population ESS. Building on Schaffer's finite population model, we define the large population ESS as a strategy which cannot be invaded by any finite number of mutants, as long as the population size is sufficiently large. The large population ESS is not equivalent to the infinite population ESS: we give examples of games in which a large population ESS exists but an infinite population ESS does not, and vice versa. Our main contribution is a simple set of two criteria for a large population ESS, which are similar (but not identical) to those originally proposed by Maynard Smith for infinite populations.