原核生物DNA的程序性复制

原核生物ＤＮＡ的程序性复制薛建华明镇寰（杭州大学生命科学学院,杭州３１００１２）关键词聚合酶Ⅲ全酶复制叉程序性ＤＮＡ的复制是一个复杂的过程,需要各种蛋白质、酶之间及蛋白质与ＤＮＡ之间的相互作用,才能顺利完成。原核生物（如Ｅ．ｃｏｌｉ）是以ＤＮＡ聚合酶...     

HMG蛋白质与人ε-珠蛋白基因5′旁侧调控元件DNA相互作用

ＨＭＧ蛋白质是一类在染色质内含量极为丰富的非组蛋白质,它们在染色质的结构与功能中起着重要的作用。本文运用凝胶电泳阻抑法和ＤＮａｓｅＩ足迹法分析了ＨＭＧ蛋白质与人ε－珠蛋白基因５′旁侧调控元件ＤＮＡ之间相互作用的情况。结果表明ＨＭＧ蛋白质（１／２）既能与两个正调控元件（－５３５～－４５３ｂｐ和－４４６～－４１９ｂｐ）ＤＮＡ相结合,也能与启动子（－１７７～＋１ｂｐ）ＤＮＡ相结合;而ＨＭＧ蛋白质（１４／１７）都不能与它们相结合。相反,ＨＭＧ蛋白质（１４／１７）能与一个负调控元件（－３９２～－１７７ｂｐ）ＤＮＡ相结合,而ＨＭＧ蛋白质（１／２）却不能与它相结合。上述的结果提示了ＨＭＧ蛋白质在人ε－珠蛋白基因时空表达过程中起着积极的调控作用。     

导入小麦加倍单倍体植株的玉米DNA在后代中的遗传及序列同源性分析

作者曾报道了一个玉米（Ｚｅａ ｍａｙｓ Ｌ．）基因组特异的重复序列ＤＮＡ（克隆ＭＲ６４）通过小麦（Ｔｒｉｔｉｃｕｍ ｐｅｒｓｉｃｕｍ Ｖａｖ．ｅｘ Ｚｈｕｋ．）与玉米杂交导入一个小麦加倍单倍体（ＤＨ）植株中。利用分析主宰该重复ＤＮＡ序列可以稳定地传递到小麦ＤＨ３代植株。通过Ｉｎｔｅｒｍｅｔ在ＤＮＡ数据库中进行序列相似性搜寻和同源性比较,结果显示,ＭＲ６４的ＤＮＡ序列和玉米的最近报道的两个逆转座子Ｐ     

由小麦＊玉米获得的普通小麦加倍单倍体后代的RFLP变异

用小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）的ｒＤＮＡ克隆ｐＴａ７１和与小麦基因组有部分同源性的玉米（ＺｅａｍａｙｓＬ．）ＤＮＡ克隆作探针,对由小麦＊玉米获得的普通小麦加倍单倍体后工群体进行ＲＦＬＰ分析。     

对EcoRI作用于DNA的新认识

对ＥｃｏＲＩ作用于ＤＮＡ的新认识邹国林皮新春（武汉大学生物化学与生物物理学系,武汉４３００７２）关键词蛋白质与核酸相互作用ＥｃｏＲＩ模体蛋白质与核酸的相互作用是分子生物学研究的中心问题之一。当前以阐明ＤＮＡ结合蛋白的结构与蛋白质－ＤＮＡ复合物的结构为...     

DNA序列信息的一种新的测度

根据信息理论给出了测度ＤＮＡ序列信息的一种新的方法,获得ＤＮＡ序列４个层次的信息量测度：Ｉｂ,Ｉｆ（１）,Ｉｆ（２）ａｎｄＩｆ（３）,这４种信息测度可分别用来测度ＤＮＡ的碱基序列、密码子序列、编码蛋白质序列和功能蛋白质序列的信息量。从Ｍ．ｅｄｕｌｉｓ的线粒体基因组中两个较短的编码蛋白质的ＤＮＡ序列和使用具有不同倍性的间并密码子组组成的模拟ＤＮＡ序列中所获得计算结果表明,这些信息测度确实能用来揭示所     

反平行β－折叠──蛋白质与DNA结合的新模式

反平行β－折叠──蛋白质与ＤＮＡ结合的新模式史永昶（扬州大学农学院生化教研室,扬州２２５００９）关键词蛋白质－ＤＮＡ结合模式,反平行β－折叠,阻遏蛋白,ＨＵ蛋白蛋白质与ＤＮＡ的相互作用涉及发生在细胞中的许多基本过程,尤其与基因表达和细胞分化密切相关。...     

玉米苹果酸脱氢酶基因的分离与结构分析

以一个玉米（ＺｅａｍａｙｓＬ．）杂种一代超亲表达的ｃＤＮＡ片段为探针,从玉米幼苗期ｃＤＮＡ文库中筛选到一个全长１２８７ｂｐ的ｃＤＮＡ克隆。序列分析表明,该ｃＤＮＡ编码细胞质苹果酸脱氢酶,推导的氨基酸序列与龙须海棠（Ｍｅｓｅｍｂｒｙａｎｔｈｅｍｕｍ　ｃｒｙｓｔａｌｌｉｕｍ　Ｌ．）及拟南芥（Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）同一编码基因的氨基酸序列同源性分别为９０％和８４％。这是禾谷类作物中首次克隆的编码细胞质苹果酸脱氢酶的完整基因。     

HMG蛋白质与人ε—珠蛋白基因5‘旁侧调控元件DNA相 …

ＨＭＧ蛋白质是一类在染色质内含量极为丰富的非组蛋白质,它们在染色质的结构与功能中起着重要的作用。本文运用凝胶电泳阻抑法和ＤＮａｓｅＩ足迹法分析了ＨＭＧ蛋白质与人ε－珠蛋白基因５＇旁侧调控元件ＤＮＡ之间相互作用的情况。结果表明ＨＭＧ蛋白质（１／２）既能与两个正调控元件（－５３５￣－４５３ｂｐ和－４４６￣－４１９ｂｐ）ＤＮＡ相结合,也能与启动子（－１７７￣＋１ｂｐ）ＤＮＡ相结合;而ＨＭＧ蛋白质（１４／     

真核生物转录因子对DNA序列的识别

真核生物转录因子对ＤＮＡ序列的识别杨岐生（浙江大学生物科学与技术系,杭州３１００２７）关键词真核生物转录因子,蛋白质－ＤＮＡ识别研究蛋白质和ＤＮＡ两类生物大分子的相互作用,以阐明基因表达、调控及信息传递的分子机制,是认识生命活动本质的核心问题。本文介...     

蓝光对绿豆下胚轴愈伤组织形成和生长过程中蛋白质代谢的影响

蓝光、白光和黑暗对绿豆下胚轴愈伤组织形成和生长过程中蛋白质代谢的影响不同。培养后３～１８ ｄ ,蓝光处理材料的可溶性蛋白质含量明显高于白光处理,更高于黑暗培养的材料。蓝光和白光明显促进３Ｈ亮氨酸掺入蛋白质,而蓝光和白光处理后游离氨基酸含量与黑暗对照相比,下降时间早,幅度大。在培养过程中,蛋白酶活性的变化与游离氨基酸相似。蛋白质合成抑制剂环己酰亚胺（ＣＨＭ） 抑制愈伤组织生长,其中以蓝光最大,白光次之,黑暗最小。在培养基中加入ＣＨＭ 愈早,抑制程度愈大。实验表明,ＣＨＭ 抑制愈伤组织蛋白质合成,也是以蓝光最甚。由此可见,蓝光促进绿豆下胚轴愈伤组织的形成、生长和蛋白质合成。     

In-vivo control of valine and leucine synthesis in the duckweed Spirodela polyrhiza

Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 M l-[¹⁴C]leucine or with 25 M l-[¹⁴C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg^–1 dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg^–1 dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.     

Isoleucine, a potent plasma glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes

To examine which branched-chain amino acids affect the plasma glucose levels, we investigated the effects of leucine, isoleucine, and valine (0.3 g/kg body weight p.o.) in normal rats using the oral glucose tolerance test (OGTT, 2 g/kg). A single oral administration of isoleucine significantly reduced plasma glucose levels 30 and 60 min after the glucose bolus, whereas administration of leucine and valine did not produce a significant decrease. Oral administration of valine significantly enhanced the plasma glucose level at 30 min after the glucose administration and leucine had a similar effect at 120 min. At each measurement timepoint, the insulin levels of the treated groups were lower than that of the control group. We then investigated the effects of leucine, isoleucine or valine at the same concentration (1 mM) on glucose metabolism in C(2)C(12) myotubes in the absence of insulin. Glucose consumption was elevated by 16.8% in the presence of 1 mM isoleucine compared with the control. Conversely, 1 mM leucine or valine caused no significant changes in glucose consumption in the C(2)C(12) myotubes. The 2-deoxyglucose uptake of C(2)C(12) myotubes significantly increased upon exposure to 1-10 mM isoleucine and 5-10 mM leucine. However, isoleucine caused no significant difference in glycogen synthesis in C(2)C(12) myotubes, although leucine and valine caused a significant increase in intracellular glycogen compared with the control. The isoleucine effect on glucose uptake was mediated by phosphatidylinositol 3-kinase (PI3K), but was independent of mammalian target of rapamycin (mTOR). These results suggest that isoleucine stimulates the insulin-independent glucose uptake in skeletal muscle cells, which may contribute to the plasma glucose-lowering effect of isoleucine in normal rats.     

Regulation of Transaminase C Synthesis in Escherichia coli: Conditional Leucine Auxotrophy

The regulation of synthesis of the valine-alanine-alpha-aminobutyrate transaminase (transaminase C) was studied in Escherichia coli mutants lacking the branched-chain amino acid transaminase (transaminase B). An investigation was made of two strains, CU2 and CU2002, each carrying the same transaminase B lesion but exhibiting different growth responses on a medium supplemented with branched-chain amino acids. Both had the absolute isoleucine requirement characteristic of ilvE auxotrophs, but growth of strain CU2 was stimulated by valine, whereas that of strain CU2002 was markedly inhibited by valine. Strain CU2002 behaved like a conditional leucine auxotroph in that the inhibition by valine was reversed by leucine. Results of enzymatic studies showed that synthesis of transaminase C was repressed by valine in strain CU2002 but not in strain CU2. Inhibition by valine in strain CU2002 appears to be the combined effect of repression on transaminase C synthesis and valine-dependent feedback inhibition of alpha-acetohydroxy acid synthase activity, causing alpha-ketoisovalerate (and hence leucine) limitation. The ilvE markers of strains CU2 and CU2002 were each transferred by transduction to a wild-type genetical background. All ilvE recombinants from both crosses resembled strain CU2002 and were inhibited by valine in the presence of isoleucine. Thus, strain CU2 carries an additional lesion that allows it to grow on a medium containing isoleucine plus valine. It is concluded that conditional leucine auxotrophy is characteristic of mutants carrying an ilvE lesion alone.     

O-Methylthreonine Inhibition of Growth and of Threonine Deaminase in Escherichia coli

O-methylthreonine (OMT), an isosteric analogue of isoleucine, markedly inhibited growth of Escherichia coli 15. This inhibition was overcome most effectively by addition of isoleucine, valine, or leucine to the medium and less effectively by addition of threonine. The dipeptide, valylleucine, also relieved the OMT-induced inhibition but only after a lag period, suggesting that valine and leucine, liberated by dipeptidase action, compete with OMT for entry into the cell. OMT was activated and transferred to transfer ribonucleic acid (RNA) by isoleucyl-RNA synthetase in vitro. The rate of OMT incorporation into protein of intact cells was comparable to that of isoleucine. In contrast to isoleucine, very high concentrations of OMT were required to inhibit threonine deaminase, and the inhibition was strictly competitive with threonine. In addition, OMT inhibited a threonine deaminase preparation desensitized to isoleucine inhibition.     

Fatty acid synthesis from amino acids in sheep adipose tissue

The rates of incorporation of 14C from 14C labelled acetate, glucose, alanine, leucine, isoleucine and valine into fatty acids has been measured in perirenal adipose tissue from foetal lambs and 8-month-old sheep, and into both fatty acids and acylglycerol glycerol in adipose tissue from 3-year-old sheep and 220-240 g female rats. Rates of incorporation of 14C from amino acids into fatty acids were much lower in adipose tissue from sheep (at all three ages) than from rats, whereas rates of incorporation of 14C into acylglycerol glycerol were either greater in sheep adipose tissue or the same as in rat adipose tissue. The rate of incorporation of 14C from amino acids into fatty acids decreased in the order leucine greater than alanine greater than isoleucine greater than valine in adipose tissue from rats and foetal lambs, and in the order leucine greater than alanine = isoleucine greater than valine in adipose tissue from 8-month- and 3-year-old sheep. Amino acids make a very small contribution to fatty acid synthesis in adipose tissue from sheep at all stages of development examined while fatty acids are a minor product of amino acid metabolism in sheep adipose tissue. The study provides further evidence for an important role for ATP-citrate lyase in restricting the utilization of acetyl-CoA generated in the mitochondria for fatty acid synthesis.     

Cell cycle inhibition of potato root tips treated with Imazethapyr

Imazethapyr inhibits the progression of the cell cycle in potato (Solanum tuberosum L.) root tips. A variety of existing methods were developed and adapted for the determination of mitotic index in potatoes. Significantly lower numbers of mitotically dividing cells were recorded after just 30 min of incubation with imazethapyr (0.35 μM) and the effect was characterised as an inhibition of mitotic entry. Imazethapyr inhibits a key enzyme in the biosynthesis of the branched-chain amino acids, valine, leucine and isoleucine. The inhibition of mitotic entry can be considered to be a secondary manifestation of a primary metabolic change induced by imazethapyr.     

Manganese mutagenesis in yeast

Summary A medium was found in which manganese efficiently induces erythromycin-resistant mitochondrial mutations, and which is suitable for measuring Mn²⁺ uptake and the labelling of DNA (Fig. 1). Mn²⁺ uptake is stimulated by glucose and slowed down by cycloheximide (Fig. 2). Mg²⁺ competes with Mn²⁺ uptake much stronger than does Zn²⁺ (Fig. 3). All of the conditions which favour Mn²⁺ uptake also favour induction of erythromycin-resistant mutations (Tables 3, 4).Mn²⁺ strongly inhibits protein synthesis (Table 1). Nuclear DNA replication is also strongly inhibited by this cation, while mitochondrial DNA replication is only weakly inhibited during the first 3 h of labelling, but there is small if any increase of the label incorporation between the 3rd and 6th h of labelling (Table 2). The relation between label incorporation into mitDNA and mutation induction by manganese is not straightforward (Table 5).From among 11 divalent cations tested, only Mn²⁺ was capable of inducing mitochondrial erythromycin-resistant mutations (Table 6).     

Specificity of the effects of leucine and its metabolites on protein degradation in skeletal muscle.

The effects of leucine, its metabolites, and the 2-oxo acids of valine and isoleucine on protein synthesis and degradation in incubated limb muscles of immature and adult rats were tested. Leucine stimulated protein synthesis but did not reduce proteolysis when leucine transamination was inhibited. 4-Methyl-2-oxopentanoate at concentrations as low as 0.25 mM inhibited protein degradation but did not change protein synthesis. The 2-oxo acids of valine and isoleucine did not change protein synthesis or degradation even at concentrations as high as 5 mM. 3-Methylvalerate, the irreversibly decarboxylated product of 4-methyl-2-oxopentanoate, decreased protein degradation at concentrations greater than or equal to 1 mM. This was not due to inhibition of 4-methyl-2-oxopentanoate catabolism, because 0.5 mM-3-methylvalerate did not suppress proteolysis, even though it inhibited leucine decarboxylation by 30%; higher concentrations of 3-methylvalerate decreased proteolysis progressively without inhibiting leucine decarboxylation further. During incubation with [1-14C]- and [U-14C]-leucine, it was found that products of leucine catabolism formed subsequent to the decarboxylation of 4-methyl-2-oxopentanoate accumulated intracellularly. This pattern was not seen during incubation with radiolabelled valine. Thus, the effect of leucine on muscle proteolysis requires transamination to 4-methyl-2-oxopentanoate. The inhibition of muscle protein degradation by leucine is most sensitive to, but not specific for, its 2-oxo acid, 4-methyl-2-oxopentanoate.     

赤霉素和光对拟南芥种子萌发和幼苗生长的影响

以拟南芥的赤霉素 (GA)缺陷型突变体ga 1,ga 2 ,ga 3和GA不敏感型突变体ga i为材料 ,研究了光和 4种GA对拟南芥种子萌发和幼苗生长影响的相互关系。结果表明 :(1)烯效唑对ga i种子萌发的抑制在光下可明显被GA恢复 ,而在黑暗中GA的作用不明显。 (2 )在光下低浓度的外源GA3 可使ga 1,ga 2和ga 3的种子萌发 ,而在黑暗中同样浓度的GA3 则难以使种子萌发。 (3)光可以降低种子萌发所需求的GA的剂量。 (4 )ga i和ga 1的幼苗的呼吸代谢有明显差异。以上结果说明 :光对拟南芥种子萌发的促进主要是提高了种子对GA反应的敏感性而不是增加GA的生物合成