Mutational analysis of a baculovirus major late promoter

We have used a linker-scan mutation strategy to analyze P_cap99, the proximal promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) gene encoding the major capsid protein. A series of recombinant viruses expressing the cat reporter gene under the control of selected mutants of this promoter was constructed. Only mutations that altered bases within a region extending from 8 bp upstream to 6 bp downstream from a TAAG sequence had a significant effect on expression from the late gene promoter. A synthetic promoter consisting of only these 18 bp (P_capmin) was sufficient to direct late expression. Aside from this small region surrounding the TAAG, no evidence for distinct late activating or repressing sequence elements was obtained. Experiments comparing and combining late and very late gene promoter sequences suggest that late expression is intrinsically determined by the presence and immediate context of a TAAG sequence and that very late expression [as previously shown in Ooi et al. J. Mol. Biol. 210 (1989) 721–736] results from additional modulation of TAAG-dependent expression by downstream promoter elements placed in an appropriate context. A compact combination promoter (95 bp), constructed by fusing P_capmin to linker-modified very late polyhedrin promoter, directs strong expression at late and very late times post-infection.     

CO2浓度增加和水分亏缺对典型绿肥植物光合性能及水分利用效率的影响

探讨大气CO₂浓度和水分变化对3种典型绿肥植物光合性能及水分利用效率的影响,可为未来气候变化情形下草地生态系统适应性管理提供理论支持。本试验利用可精准控制CO₂浓度的人工气候室,设置400(自然大气)和800 μmol·mol^-1(倍增)两个CO₂浓度,80%土壤田间持水量(FC)(充分灌水对照)、55%～60%FC(轻度水分亏缺)、35%～40%FC(中度水分亏缺)、<35%FC(重度水分亏缺)4个水分梯度,研究CO₂浓度增加和水分亏缺对甘蓝型油菜、白三叶和紫花苜蓿叶绿素含量、气体交换参数及水分利用效率(WUE)的影响。结果表明: 同一CO₂浓度下,与充分灌水对照相比,当土壤水分<40%FC时,3种植物的叶绿素含量和气体交换参数均显著降低;土壤水分为55%～60%FC时,3种植物的叶绿素总含量无显著变化,而白三叶和紫花苜蓿的光合速率(P_n)、蒸腾速率(T_r)降低了6%～25%,但WUE无显著性差异。与大气CO₂浓度相比,CO₂浓度倍增使充分灌水处理下甘蓝型油菜的P_n显著降低了21.5%,而显著增加了轻度水分亏缺下3种植物的P_n,且增加了中度水分亏缺下甘蓝型油菜和紫花苜蓿的P_n,但只对重度水分亏缺下紫花苜蓿的P_n有所改善;CO₂浓度倍增显著增加了白三叶和紫花苜蓿在所有水分处理下的WUE,但只增加了甘蓝型油菜在轻度水分亏缺下的WUE。CO₂浓度和水分的交互作用对3种植物的P_n均有显著影响,但仅对甘蓝型油菜的WUE有显著影响。综上,3种植物对大气CO₂浓度倍增和水分亏缺的响应存在明显差异,CO₂浓度升高能改善轻度水分亏缺对3种植物光合性能和WUE的不利影响,但只改善了重度水分亏缺下紫花苜蓿的光合性能。     

Malic enzyme cofactor and domain requirements for symbiotic N2 fixation by Sinorhizobium meliloti

The NAD(+)-dependent malic enzyme (DME) and the NADP(+)-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N(2) (Fix(-)) in alfalfa root nodules, whereas tme mutants are unimpaired in their N(2)-fixing ability (Fix(+)). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N(2) fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N(2) fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD(+)-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N(2) fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + H(+) to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N(2)-fixing bacteroids.     

Molecular biology of rat steroid 11 beta-hydroxylase [P450(11 beta)] and aldosterone synthase [P450(11 beta, aldo)].

The molecular features of rat steroid 11ß-hydroxylase [P450(11ß)] and aldosterone synthase [P450(11ß, aldo)] are discussed. P450(11ß) is biosynthesized as a precursor form composed of 499 amino acids, having a 24-amino acid extension peptide. Two species of P450(11ß, aldo) were identified; a precursor form of P450(11ß, aldo)-1 is 510 amino acids long and has a 34-amino acid extension peptide, while that of P450(11ß, aldo)-2 is 500 amino acids long and has a 24-amino acid extension peptide. The 286th amino acid of P450(11ß, aldo)-1 is Glu, while that of P450(11ß, aldo)-2 is Lys. The cDNA-expression studies showed that P450(11ß, aldo)-1 had the aldosterone producing activity whereas P450(11ß, aldo)-2 had no activity, suggesting that Glu286 of P450(11ß, aldo) plays an important role in the catalysis. The amino acid sequence of a region in P450(11ß) from Leu337 through Pro352 is highly conserved among the steroidogenic P450s. Functional expression studies on the cDNAs for two P450(11ß)s showed that P450(11ß) catalyzes the 11ß-, 18- and 19-hydroxylations of 11-deoxycorticosterone, but not the aldosterone synthesis. P450(11ß, aldo), on the other hand, catalyzes the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. The two P450(11ß)s were also shown to catalyze the conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol and cortisone.     

Periplasmic PQQ-Dependent Glucose Oxidation in Free-Living and Symbiotic Rhizobia

The expression of the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) of Rhizobium tropici CIAT899 and Sinorhizobium meliloti RCR2011 was investigated under different nutrient-limiting conditions in continuous cultures, under different conditions of phosphate availability, and in S. meliloti bacteroids. The presence of free PQQ in alfalfa root exudates has also been assayed. It was shown that apo-GDH or holoenzyme was actively synthesized by these rhizobia, with the concomitant production of gluconate from glucose, under certain environmental conditions. GDH activity was also detected in bacteroids from alfalfa root nodules inoculated with either S. meliloti RCR2011 or 102F34. It was also shown that free PQQ was present in root exudates of alfalfa, but its production is ascribed to the activity of Erwinia sp., a normal contaminant of these seeds. Received: 28 August 2000 / Accepted: 2 October 2000     

Use of deciduous woody species as a diet supplement for goats grazing Mediterranean shrublands during the dry season

Nutritional stress during the dry summer period is considered as a primary factor limiting goat production in the Mediterranean region. This study was conducted to determine if the browse of deciduous woody fodder plants is useful as a supplement for goats grazing kermes oak shrublands during July and September. Browses used were of Amorpha fruticosa L., Carpinus orientalis Mill., Colutea arborescens L., Fraxinus ornus L., Ostrya carpinifolia Scop. and Robinia pseudoacacia L.. Treatments included: (1)–(6) kermes oak (Quercus coccifera L.) shrubland plus fresh branches (2 kg/animal/day) of one of the above fodder plants as browse supplement, (7) kermes oak shrubland plus 0.25 (July) or 0.5 (September) kg alfalfa pellets/animal/day and (8) grazing in kermes oak shrubland as a control. Fodder plants produced high amounts of grazable material during summer whose quality ranged from medium (Carpinus, Fraxinus, Ostrya) to high (Amorpha, Colutea and Robinia). Goats ingested higher (p ≤ 0.05) browse of Fraxinus (268 g DM) and Ostrya (285 g DM) than the remaining browses (22–177 g DM) and alfalfa pellets (170 g DM) during July, but of Robinia and alfalfa (469 and 434 g DM, respectively) in September. During September, intake of all supplement feeds increased compared to July except for Ostrya, which remained constant. Goats supplemented by alfalfa pellets and browse supplements gained weight in both these periods in contrast to the animals grazing only kermes oak shrubland, except for Amorpha and Colutea in July. It is concluded that during summer, when the forage quality of kermes oak shrubland declines, browse supplement with certain deciduous woody fodder plants allows weight gains similar to those of alfalfa pellets; thereby, improving grazing animal performance.     

Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium

Cellular ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from soybean root nodules. Increased gaseous O₂ tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O₂ carrier rather than terminal oxidase. A less efficient oxidase appears to function when cytochrome P-450 is inhibited.     

Regulation of cytochrome P450 in the central nervous system

The role of brain P450 in the physiology, pharmacology and toxicology of the brain is the subject of this study. Cytochrome P450 was isolated from the brains of rats and quantitated spectrally. The contribution of the known hepatic forms of the enzyme to the forms constitutive in the brain as well as those which are induced by hormones are xenobiotics were characterized on Western blots. We have found that the level of P450 in the brain is increased during pregnancy and lactation, by partial hepatectomy and by ethanol. In each case the profile of P450s induced is different. In pregnancy and lactation the P450 content of the hypothalamic preoptic area and olfactory lobes were increased up to 10-fold and the only subfamily identified on Western blots was 4A. There was no detectable 1A, 2A, 2B, 2C, or 2E1. Ethanol increases the level of brain P450 3- to 5-fold and P450 2C, 2E1 and 4A are induced. Upon partial hepatectomy P450 1A, 2C and 4A were detected on Western blots but there was no 2E1. The inducibility of these forms of P450 in the brain suggests that there is in situ metabolism of steroids, fatty acids, prostaglandins, ethanol and other xenobiotics in the brain and raises questions about the role of brain P450 in the development of tolerance to drugs and the neurotoxicity of xenobiotics. More importantly, the action of neurotransmitters such as dopamine which utilize fatty acid metabolites as intracellular mediators, could be influenced by the levels of 2C and 4A P450s.     

肺形侧耳低温胁迫时期的转录组分析

本文以肺形侧耳栽培菌株X57为研究对象,利用高通量RNA测序技术对4℃低温处理0h、6h和12h后的肺形侧耳进行基因表达分析来探讨肺形侧耳低温应答机制。分析结果筛选出低温处理6h差异表达基因742个,其中上调表达基因374个,下调表达基因368个;低温处理12h差异表达基因1 489个,其中上调表达基因占53%,下调表达基因占47%。Gene Ontology（GO）功能聚类分析表明,差异表达基因主要富集在结合、催化分子功能组和代谢过程、细胞过程生物学过程中。KEGG功能富集分析结果显示,差异基因主要富集在氨基酸、核糖体和类固醇生物合成,及氮类物质代谢的通路上,富集到与低温胁迫相关通路MAPK signaling pathway-yeast上的基因表达随低温处理时间的增加呈上调趋势。已报道HOG-MAPK通路是MAPK途径研究较为明确、信号传递单一的真菌低温胁迫中重要的通路,本文运用生物信息学软件构建肺形侧耳的HOG-MAPK通路,并利用荧光定量PCR对相关基因表达进行验证,结果显示以低温处理0h为参照,随着低温胁迫时间增加通路上大部分基因的表达量持续上升,且差异显著,与RNA‐Seq分析结果一致。本文通过全面分析肺形侧耳低温胁迫时期基因表达情况,为进一步研究肺形侧耳低温胁迫相关重要基因的功能鉴定与信号通路调控机理提供基础。     

Molecular genetic studies on the biosynthesis of aldosterone in humans

Corticosterone methyl oxidase Type I (CMO I) and II (CMO II) have been postulated to be the enzymes involved in the final two steps of aldosterone biosynthesis in humans. We have isolated human cDNAs for P450c11 and P450c18 as well as the corresponding genes, CYP11B1 and CYP11B2. Both protein products of these two genes as expressed in COS-7 cells exhibit steroid 11β-hydroxylase activity, but only P450c18, a product of CYP11B2, carried steroid 18-hydroxylase activity to form aldosterone. These results indicate that CYP11B2 encodes CMO, the actual catalytic function of which is retained by P450c18, a multifunctional enzyme. This conclusion is further supported by the finding that the P450c18 gene, CYP11B2, is mutated at several different loci in patients deficient in CMO I or II.     

Cytochrome P450s in the guinea pig adrenal that are immunologically similar to liver forms: estrogen suppression explains male-female differences

Several cytochrome P450s have been identified in guinea pig adrenal microsomes which are distinct from the known steroidogenic P450s, c17 and c21, and are immunochemically related to cytochrome P450s found in liver. One, a 52 K protein related to P450 I (CYP1), occurs almost exclusively in males, is localized to the inner zone, and is suppressed by ACTH. Its levels correlate with microsomal capacity for xenobiotic metabolism. The others, related to P450s II and III (CYP2 and 3), are more predominant in males, but not exclusive to them, are found in both the inner and outer zones, and are not suppressed by ACTH. Their functions remain to be elucidated. The male predominance of the CYP1-related protein has recently been shown to be due to suppression of the protein in females by estrogen. To determine if estrogen is also involved in the regulation of the CYP2-related proteins, ovariectomized and sham-operated animals were treated with a long-acting estrogen, estradiol valerate, or with the vehicle alone. These P450s reached male levels in ovariectomized females treated only with the vehicle. Their enhanced levels were suppressed by treatment with estrogen. Estrogen treatment also suppressed the levels of the P450s seen in sham-operated females. Endogenous estrogen produced similar effects. In hemi-ovariectomized females the contralateral ovary hypertrophied, a state in which estrogen levels would be maintained or increased. In these females no increase occurred in the immunodetectable P450s. In normal females, estrogen levels are low in prepubertal animals, rise at the time of puberty and drop again after ovarian cycling is completed. The CYP2-related proteins were present in adrenal microsomes of prepubertal females, but were suppressed after puberty. On the other hand, post-estrous females, in whom estrogen levels would be low, acquired male levels of these proteins in their adrenal microsomes. P450c17 and P450c21, as well as 3β-hydroxysteroid dehydrogenase, were not affected by surgery or estrogen. Taken together, these experiments indicate that suppression by estrogen in females can account, in large part, for the predominance of several immunochemical homologs of liver P450s in adult male guinea pig adrenals.     

刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究

目的: 核糖体蛋白(RPs)属于多功能蛋白,能够参与调控细胞生长和响应胁迫条件。RpRPL22是一个从豆科植物刺槐中分离得到的结瘤相关基因,通过序列比对发现其与核糖体大亚基蛋白RPL22高度同源。对其如何通过调控根瘤菌侵染而在共生结瘤过程中发挥重要作用进行了较为深入的探索。方法: 利用实时荧光定量PCR技术(qRT-PCR)分析RpRPL22在接菌后不同时间及不同植物组织的表达变化。利用cDNA末端快速扩增技术(RACE)获得目的基因cDNA全长。通过GFP报告基因进行RpRPL22亚细胞定位分析。通过Gateway BP重组技术构建RNA干扰(RNAi)重组载体,借助电转化法将重组载体转至农杆菌K599,利用农杆菌介导植物根部,接菌后观察和测量植株表型。首先从宏观水平统计观察目的基因是否对结瘤过程有影响,其次从分子水平揭示目的基因在共生结瘤过程的重要功能。结果: 不同接菌时期、不同植物组织目的基因qRT-PCR相对表达量结果显示,几乎在所有取样的接菌时间,目的基因RpRPL22在接菌根中的相对表达量都低于未接菌对照根,只有接菌后第25天除外。在成熟的根瘤中,接菌后第25天该基因的表达量也最高。洋葱表皮和毛状根亚细胞定位结果均显示在椰菜花叶病毒(CaMV)的35S启动子控制下,RpRPL22融合绿色荧光蛋白GFP的荧光信号在细胞核和细胞质有明显的表达。RNAi转化植株的表型统计观察结果,比如植株鲜重、植株的有效结瘤数目较对照组均有明显的降低;同时RNAi转化植株在根瘤菌侵染过程形成的侵染线数目和根瘤原基数目较对照均显著降低。根瘤切片实验用于观察根瘤显微超微结构,结果显示RNAi植株根瘤中固氮区的受菌侵染细胞数目与对照相比明显减少。电镜观察根瘤单个受菌侵染细胞中类菌体形态显示,RNAi根瘤中类菌体侵染细胞胞体多呈不规则形状,皱缩变形严重,环类菌体周间隙空间增大,多共生体融合,表现出细胞凋亡的迹象。对照根瘤中的受菌侵染细胞胞体多呈圆形椭圆形,胞质饱满丰富且分布均匀,细胞发育正常,表明RNAi植株根瘤发育过程明显受阻。结论: 核糖体蛋白(RP)能够参与调控豆科植物共生结瘤过程,相关同源基因RpRPL22可能在起始根瘤菌侵染植物和阻止类菌体降解过程中起重要作用。     

Sequential endocrine changes and behaviour during oestrus and metoestrus in repeat breeder and virgin heifers

The aim of the experiment was to study the oestrous behaviour and the peripheral blood plasma profiles of luteinizing hormone (LH), progesterone and the prostaglandin metabolite, 15-keto-13,14-dihydro-PGF₂, during oestrus and metoestrus in repeat breeder (RBH) and virgin heifers (VH). Ten animals of each category were utilized. The RBH had a history of at least three inseminations without conception, and the VH were sexually mature animals not previously inseminated or mated. Oestrous symptoms were recorded and blood collected from the onset of prooestrus to 7 days after oestrus. The animals were inseminated during oestrus and their embryos were collected by a nonsurgical technique 7 days after insemination. The morphology of the embryos was evaluated.

The duration of oestrus was longer (P < 0.05) in the RBH (31.5 ± 3.6 h) than in the VH (23.8 ± 2.0 h). No differences in duration of prooestrus or in the interval from the end of oestrus to postoestrous bleeding were found between the heifer categories. The interval from the onset of oestrus to the preovulatory LH peak was longer (P < 0.05) in the RBH (12.2 ± 2.8 h) than in the VH (4.8 ± 1.5 h). There was a lower LH release in the RBH than in the VH, measured as the magnitude of the preovulatory LH peak (P < 0.05; 28.0 ± 4.0 vs. 40.7 ± 3.6 μg/l) or as the area under the curve of the LH peak (P < 0.01; 1141 ± 164 vs. 1765 ± 144 mm²). The progesterone levels were higher (P < 0.05) in the RBH than in the VH during the interval 5–48 h and lower (P < 0.05) during the interval 121–168 h after the LH peak. Peaks of the prostaglandin metabolite were seen during oestrus in both heifer groups. There were more prostaglandin metabolite peaks in the RBH than in the VH during the interval 13–24 h after the LH peak. Fewer normal embryos (P < 0.05) and more degenerated embryos (P < 0.01) were found in the RBH than in the VH group 7 days after insemination. No apparent relation was found between the morphology of the embryos and the hormonal changes.

The result of the study indicates a hormonal imbalance in the RBH. The hormonal asynchronism starts before or early in oestrus, which presumably leads to a sequence of improper hormonal changes responsible for the elevated embryonic loss in repeat breeder animals.