Two new mutations of the human BCHE gene (IVS3-14T>C and L574fsX576)

The genetic variation of human butyrylcholinesterase is associated with the majority of prolonged cases of apnea in patients submitted to the muscle relaxant succinylcholine. The present study reports two new mutations of the BCHE gene in 346 Euro-Brazilians: IVS3-14T>C found in five heterozygotes (allele frequency: 0.72 ± 0.32%) and L574fsX576 found in one heterozygote (allele frequency: 0.14 ± 0.14%). These two variants were not found in 85 Guarani Amerindians. It is not expected that the IVS3-14T>C mutation may interfere in the splicing process and that the mutation found in exon 4 (L574fsX576) may disturb BChE tetramerization and activity.     

A −1 frameshift in the HIV-1 env gene is enhanced by arginine deficiency via a hungry codon mechanism

Ribosomal frameshifting is used by various organisms to maximize protein coding potential of genomic sequences. It is commonly exploited by RNA viruses to overcome the constraint of their limited genome size. Frameshifting requires specific RNA structural features, such as a suitable heptanucleotide “slippery” sequence and an RNA pseudoknot. Previous genomic analysis of HIV-1 indicated the potential for several hidden genes encoded through frameshifting; one of these, overlapping the envelope gene, has an RNA pseudoknot just downstream from a slippery sequence, AAAAAGA that features an adenine quadruplet prior to a potential hungry arginine codon (AGA). This env-frameshift (env-fs) gene has been shown to encode a truncated glutathione peroxidase homologue, with both antioxidant and anti-apoptotic activities in transfected cells. Using a dual reporter cell-based frameshift assay, we demonstrate that the env-fs frameshift sequence is active in vitro. Furthermore, in arginine deficient media, env-fs frameshifting increased over 100% (p < 0.005), consistent with the hypothesized hungry codon mechanism. As a response to arginine deficiency, increased expression of the antioxidant viral GPx gene (env-fs) by upregulation of frameshifting could be protective to HIV-infected cells, as a countermeasure to the increased oxidative stress induced by arginine deficiency (because NO is a known scavenger of hydroxyl radical).     

Comparison of variations between percentage of body fat, body mass index and daily physical activity among young Japanese and Thai female students

ABSTRACT: BACKGROUND: In our series of investigations concerning the causes of seasonal change in fat accumulation in young university students, we could not find any contribution of seasonal variation in the ratio of carbohydrate and fat metabolism to that of body fat percentage in Japanese and Thai participants. After our previous study, we examined the effect of daily physical activity on body fat percentage to look for the major causes of seasonal change in fat accumulation in young university students. FINDINGS: In this study, we measured participants' (young Japanese and Thai university students) daily physical activity by a uniaxial accelerometer in addition to the measurements of body fat percentage and body mass index by a bioelectrical impedance meter. We found that there was significant and moderate negative correlation between body fat percentage and daily step counts among Japanese but not Thai participants. We observed significant, moderate and positive correlations between the percentage of body fat and body mass index among Japanese and Thai participants. CONCLUSIONS: Daily physical activity plays an important role in the seasonal variation of body fat percentage of Japanese female students. Our present study also confirmed the importance of daily physical activity for controlling body mass index and for the prevention of obesity.     

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli

LacI⁻ mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G: C→C: G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G: C→C: G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication mechanisms.     

CaXMT和TCS1突变体基因串联共表达及体外酶活检测分析

咖啡碱和可可碱是茶叶生物碱的主要组分,且咖啡碱是茶叶重要的滋味物质,随着咖啡碱在食品和药物领域的应用愈发广泛,咖啡碱的生物合成成为新的研究热点.目前市场上的咖啡碱主要靠化学合成,为了探索其生物合成途径,该研究将咖啡黄嘌呤核苷甲基转移酶(coffee xanthosine methyltransferase,CaXMT)基因和茶树咖啡碱合成酶(tea caffeine synthase,TCS1)基因的4个突变体分别串联至同一大肠杆菌表达载体pMAL-c5X,诱导融合蛋白共表达,并进行SDS-PAGE凝胶电泳分析.结果表明:目的蛋白成功表达后,应用超声破碎法制备含有目的蛋白的粗酶液,添加底物黄嘌呤核苷(xanthosine,XR)和甲基供体S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM)进行体外酶促反应,将反应产物进行高效液相色谱检测.检测结果显示,pMAL-CaXMT-TM2/3/4的体外酶促反应产物仅有可可碱生成,均未见咖啡碱生成.该研究结果为构建生物合成咖啡碱和可可碱的串联共表达载体奠定了基础,也为进一步研究生物合成咖啡碱和可可碱提供了新思路.     

Functional interactions between the gene tetanic and the Shaker gene complex of Drosophila

Different phenotypes associated with the tetanic (tta) mutation such as appendage contraction, maternal effect and low viability and fertility are enhanced by one extra dose of the Shaker gene complex (ShC). The tta mutation is lethal with two extra doses of ShC. In addition, tta embryos have a defective nervous system. In this paper, I analyse the interaction between tta and ShC to gain insight into their relationship. Aneuploid analysis suggests that the lethality is due to an interaction of the tta mutation with the maternal effect (ME) region of this gene complex. Mutations in the ME region of ShC partially suppress this interaction. Trans-heterozygous combinations of MEI[l(1)305] and MEIII [l(1)459] mutations causes dominant lethality in a tta background. Trans-heterozygous combinations of an MEII [l(1)1359] mutation with the cited MEI and MEIII mutations are lethal in a tta background. Double mutant combinations and gene dosage experiments, suggest that tta also interacts with the viable (V) region of ShC. These specific genetic interactions indicate that tta and the ME and V regions of ShC are functionally related. These results, together with the previous electrophysiological, molecular and biochemical studies on these mutants suggest an interaction at the protein level. Thus, in the case of the V region, the tta gene product may modulate the activity of the K⁺ channels encoded in this region. Furthermore, the extreme dosage sensitivity of the interaction between tta and ShC suggests a stoichiometric requirement for the different gene products involved, which might be physically associated and form heteromultimers.     

The influence of environmental conditions on the body mass of Barn Swallows (Hirundo rustica) during spring migration

The present study examines the effects of seven microclimatic factors on the arrival body mass of migrant Barn Swallows (Hirundo rustica) in the Aiguamolls de l’Empordà Natural Park, a site located in northeastern Spain. We used principal component analysis, followed by a general linear model procedure, to create a model that examines the relationships between body mass and weather-related events recently experienced during the spring migration. Our results suggest that local weather variation during the migratory flight clearly affects the body mass of Barn Swallows on a short time scale. This effect demonstrates the importance of the environmental conditions en route for migrating swallows and how these conditions might influence diverse events of the life cycle, such as the arrival time at the breeding grounds or the reproductive success.     

环链虫草Cordyceps cateniannulata对番茄生长和抗氧化酶活性的影响

【目的】评估环链虫草Cordyceps cateniannulata对植物促生和植物抗氧化酶活性的影响。【方法】本研究利用浸种法将环链虫草接种于番茄植物体,在接种后的第30天和60天,通过番茄株高、根长、地上和地下部分的干鲜重指标评价其对番茄生长的影响;在接种后第10、20、30、60和90天,通过选择性培养基分析其在番茄不同组织中的生存情况,使用形态学及DNA序列比对的方法检验所分离菌株与原有菌株的一致性。在处理后的第30天,检测番茄叶片中的过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量,观察环链虫草对番茄的抗氧化酶活性影响。【结果】环链虫草可定殖于番茄幼苗且对番茄生长有显著促进作用,菌株对植株的定殖偏好性分别为根部>茎部>叶部。酶活检测结果表明,处理组番茄叶片防御酶活性均呈显著升高的趋势,其中POD、CAT、SOD活性分别比对照增加了52.21%、75.31%和158.59%,MDA含量下降了35.15%。【结论】环链虫草可以通过浸种的方法感染并定殖番茄幼苗的根、茎、叶,促进番茄幼苗的生长并提高番茄抗氧化酶活性,具有较好的田间...     

红曲霉繁殖相关brlM基因鉴定及功能分析

brlA作为曲霉分生孢子形成的中心调控路径中最上游的转录因子,能够诱导下游特异基因的表达调控分生孢子的产生,brlA缺失将导致曲霉无法产生分生孢子,同时生长代谢发生改变,但对不同菌种的影响有显著差异。【目的】探究brlA同源基因brlM在红曲霉中的基因功能,探索红曲霉繁殖调控机制。【方法】从紫色红曲霉Mp-21菌株中克隆了brlA同源基因brlM。利用同源重组原理,用农杆菌介导转化法构建了brlM基因缺失突变株(△brlM),分析△brlM和野生菌株Mp-21在菌落表型、显微结构、生长速率和次生代谢产物等方面的差异,明确brlM基因在红曲霉中的主要功能。【结果】在形态水平上,brlM基因缺失菌株导致菌丝生长更加旺盛,赋予菌落更加蓬松的表型;显微观察发现△brlM失去了有性繁殖产生闭囊壳的能力,但却提升了无性繁殖产生分生孢子的能力;同时代谢产物红曲色素、莫纳可林K和桔霉素产量显著下降。【结论】红曲霉brlM基因的功能与曲霉属中的brlA并不相同,brlM基因在红曲霉有性繁殖中的作用不可替代。本研究的结果对进一步探索丝状真菌繁殖调控机制提供了新的思路。     

Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation

Summary The Escherichia coli gene ssyB was cloned and sequenced. The ssyB63 (Cs) mutation is an insertion mutation in nusB, while the nusB5 (Cs) mutation suppresses secY24, indicating that inactivation of nusB causes cold-sensitive cell growth as well as phenotypic suppression of secY24. The correct map position of nusB is 9.5 min rather than I I min as previously assigned. It is located at the distal end of an operon that contains a gene showing significant homology with a Bacillus subtilis gene involved in riboflavin biosynthesis.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Effects of temperature acclimation on body mass and energy budget in the Chinese bulbul Pycnonotus sinensis

Chinese bulbuls(Pycnonotus sinensis) are small passerine birds that inhabit areas of central, southern and eastern China. Previous observations suggest that free–living individuals of this species may change their food intake in response to seasonal changes in ambient temperature. In the present study, we randomly assigned Chinese bulbuls to either a 30 °C or 10 °C group, and measured their body mass(BM), body temperature, gross energy intake(GEI), digestible energy intake(DEI), and the length and mass of their digestive tracts over 28 days of acclimation at these temperatures. As predicted, birds in the 30 °C group had lower body mass, GEI and DEI relative to those in the 10 °C group. The length and mass of the digestive tract was also lower in the 30 °C group and trends in these parameters were positively correlated with BM, GEI and DEI. These results suggest that Chinese bulbuls reduced their absolute energy demands at relatively high temperatures by decreasing their body mass, GEI and DEI, and digestive tract size.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Ecophysiological responses of the euhalophyte Suaeda salsa to the interactive effects of salinity and nitrate availability

Effects of salinity and nitrate nitrogen (NO₃⁻-N) on ion accumulation and chlorophyll fluorescence were monitored for two populations of Suaeda salsa grown from seeds in a greenhouse experiment. One population inhabits the intertidal zone and the other occurs on inland saline soils. Ion contents in soils and in leaves of the two populations were also investigated in field. In the greenhouse, seedlings were exposed to a NaCl concentration of 0.6 and 35.1 ppt, with 0.1 or 5 mM NO₃⁻-N treatments for 20 days. The contents of Na⁺ and Cl⁻ were higher, but NO₃⁻ was lower in soils of the intertidal zone than at the inland site. In the field, ion concentrations and the estimated contribution of these ions to osmotic potential in leaves showed no difference between the two populations, except that the estimated contribution of Na⁺ to osmotic potential in leaves of the intertidal population was lower than that in the inland population. In the greenhouse, in contrast, the concentration of Cl⁻ was lower, but NO₃⁻ concentration and the estimated contribution of NO₃⁻ to osmotic potential were higher, in the leaves of plants from the intertidal zone. Salinity had no effect on the maximal efficiency of PSII photochemistry (Fv/Fm) and the actual PSII efficiency (ΦPSII). The results indicated that S. salsa from the intertidal zone was better able to regulate Cl⁻ to a lower level, and accumulate NO₃⁻ even with low soil NO₃⁻ concentrations. Tolerance of the PSII machinery to high salinity stress may be an important characteristic for the studied species supporting growth in highly saline environments.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

The sequence and phylogenesis of the α-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion)

In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked ^Iα and ^IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the ^Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.     

三孢布拉霉crgA启动子的克隆和活性分析

【背景】CrgA是三孢布拉霉(Blakesleatrispora,Bt)中调控类胡萝卜素合成的关键负调控因子,其表达水平会影响类胡萝卜素的合成。【目的】克隆三孢布拉霉crgA启动子并分析其活性,为进一步解析CrgA表达调控机制奠定基础。【方法】通过综合微生物基因组(integrated microbial genomes, IMG)数据库提供的基因组序列,克隆crgA翻译起始位点上游2 000 bp序列,分析其顺式调控元件和转录起始区域预测,通过RT-qPCR分析不同光照时间对三孢布拉霉crgA相对转录水平的影响;构建4个不同长度的crgA启动子截短序列驱动的GUS-mGFP5重组表达载体p1303-procrgAF、F1、F2和F3,利用农杆菌侵染整合到三孢布拉霉基因组中,在黑暗和光照条件下测定β-D-葡萄糖苷酸酶(β-D-glucuronidase,GUS)酶活性并观察荧光信号。【结果】crgA启动子不仅包含基础的TATA-box和CAAT-box元件,还包括多个与光响应相关的元件。观察荧光结果显示CaMV35S和构建的4个突变启动子均能在三孢布拉霉体内驱动下游基因表达,检测GUS...