Functional analysis of microtubule-binding domain of bovine MAP4

Bovine microtubule-associated protein 4 (MAP4) consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain of MAP4 is further divided into three subdomains: a region rich in proline and basic residues (Pro-rich region), a region containing four repeats of an assembly-promoting (AP) sequence, which consists of 22 amino acid residues (AP sequence region), and a hydrophobic tail region (Tail region). The subdomain structure of MAP4 microtubule binding domain is similar to those of other MAPs (MAP2 and tau). In order to study the function of each subdomain per se of bovine MAP4 microtubule-binding domain, we purified a series of truncated fragments of MAP4, expressed in Escherichia coil. Binding affinity of the PA4T fragment (containing the Pro-rich region, the AP sequence region and the Tail region) is only four times higher than that of the A4T fragment (containing the AP sequence region and the Tail region), while the microtubule nucleating activity of the PA4T fragment is far greater. We propose that the Pro-rich region promotes the nucleation of microtubule assembly. The A4 fragment (corresponding to the AP sequence region) stimulated the assembly of tubulin into coldstable amorphous aggregates. The AP sequence region of MAP4 failed to promote microtubule assembly. On the other hand, the fragment has an activity to stimulate microtubule elongation. The function of the MAP4 Tail region is not clear at present. The A4T fragment (containing the AP sequence region and the Tail region) promote both microtubule nucleation and elongation step, but the A4 fragment only promotes microtubule elongation, suggesting that the Tail region is indispensable for the nucleation step. However, the fragment containing only the Tail region could not bind to microtubule. Although MAP4 was considered to be long, thin and flexible molecule, never the Tail region may contribute to be the proper folding of MAP4, and/or may interact with other molecules. We concluded that both the Pro-rich region and the AP sequence region take part in the promotion of tubulin polymerization, and that the former is important for the lateral protofilament-protofilament interaction, and the latter is important for the longitudinal affinity between each tubulin dimer in a protofilament.     

中国生态地理区城市水资源利用效率时空分异特征

我国水资源面临的形势十分严峻,水资源短缺、水污染严重、水生态环境恶化等问题突出,已成为制约社会经济可持续发展的主要瓶颈。基于分类数据包络分析（Categorical DEA）对2007-2016年中国生态地理区城市水资源利用效率进行测算,对其时间变化、空间分布以及空间收敛性进行分析。结果表明：1）2007-2016年期间全国城市水资源利用效率总体处于低效率状态,随时间高低交错波动式发展,整体呈先降后升势态;2）生态地理区城市水资源利用综合效率由高到低依次为温带干旱区、温带半干旱区、温带半湿润区、温带湿润区、亚热带湿润区,呈西北向东南逐渐递减的空间分布格局,生态地理区纯技术效率区间差异较大,但空间分布规律不显著,区域规模效率间差距小,且无明显空间分布规律;3）纯技术效率低效是制约全国水资源利用综合效率提升的主要因素,生态地理区间纯技术效率巨大差距导致全国纯技术效率整体低效,也是全国水资源利用综合效率低效的根本原因;4）生态地理区水资源利用综合效率区域差距逐渐缩小,主要是因为高效率区域效率值向低效率区域靠拢,区域间低效向高效的"追赶效应"不明显。     

香蕉果实特异性ACC合酶基因启动子区的克隆及其功能初探

根据本实验室所获得的香蕉果实特异性ACC合酶cDNA序列,以改进的接头连接PCR方法通过两次步行从香蕉基因组中分别扩增并克隆了其基因5′旁侧区近端1.2kb和远端1.6kb的片段。通过拼接,构建出含有2505bp启动子区和转录起始位点下游86bp的共2591bp的基因5′旁侧区片段;其启发性动子区中34至28为推测的TATA盒序列,158至146为推测的CCAAT盒,与其它植物基因启动子结构相类似。将2.5kb启动子片段与β-葡糖苷酸酶(GUS)基因编码序列融合,用基因枪法将构建的嵌合基因转入香蕉叶、根和果实的细胞后,只在果实细胞中观察到报告基因的瞬时表达,从功能上证明了此25kb的启动子片段具有指导报告基因在香蕉果实中特异性表达的作用。同时构建5个含不同5′端缺失启动子与GUS融合基因的表达载体。瞬时表达结果表明可能负责果实特异性表达的调控区存在于转录起始位点至-1111的启动子区中,而在-1111至-608区间可能存在一个正控制区。     

The accurate replacement of long genome region more than several hundreds kilobases in Bacillus subtilis

Competent cell transformation with DNA obtained by the gentle lysis of protoplasts (LP transformation) was used to replace a large genomic region in this study. Discontinuity was detected in the replacement of the donor region tested, probably due to multiple crossover events involving a single donor genome fragment. To overcome discontinuous replacement, we inverted the genomic region to be replaced in the donor used for LP transformation. The replaced region in the transformant was identified to have a continuous genomic region originating from the donor genome. Furthermore, the genome region to be replaced was inverted in the recipient, and the same region and the flanking 10 kb region of both ends was inverted in the donor genome. LP transformation was conducted with the two inversion mutants and it is possible to restrict homologous recombination to the 10 kb flanking regions. Using this method, the 99 kb yxjG-yxbA region, the 249 kb pbpG-yxbA region and the 602 kb yvfT-yxbA region were suggested to be replaced continuously and accurately.     

Nucleotide sequence of the promoter region of the melibiose operon of Escherichia coli

The nucleotide sequence of the promoter region of the melibiose operon of E. coli was determined. Consensus sequences for the -35 region, the Pribnow box and the binding site for cyclic AMP receptor protein were found in this region. The possible secondary structure of this DNA region was very similar to that of the promoter region of the lactose operon. A possible initiation ATG preceded by a Shine-Dalgarno sequence with proper spacing was present just downstream of the promoter region. The possible sequence of 52 amino acid residues in the NH2 terminus of the alpha-galactosidase were determined.     

A physical map of the Q region of B10.P

The murine class I MHC Q region is part of a large complex multigene family whose members have various peptide-binding functions. The structure of the Q region is complex, varying extensively in the b, d, k, and q haplotypes so far examined. To better understand the structural heterogeneity, we examined the Q region of B10.P, a strain whose immunological characteristics are distinct from other haplotypes. A total of 89 cosmids were isolated from genomic DNA. The B10.P Q region was found to contain seven genes in a 190-kb cluster linked to D^p and two additional Q genes in a separate 55-kb cluster. The gene arrangement in this haplotype was unique and did not correspond to any other haplotype; this underscores the complexity of chromosomal structure in this region. In addition to the Q region clusters, Tla region was tentatively aligned in five clusters spanning approximately 300 kb. One 37-kb M region cosmid was also identified. Received: 24 February 1995 / Accepted: 10 October 1995     

干旱沙区不同种源的绵毛优若藜表现性比较

在民勤沙生植物园．通过室内和田间试验,对引自美国西部沙漠区和草原区的绵毛优若藜的物候期、种子发芽温度、植物的生长和地上部分营养成分进行了观测与测定。在昼夜变温条件下,草原区种子对温度的反应较迟钝．适宜的发芽温度应为5～25C;沙漠区种子发芽适宜的温度为lO～30C。两个种源的绵毛优若藜均在6～7月间生长量最大。草原区种源的植株月平均生长量大于沙漠区种源的生长量。不同种源植物的物候期相差较小,除开花期外,沙漠区植物的展叶、孕蕾、结果和叶变色均早于草原区。在民勤沙区,绵毛优若藜表现为准常绿性。沙漠区植株的营养成分含量略低于草原区植株。     

中国西南纵向岭谷区外来入侵植物对生物多样性的影响

论述中国西南纵向岭谷区外来入侵植物对生物多样性的影响。中国西南纵向岭谷区是我国生物多样性保护的关键地区。目前,该区域已成为外来植物入侵的重灾区,外来入侵植物种类多达70余种,其中,紫茎泽兰Ageratina adenophora（Eupatoriumadenophorum）、飞机草Chromolaena odorata（Eupatorium odoratum）和肿柄菊Tithonia diversifolia等在该区域危害严重,该区域的生态系统的结构与功能遭受破坏,本地物种面临灭绝,农、林、牧生产乃至区域的生态安全受到威胁。该区域的外来入侵植物的危害居全国之冠。研究该区域的外来入侵植物,揭示其危害规律,遏制其危害速度,最终清除该区域的外来入侵植物,恢复该区域的生态平衡,具有十分重要的现实意义和科学价值。文中详细论述了外来入侵植物对植物物种多样性、植物群落多样性、生态系统多样性、农牧业及林业的影响,最后指出消除和控制外来入侵植物的途径。     

Reassessing the role of region A in Pit1-mediated viral entry

The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.     

Molecular dynamics study of deformation mechanisms of poly(vinyl alcohol) hydrogel

Molecular dynamics (MD) simulations have been performed on the physically crosslinking poly(vinyl alcohol) (PVA) hydrogel to study the deformation mechanisms under uniaxial tensile conditions. The distributions of hydroxyl oxygens and dihedral angle and the number of hydrogen bonds have been analysed to study the structure of the hydrogel. The water content and temperature dependency of mechanical properties have been investigated. The energy contributions from the partially united atom potential have been calculated as a function of strain. It is found that the stress–strain curve comprises toe region, linear region and yield and failure region which is close to most biomaterials. In the toe and yield region, all the contributions to the internal energy change a little. However, in the linear region, the bond stretching and angle bending energy increase rapidly and mainly dominate the region, and the energy increases more rapidly with the increasing water content but the decreasing temperature. The degree of crosslinking decreases with the increasing deformation. The polymer chains occur significant torsional activity in the toe region. Hydrogen bonds are stable in the toe and yield region, but the hydrogen bonds between hydroxyl groups and waters decrease in the linear region.     

气候变化对6种荒漠植物分布的潜在影响

分析气候变化对植物分布的影响,对保护生物多样性具有重要意义。利用CART（分类和回归树）模型及A2和B2情景,分析了气候变化对短叶假木贼（Anabasis brevifolia）、裸果木（Gymnocarpos przewalskii）、梭梭（Haloxylon ammoden-dron）、膜果麻黄（Ephedra przewalskii）、驼绒藜（Ceratoides latens）和喀什膜果麻黄（Ephedra przewalskii var.kaschgarica）分布范围及空间格局的影响。结果表明：气候变化下,这些植物目前适宜分布范围减小;从新适宜及总适宜分布范围而言,短叶假木贼和梭梭从1991-2020年到2051-2080年时段增加,之后减小,其它植物从1991-2020年到2081-2100年时段减小;喀什膜果麻黄和驼绒藜适宜分布范围减小并破碎化,其它植物向目前适宜分布的西部、西北部（或青海西南部）、昆仑山、阿尔金山和祁连山区扩展;除驼绒藜和喀什膜果麻黄与年均气温变化具显著相关性外,其它植物分布范围与年均气温和降水量变化的相关性较弱（P〉0.05）,除驼绒藜、喀什膜果麻黄和裸果木目前分布范围与年均气温和降水量变化的回归关系较强外,其它植物分布范围与年均气温和年降水量变化多元线性回归关系较弱。上述研究结果表明,气候变化下,这些植物空间分布格局改变,目前分布范围减少,新适宜及总适宜分布范围近期增加,随着气候变化程度的增强,又逐渐减小。     

Positional control of the fates of nuclei produced after meiosis in Paramecium caudatum: Analysis by nuclear transplantation

The survival or degeneration of nuclei produced by meiosis in Paramecium caudatum depends on their position in the cytoplasm. The surviving nucleus always lies in the special region of the cytoplasm called the paroral region, which is the region around the cytostome. The remaining three degenerate outside the region. The mechanism controlling the survival or degeneration of meiotic nuclei was analyzed microsurgically. When the nucleus in the early conjugating cells (stage II) was transplanted into the cell at the stage when three meiotic nuclei were degenerating, it did not degenerate, but divided. When one of the meiotic nuclei which was outside the paroral region and destined to degenerate was transplanted into the cell in the meiotic prophase (stage IV), it did not survive but degenerated. When the surviving nucleus in the paroral region was removed microsurgically, one of the three meiotic nuclei lying outside the paroral region and destined to degenerate moved into the paroral region and survived. These results suggest that the nuclei after meiosis are destined to degenerate but can be rescued from degeneration by the special environment of the paroral region.     

Micturition-suppressing region in the periaqueductal gray of the mesencephalon of the cat

The periaqueductal gray (PAG) of the mesencephalon has been implicated to be involved in the control of micturition. We investigated the micturition-suppressing region in the PAG of the cat. Decerebrated 27 adult cats were used. A microelectrode was inserted stereotaxically into the PAG, and a region was searched where electrical stimulation suppressed isovolumetric bladder contractions. Simultaneous stimulation of the pontine micturition center (PMC) and micturition-suppressing region in the PAG was performed before and after an injection of bicuculline (GABA(A) blocker) into the PMC. The micturition-suppressing region was found at the dorsolateral margin of the rostral PAG. Bladder contractions were not provoked by simultaneous stimulation of the PMC and micturition-suppressing region in the PAG. However, after bicuculline injection into the PMC, partial bladder contractions were provoked by simultaneous stimulation of the PMC and the micturition-suppressing region in the PAG. These results suggest that the dorsolateral margin of the rostral PAG includes the micturition-suppressing region that seems to have neural connections with the PMC. GABA is assumed to be one of the neurotransmitters that are involved in the PMC inhibition from the micturition-suppressing region in the PAG.     

Characterization of the cis-acting contributions to avian hepadnavirus RNA encapsidation

Previous analysis of duck hepatitis B virus (DHBV) indicated the presence of at least two cis-acting sequences required for efficient encapsidation of its pregenomic RNA (pgRNA), epsilon and region II. epsilon, an RNA stem-loop near the 5' end of the pgRNA, has been characterized in detail, while region II, located in the middle of the pgRNA, is not as well defined. Our initial aim was to identify the sequence important for the function of region II in DHBV. We scanned region II and the surrounding sequence by using a quantitative encapsidation assay. We found that the sequence between nucleotides (nt) 438 and 720 contributed to efficient pgRNA encapsidation, while the sequence between nt 538 and 610 made the largest contribution to encapsidation. Additionally, deletions between the two encapsidation sequences, epsilon and region II, had variable effects on encapsidation, while substitutions of heterologous sequence between epsilon and region II disrupted the ability of the pgRNA to be encapsidated efficiently. Overall, these data indicate that the intervening sequences between epsilon and region II play a role in encapsidation. We also analyzed heron hepatitis B virus (HHBV) for the presence of region II and found features similar to DHBV: a broad region necessary for efficient encapsidation that contained a critical region II sequence. Furthermore, we analyzed variants of DHBV that were substituted with HHBV sequence over region II and found that the chimeras were not fully functional for RNA encapsidation. These results indicate that sequences within region II may need to be compatible with other viral components in order to function in pgRNA encapsidation.     

Morphometric testing of structural hypotheses of the supraorbital region in modern humans

Our understanding of the functional morphology of the primate supraorbital region is based largely on previous morphometric and in vivo mechanical tests of hypotheses in non-human anthropoids. Prior tests of two structural hypotheses explaining morphological variation in the supraorbital region, the craniofacial size hypothesis and the spatial hypothesis, did not fully consider modern humans. We extend these previous findings to include modern humans by conducting morphometric tests of these two hypotheses in a sample of adult Melanesian crania. Morphometric correlates of structural predictions for the craniofacial size and spatial hypotheses were developed and compared to measurements of the supraorbital region via bivariate product-moment correlations. Measurements of the supraorbital region are significantly correlated with a craniofacial size estimate across individuals from this Melanesian sample. This result supports the prediction of the craniofacial size hypothesis that the magnitude of the supraorbital region is proportional to craniofacial size. The predicted link between the degree of neural-orbital disjunction and the magnitude of the supraorbital region, explicated in the spatial hypothesis, receives mixed support in the correlation analysis. These two results agree with previous research indicating that support for the craniofacial size and spatial hypotheses can be found across and within anthropoid primate species, including modern humans. Correlational support for both the craniofacial size and spatial hypotheses suggests multiple factors influence variation in the modern human supraorbital region. Thus, a single hypothesis cannot fully account for modern human variation in this region. The low bivariate correlation coefficients in this study further question whether existing hypotheses can adequately explain morphological variation in the supraorbital region in a primate population sample. Novel functional, structural, behavioral and developmental ideas must be explored if we are to better understand morphological variation in the modern human supraorbital region.     

Sequencing of selected regions of the human immunoglobulin heavy-chain gene locus that completes the sequence from JH through the delta constant region.

Much of the nucleotide sequence between the start of the joining region and the end of the immunoglobulin heavy chain delta gene has already been determined. However, two gaps existed in potentially functionally important regions in this sequence: the region between the 3' end of the joining region and the heavy chain enhancer region and that between the enhancer and the mu constant region. We have determined the nucleotide sequences of these regions. The 734 bp between the joining and enhancer regions contained no additional joining regions. The 4525 bp region between the heavy chain enhancer and the mu constant region contains the mu switch region, which consists of pentameric repeats. Approximately 60% of these repeats are GGGCT and GAGCT. With the determination of these sequences, the entire region of the heavy chain locus starting upstream of the joining region to downstream of the last exon of the delta constant region (a total of more than 29 kb) has now been sequenced.     

Linker region of a halobacterial transducer protein interacts directly with its sensor retinal protein

pHtrII, a pharaonis halobacterial transducer protein, possesses two transmembrane helices and forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, NpSRII) within the halobacterial membrane. This complex transmits a light signal to the sensory system located in the cytoplasm. It has been suggested that the linker region connecting the transmembrane region and the methylation region of pHtrII is important for binding to ppR and subsequent photosignal transduction. In this study, we present evidence to suggest that the linker region itself interacts directly with ppR in addition to the interaction in the membrane region. An in vitro pull-down assay revealed that the linker region bound to ppR, and its dissociation constant (K(D)) was estimated to be approximately 10 microM using isothermal titration calorimetry (ITC). Solution NMR analyses showed that ppR interacted with the linker region of pHtrII (pHtrII(G83)(-)(Q149)) and resulted in the broadening of many peaks, indicating structural changes within this region. These results suggest that the pHtrII linker region interacts directly with ppR. There was no demonstrable interaction between the C-terminal region of ppR (ppR(Gly224)(-)(His247)) and either the linker region (pHtrII(G83)(-)(Q149)) or the transmembrane region (pHtrII(M1)(-)(E114)) of pHtrII. On the basis of the NMR, CD, and photochemical data, we discuss the structural changes and role of the linker region of pHtrII in relation to photosignal transduction.