Modes of binding of guanosine monophosphates to ribonuclease T1 - A computer modelling study

Computer-modelling studies on the modes of binding of the three guanosine monophosphate inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to ribonuclease (RNase) T1 have been carried out by energy minimization in Cartesian-coordinate space. The inhibitory power was found to decrease in the order 2'-GMP > 3'-GMP > 5'-GMP in agreement with the experimental observations. The ribose moiety was found to form hydrogen bonds with the protein in all the enzyme-inhibitor complexes, indicating that it contributes to the binding energy and does not merely act as a spacer between the base and the phosphate moieties as suggested earlier. 2'-GMP and 5'-GMP bind to RNase T1 in either of the two ribose puckered forms (with C3'-endo more favoured over the C2'-endo) and 3'-GMP binds to RNase T1 predominantly in C3'-endo form. The catalytically important residue His-92 was found to form hydrogen bond with the phosphate moiety in all the enzyme-inhibitor complexes, indicating that this residue may serve as a general acid group during catalysis. Such an interaction was not found in either X-ray or two-dimensional NMR studies.

Computer modelling studies of ribonuclease A-pyrimidine nucleotide complexes

Different modes of binding of pyrimidine monophosphates 2'-UMP, 3'-UMP, 2'-CMP and 3'-CMP to ribonuclease (RNase) A are studied by energy minimization in torsion angle and subsequently in Cartesian coordinate space. The results are analysed in the light of primary binding sites. The hydrogen bonding pattern brings out roles for amino acids such as Asn44 and Ser123 apart from the well known active site residues viz., His12,Lys41,Thr45 and His119. Amino acid segments 43-45 and 119-121 seem to be guiding the ligand binding by forming a pocket. Many of the active site charged residues display considerable movement upon nucleotide binding.

Computer modeling studies on the binding of 2',5'-linked dinucleoside phosphates to ribonuclease T1-influence of subsite interactions on the substrate specificity

The modes of binding of Gp(2',5')A, Gp(2',5')C, Gp(2',5')G and Gp(2',5')U to RNase T1 have been determined by computer modelling studies. All these dinucleoside phosphates assume extended conformations in the active site leading to better interactions with the enzyme. The 5'-terminal guanine of all these ligands is placed in the primary base binding site of the enzyme in an orientation similar to that of 2'-GMP in the RNase T1-2'-GMP complex. The 2'-terminal purines are placed close to the hydrophobic pocket formed by the residues Gly71, Ser72, Pro73 and Gly74 which occur in a loop region. However, the orientation of the 2'-terminal pyrimidines is different from that of 2'-terminal purines. This perhaps explains the higher binding affinity of the 2',5'-linked guanine dinucleoside phosphates with 2'-terminal purines than those with 2'-terminal pyrimidines. A comparison of the binding of the guanine dinucleoside phosphates with 2',5'- and 3',5'-linkages suggests significant differences in the ribose pucker and hydrogen bonding interactions between the catalytic residues and the bound nucleoside phosphate implying that 2',5'-linked dinucleoside phosphates may not be the ideal ligands to probe the role of the catalytic amino acid residues. A change in the amino acid sequence in the surface loop region formed by the residues Gly71 to Gly74 drastically affects the conformation of the base binding subsite, and this may account for the inactivity of the enzyme with altered sequence i.e., with Pro, Gly and Ser at positions 71 to 73 respectively. These results thus suggest that in addition to recognition and catalytic sites, interactions at the loop regions which constitute the subsite for base binding are also crucial in determining the substrate specificity.

Accurate transcription initiation by RNA polymerase II from Candida utilis

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis

Molecular dynamics studies on nucleoside 2',3'-cyclic phosphates

2',3'-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3'-GMP, 3'-UMP, A>p, G>p, U>p, C>p, GpA>p and UpA>p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.

Essential dynamics and sidechain hydrogen bond cluster studies on eosinophil cationic protein

Eosinophil Cationic Protein (ECP) is a member of RNase A superfamily which carries out the obligatory catalytic role of cleaving RNA. It is involved in a variety of biological functions. Molecular dynamics simulations followed by essential dynamics analysis on this protein are carried out with the goal of gaining insights into the dynamical properties at atomic level. The top essential modes contribute to subspaces and to the transition phase. Further, the sidechain-sidechain/sidechain-mainchain hydrogen bond clusters are analyzed in the top modes, and compared with those of crystal structure. The role of residues identified by these methods is discussed in the context of concerted motion, structure and stability of the protein.

A molecular dynamics study based post facto free energy analysis of the binding of bovine angiogenin with UMP and CMP ligands

Angiogenin is a protein belonging to the superfamily of RNase A. The RNase activity of this protein is essential for its angiogenic activity. Although members of the RNase A family carry out RNase activity, they differ markedly in their strength and specificity. In this paper, we address the problem of higher specificity of angiogenin towards cytosine against uracil in the first base binding position. We have carried out extensive nano-second level molecular dynamics(MD) computer simulations on the native bovine angiogenin and on the CMP and UMP complexes of this protein in aqueous medium with explicit molecular solvent. The structures thus generated were subjected to a rigorous free energy component analysis to arrive at a plausible molecular thermodynamic explanation for the substrate specificity of angiogenin.

Cytokine control of neuronal phenotype

Diffusible proteins regulate neural development at a variety of stages. Using a novel neuronal culture assay, I have identified several cytokines that regulate the expression of neurotransmitters and neuropeptides in sympathetic neurons. These cytokines fall into two families. The first group is termed the neuropoietic cytokines, while including CDF/LIF, CNTF, OSM and GPA, induces expression of the same set of neuropeptide mRNAs in cultured sympathetic neurons. These four factors not only exhibit similar biological activities; they also share a predicted secondary structure and bind to a signal-transducing receptor subunit in common with IL-6 and IL-11. The latter two cytokines display a weaker activity in this assay. In addition, I find that several members of the TGF-β superfamily, activin A, BMP-2, and BMP-6, have a selective overlap with the neuropoietic family in the spectrum of neuropeptides that these cytokines induce in sympathetic neurons. Different patterns of neuropeptides induced by the TGF-β family members, however, demonstrate that the activities of these cytokines are distinct from those of the neuropoietic family. Another 30 cytokines are without detectable effect in this neuronal assay.

Activin A induces a set of neurotransmitters and neuropeptides that is somewhat similar to the phenotype of sympathetic neurons innervating sweat glands in rat footpads. In situ hybridization and RNase protection were carried out to test whether activins were involved in the phenotypic transition when sympathetic neurons contact sweat glands. I find that activin mRNA is present in both cholinergic and noradrenergic targets. Moreover, homogenates of footpads do not contain activin-like activity in the neuronal assay in vitro. Taken together, these data do not support activins as the best candidates for the sweat gland factor.

Several novel factors that regulate neuropeptide expression exist in heart cell conditioned medium. I attempted to purify these factors in collaboration with Dr. Jane Talvenheimo. Our results suggest that these factors are sensitive to the storage conditions used. Several modifications of purification strategy are discussed.

Studies on 4S and 5S RNA of HeLa cells

SECTION I

Section I is concerned with a partial sequence analysis conducted on 5S RNA from HeLa cells. Analysis of the oligonucleotide pattern after pancreatic ribonuclease digestion of a highly-purified preparation of 5S RNA gave results which were in general agreement with those published for KB cells, both with respect to the identity and the frequency of the partial sequences. However, the presence of a trinucleotide not found in the KB 5S pattern, together with the reproducibly much lower than expected molar yield of the larger oligonucleotides strongly suggested the occurrence of alternate sequences at various sites in the 5S molecules of human cells. The presence of ppGp and pppGp at the 5'-terminus of HeLa 5S RNA was clearly demonstrated. The implications of this finding with regard to the origin of 5S RNA are discussed.

SECTION II

In Section II the proportion of the HeLa cell genome complementary to tRNA was investigated by using RNA- DNA hybridization. The value for saturation of the HeLa DNA by tRNA was found to be 1.1 x 10^-5, which corresponds to about 4900 sites for tRNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized tRNA revealed significant differences from the nucleotide composition of the input tRNA, with the purine to pyrimidine ratio indicating, however, that these differences were not produced by excessive RNase attack of the hybrid. The size of the hybridized tRNA was only moderately smaller than that of the input RNA; the average S value in formaldehyde was 2.7 (corresponding to a length of about 65 nucleotides), suggesting that a relatively small portion near the ends of the hybridized 4S chains had been removed by RNase.

SECTION III

The proportion of the HeLa cell genome complementary to 5S RNA was investigated by using RNA-DNA hybridization. The value for saturation of the HeLa DNA by 5S RNA was found to be 2.3 x 10^-5, which corresponds to about 7,000 sites for 5S RNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized 5S RNA revealed no significant difference from the nucleotide composition of the input RNA. At the RNA to DNA input ratio of 1:1000, the average S value in formaldehyde of the hybridized 5S RNA corresponded to a polynucleotide chain about two-thirds the size of the input RNA.

Studies on rabbit reticulocyte ribosomes and polyribosomes and their relation to hemoglobin synthesis

This dissertation is divided into three parts.

The first section is concerned with protein synthesis in cellfree systems from reticulocytes. The sub-cellular reticulocyte fractions, reagents, etc. have been examined for the presence of traces of ribonuclease, using. an assay based upon the loss of infectivity of RNA fran bacteriophage MS2. This assay is sensitive to 5 x 10^-7 γ RNase/ml. In addition, the loss of synthetic capacity of an 80S ribosome on dissociation has been studied, and can be attributed to loss of messenger RNA when the monomer is separated into subunits. The presence of ribonuclease has been shown to be a major cause of polyribosome disintegration during cell-free protein synthesis.

The second section concerns the changes in ribosomes and polyribosomes which occur during the maturation of a reticulocyte into an erythrocyte. With increasing age, the cells lose a large proportion of the ribonucleoprotein, but the percentage of ribosomes present as polyribosomes is only slightly altered. The loss of hemoglobin synthesis on maturation is probably due to both the loss of total ribosomes and to the lessened specific activity of the polyribosomes.

The third section contains analytical ultracentrifugation data on 80S ribosomes, polyribosomes, and ribosomal RNA from reticulocytes. The 60s and 40s subunits, obtained by dissociation of the 80s particle with inorganic pyrophosphate, were also studied. The RNA from reticulocyte ribosomes has been examined under a variety of denaturing conditions, including dimethyl sulfoxide treatment, formaldehyde reaction and thermal denaturation. From these studies we can conclude that the 28S and 16S RNA's are single polynucleotide chains and are not made up of smaller RNA subunits hydrogen-bonded together.

Adaptive Evolution of Digestive RNASE1 Genes in Leaf-Eating Monkeys Revisited: New Insights from Ten Additional Colobines

Pancreatic RNase genes implicated in the adaptation of the colobine monkeys to leaf eating have long intrigued evolutionary biologists since the identification of a duplicated RNASE1 gene with enhanced digestive efficiencies in Pygathrix nemaeus. The recent emergence of two contrasting hypotheses, that is, independent duplication and one-duplication event hypotheses, make it into focus again. Current understanding of Colobine RNASE1 gene evolution of colobine monkeys largely depends on the analyses of few colobine species. The present study with more intensive taxonomic and character sampling not only provides a clearer picture of Colobine RNASE1 gene evolution but also allows to have a more thorough understanding about the molecular basis underlying the adaptation of Colobinae to the unique leaf-feeding lifestyle. The present broader and detailed phylogenetic analyses yielded two important findings: 1) All trees based on the analyses of coding, noncoding, and both regions provided consistent evidence, indicating RNASE1 duplication occurred after Asian and African colobines speciation, that is, independent duplication hypothesis; 2) No obvious evidence of gene conversion in RNASE1 gene was found, favoring independent evolution of Colobine RNASE1 gene duplicates. The conclusion drawn from previous studies that gene conversion has played a significant role in the evolution of Colobine RNASE1 was not supported. Our selective constraint analyses also provided interesting insights, with significant evidence of positive selection detected on ancestor lineages leading to duplicated gene copies. The identification of a handful of new adaptive sites and amino acid changes that have not been characterized previously also provide a necessary foundation for further experimental investigations of RNASE1 functional evolution in Colobinae.

Inhibition of no tail (ntl) gene expression in zebrafish by external guide sequence (EGS) technique

External guide sequence (EGS) technique, a branch of ribozyme strategy, can be enticed to cleave the target mRNA by forming a tRNA-like structure. In the present study, no tail gene (ntl), a key gene participating in the formation of normal tail, was used as a target for ribonuclease (RNase) P-mediated gene disruption in zebrafish in vivo. Transient expression of pH1-m3/4 ntl-EGS or pH1-3/4 ntl-EGS produced the full no tail phenotype at long-pec stage in proportion as 24 or 35%, respectively. As is expected that the full-length ntl mRNA of embryos at 50% epiboly stage decreased relative to control when injected the embryos with 3/4 EGS or m3/4 EGS RNA. Interestingly, ntl RNA transcripts, including the cleaved by EGS and the untouched, increased. Taken together, these results indicate that EGS strategy can work in zebrafish in vivo and becomes a potential tool for degradation of targeted mRNAs.

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays all important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. Coli Under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1. excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His-Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.

Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp

Background: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. Methods: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4degreesC. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 mug/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. Results: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5-38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. Conclusions: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/ chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus). Cytometry Part A 56A:46-52, 2003. (C) 2003 Wiley-Liss, Inc.

基于核酸特殊结构的分子识别及核酸相关纳米结构的设计应用

核酸为生命的最基本物质之一，是生物体遗传信息的携带者，在生长、遗传、变异等一系列重大生命现象中起决定性的作用。以核酸作为新药设计的靶分子，越来越受到人们的广泛重视。然而，不像其它靶分子如蛋白质、受体等具有特定的结构和功能，核酸结构在很多情况下是同源的，而且联系到很多人体正常的生理功能；能够与核酸结合的药物又往往不具有序列选择性，这就带来明显的毒副作用。因此，寻找和发现一些与疾病相关的核酸的特殊结构，并筛选对其有特异性结合能力的小分子，是以核酸为靶的药物研究的一个重要课题。 近年来，随着纳米科学技术的兴起，以核酸作为纳米体系的结构材料开始受到人们的广泛关注。作为一类特殊的线性高分子，核酸具有化学性质稳定，结构丰富且可控，良好的刚性和柔性，精确识别，高度生物相容性，合成方便等诸多优点，是一类优良的结构材料。目前核酸相关的纳米组装结构和器件研究还处于起步阶段，但是已经呈现出良好的发展前景。 本论文主要针对核酸特殊结构的分子识别及核酸相关功能纳米结构的设计这两方面展开了研究，全文由以下两大部分组成： 第一部分通过光谱学和生物化学等手段，研究了小分子对不同核酸结构的识别作用。借助于竞争平衡透析技术，发现一类恶嗪染料(oxazine dyes)能够与多种结构核酸结合。热变性及光谱实验结果表明，oxazine染料能够诱导杂合体三链核酸poly(rA):2poly(dT)的形成，并强烈地稳定其结构，其中以cresyl violet作用最强，是迄今发现的化合物中最强的。进一步研究发现，此类化合物以嵌插方式与杂合体三链核酸结合。RNase H酶切实验表明，杂合体三链核酸的形成能够强烈地抑制RNase H核酸酶的活性。研究了oxazine-170与三链核酸poly(dA):2poly(dT)及poly(rA):2poly(rU)的相互作用，发现oxazine-170能够强烈稳定三链DNA poly(dA):2poly(dT)的结构，而对相应双链DNA不具稳定作用；对三链RNA poly(rA):2poly(rU)及相应的双链RNA都有一定稳定作用，但作用不强。进一步研究发现，oxazine-170能够以两种结合方式与核酸结合：嵌插方式和外部静电堆积作用。研究了oxazine-170及cresyl violet与单链核酸的相互作用。研究发现oxazine-170能够序列特异性地与单链核酸poly(rA) 结合，CD光谱及AFM研究发现oxazine-170诱导poly(rA)形成新的二级结构。UV光谱、FL光谱及RLS研究发现poly(rA)促使oxazine 170形成H-aggregate，并以poly(rA)为模板自组装。而cresyl violet能够与单链核酸poly(rA)及poly(dA)结合，且采用不同的结合方式： cresyl violet能够与oxazine-170 类似地以poly(rA)为模板自组装；以嵌插方式与poly(dA)结合，并诱导其单链碱基堆积方式的改变。通过以上实验结果，我们进一步揭示了oxazine染料作为肿瘤细胞染色及光动力学治疗试剂的结构基础，对进一步设计、合成更加高效的抗肿瘤药物具有一定的指导意义。 第二部分中，我们尝试设计了几种基于核酸的纳米结构功能体系，并讨论了其相关应用。利用有机小分子coralyne能够诱导聚腺嘌呤序列反平行双链结构的形成，实现了一类新型的小分子诱导的纳米金组装结构。并以(dA)16功能化的金纳米粒子作为新型纳米探针，发展了一种简单的筛选单链核酸聚腺嘌呤序列结合分子的筛选方法。利用核酸限制性内切酶酶切位点回文序列的结构特点，设计了一种以DNA功能化的金纳米粒子组装体为酶切底物的比较通用的核酸限制性内切酶活力检测方法，并进一步用于甲基化酶活性检测及其抑制剂的筛选。基于单链DNA富胞嘧啶i-motif结构在不同pH值条件下的形成与解离，设计了一类质子驱动的DNA分子镊子，与基于链交换反应的DNA分子镊子相比，该体系更加简单，工作效率更高。随后，我们又通过合理设计，得到了两种分别能够结合与释放DNA和蛋白的分子镊子，为其应用做了一些探索。