Naturally occurring mutations in human mitochondrial pre-tRNASer(UCN) can affect the transfer ribonuclease Z cleavage site, processing kinetics, and substrate secondary structure

tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. The 5' end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3' end trailer. Long ((L)) and short ((S)) forms of the tRNase Z gene are present in the human genome. tRNase Z(L) processes a nuclear-encoded pre-tRNA approximately 1600-fold more efficiently than tRNase Z(S) and is predicted to have a strong mitochondrial transport signal. tRNase Z(L) could, thus, process both nuclear- and mitochondrially encoded pre-tRNAs. More than 150 pathogenesis-associated mutations have been found in the mitochondrial genome, most of them in the 22 mitochondrially encoded tRNAs. All the mutations investigated in human mitochondrial tRNA(Ser(UCN)) affect processing efficiency, and some affect the cleavage site and secondary structure. These changes could affect tRNase Z processing of mutant pre-tRNAs, perhaps contributing to mitochondrial disease.     

The factor V-activating enzyme (RVV-V) from Russell's viper venom. Identification of isoproteins RVV-V alpha, -V beta, and -V gamma and their complete amino acid sequences

The complete amino acid sequences of two isoproteins of the factor V-activating enzyme (RVV-V) isolated from Vipera russelli (Russell's viper) venom were determined by sequencing S-pyridylethylated derivatives of the proteins and their peptide fragments generated by either chemical (cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine) or enzymatic (trypsin, alpha-chymotrypsin, and lysyl endopeptidase) cleavages. Both enzymes, designated RVV-V alpha and RVV-V gamma, consist of 236 amino acid residues and have a N-linked oligosaccharide chain at Asn229. The six amino acid substitutions between RVV-V alpha and -V gamma are: Thr22(alpha)-Ala22(gamma), Gly29(alpha)-Ala29(gamma), Gln191(alpha)-Glu191(gamma), Ile192(alpha)-Met192(gamma), Gln193(alpha)-His193(gamma), and Asn224(alpha)-Ser224(gamma). The molecular weights were calculated as 26,182 for RVV-V alpha and 26,167 for RVV-V gamma. The sequences of the RVV-V isoproteins exhibited 62% identity with that of batroxobin, a thrombin-like enzyme present in Bothrops atrox venom, and 33% identity with that of human thrombin B chain. The most interesting difference between the structures of RVV-V and other trypsin-type serine proteases is that the conservative Ser214-Trp215-Gly216 sequence (chymotrypsinogen numbering), considered as the site of antiparallel beta-sheet formation between the protein substrate and most serine proteases, has been replaced by the corresponding sequence Ala-Gly-Gly.     

Friedolanostane, friedocycloartane and benzophenone constituents of the bark and leaves of Garcinia benthami

Friedolanostanes, (22Z,24E)-3β-acetoxy-9α-hydroxy-17,14-friedolanosta-14,22,24-trien-26-oic acid, (22Z,24E)-3β,9α-dihydroxy-17,14-friedolanosta-14,22,24-trien-26-oic acid, (22Z,24E)-9α-hydroxy-3-oxo-17,14-friedolanosta-14,22,24-trien-26-oic acid, a friedocycloartane, (22Z,24E)-3α-hydroxy-17,13-friedocycloarta-12,22,24-trien-26-oic acid, and a benzophenone, benthaphenone, together with known compounds (22Z,24E)-3α,9α-dihydroxy-17,13-friedolanosta-12,22,24-trien-26-oic acid, methyl (24E)-3α,23-dihydroxy-17,14-friedolanosta-8,14,24-trien-26-oate, glutinol, lupeol, and stigmasterol, were isolated from leaves and bark of Garcinia benthami. Their structures were elucidated using spectroscopic techniques, mainly 1-D and 2-D NMR spectroscopy, and chemical correlations.     

Fine mapping of the chromosome 11q22-23 region using PFGE, linkage and haplotype analysis; localization of the gene for ataxia telangiectasia to a 5cM region flanked by NCAM/DRD2 and STMY/CJ52.75, phi 2.22.

Using pulsed-field gel electrophoresis, and a range of different enzyme digests, we have established that both markers of each of the pairs CJ52.208/YNB3.12, NCAM/DRD2, and STMY/CJ52.75, on chromosome 11q22-23, show physical linkage on a single DNA fragment. We have also shown, using genetic linkage and haplotype analyses, that these markers lie within a region of approximately 18cM, which, it has been shown previously, is likely to contain the A-T gene. The relative positions of these marker loci, and the distance between them was determined in order to construct a detailed map which has allowed a more precise localization of the A-T gene. We have shown that in pairwise linkage analysis the strongest support for linkage to the A-T gene was with the STMY/CJ52.75 locus (Z = 5.59, theta = 0.0). A three-point analysis using the results from STMY/CJ52.75 and the closely linked marker phi 2.22 gave Z = 5.55, theta = 0.03. Despite persisting evidence of some linkage to Thy-1 our results are consistent with the existence of a single A-T locus on chromosome 11q22-23 and our best estimate of the position of this locus places it between NCAM/DRD2 and (STMY/CJ52.75, F2.22) (Z = 6.74), a region of approximately 5cM in males.     

The gene for X-linked hydrocephalus maps to Xq28, distal to DXS52.

We report the study of five independent X-linked hydrocephalus (HSAS1) families with polymorphic DNA markers of the Xq28 region. A total of 58 individuals, including 7 living affected males and 22 obligate carriers, have been studied. Maximum lod score was 7.21 at theta = 2.40% for DXS52 (St14-1). A single recombination event was observed between this marker and the HSAS1 locus. Other markers studied were DXS296 (Z = 2.02 at theta = 2.5%), DXS304 (Z = 4.37 at theta = 7.8%), DXS74 (Z = 3.50 at theta = 0%), DXS15 (Z = 1.96 at theta = 5.7%), DXS134 (Z = 3.31 at theta = 0%), and F8C (Z = 5.79 at theta = 0%). These data confirm the localization of the HSAS1 gene to Xq28 and provide evidence for genetic homogeneity of this syndrome. In addition, examination of two obligate recombinant meioses along with multipoint linkage analysis supports the distal localization of the HSAS1 locus with respect to the DXS52 cluster. These observations are of potential interest for future studies aimed at HSAS1 gene characterization.     

Of orphons and UHOs. Delimitation of the germline repertoire of human immunoglobulin kappa genes.

Two problems in defining the germline repertoire of immunoglobulin kappa genes were investigated. One concerns putative transposed V kappa genes (orphons), the other one weak hybridization signals which may or may not turn out to be V kappa genes (UHOs). It was shown by sequencing that the three V kappa genes Z2, Z3 and Z4 are very closely related to the Z1 and V118 genes and to two other genes which had been localized on chromosomes 1 and 22, i.e. outside the kappa locus on chromosome 2. It is therefore likely that also the Z2-Z4 genes are orphons and not part of the kappa locus. Two UHOs turned out not to contain V kappa-like structures. This together with previous results makes it likely that we have detected all germline V kappa genes with the available hybridization probes.     

Molecular structure, conformation and interactions of antitumor antibiotic cyanonaphthridinomycin, a covalent binder of DNA

X-ray, NMR and molecular modeling studies on cyanonaphthridinomycin (C22H26N4O5), a DNA binding antibiotic, have been carried out to study the structure, conformation and interactions with DNA. The crystals belong to the space group P21 with the cell dimensions of a = 5.934(1)b = 20.684(4), c = 16.866(3)A, gamma = 90.9 degrees and Z = 4(two molecules/asymmetric unit). The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.087 for 4061 reflections. The conformation of the molecule is compared with that of naphthridinomycin. There are differences in the orientation of the methoxyl group and the saturated oxazole ring. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. Molecular mechanics studies were carried out to obtain the energy minimized structure and its comparison with X-ray and NMR results. Molecular modelling studies were performed to propose models for drug-DNA interactions. Both partial intercalation and groove-binding models have been proposed.     

A protein, connected with the integrity of the recF gene in Escherichia coli K-12

Summary A protein of molecular weight 74,000, called protein Z, has been identified in cells of the genotype recB21 recC22 sbcB15 by SDS-polyacrylamide gel electrophoresis. This protein has not been detected in cells of the genotype recB21 recC22 sbcB15 recF144. The transductional transfer of recF144 into the rec ⁺ cells leads to the disappearance of the protein Z band. These results demonstrate that the recF gene is essential for protein Z synthesis. Of two recF ^– mutants studied, recF144 completely lacks protein Z, while recF143 preserves a functionally inactive protein Z, probably resulting from a missense mutation.The recF144 cells are characterizied by a very low frequency of genetic exchange between the donor and recipient chromosomes after conjugation. The scale of the genetic map for these cells is 3-fold higher than for wild-type cells.     

白豆杉的核型和性染色体的研究

白豆杉pseudotaxus chienii(Cheng)Cheng是我国裸子植物特有属之一,雌雄异常,根尖 细胞染色体分析表明：雌株有一对异形性染色体,异配性别,属ZW型;雄株是同配性别,属ZZ型,雌株的型为2n=2x=24=22m(2SAT ZW) 2T,雄株的核型为2n=2x=24=22m(2SAT ZZ) 2T。Giemsa C-带,显示,Z染色体长短臂均具端带,W染色体不显带。     

中华绒螯蟹幼体发育阶段对淀粉营养需要的研究

本实验研究了饲料中淀粉含量对中华绒螯蟹幼体生长发育与淀粉酶活力的影响。结果表明:饲料中不同淀粉含量对中华绒螯蟹幼体存活率、变态率和增重量的影响显著(P1、Z2、Z3、Z4)或下降(Z5、M)趋势,且饲料中淀粉含量对幼体淀粉酶活力变化有显著影响(P1、Z2、Z34、Z5、M的适宜淀粉需求量分别为18%、22%、18%、18%-22%、18%、14%-18%。       

Leaf architecture and epidermal characters in Zelkova, Ulmaceae

Zelkova , with six extant species in Eurasia - three in East Asia (Z. schneideriana, Z. serrata, Z. sinica) , one in south-western Asia (Z. carpinifolia) and two in southern Europe (Z. abelicea, Z. sicula) - was investigated with respect to leaf architecture and epidermal characters by LM and SEM. The leaf venation of Zelkova is relatively uniform, while leaf size and shape are highly variable. Characters such as teeth and epidermal cells, trichomes and stomata provide useful specific distinctions. Beginning in the Miocene, increasing aridity in Central Asia would appear to have been responsible for isolating the eastern Asiatic species from their European/western Asiatic counterparts. The European range underwent further changes in response to Quaternary climatic oscillations.     

十种姜科植物的染色体数目研究

报道了姜科3属10种植物的染色体数目。(1)姜属5种:版纳姜2n=22,弯管姜2n=22,圆瓣姜2n=22,红球姜2n=22,紫色姜2n=22;(2)舞花姜属4种:舞花姜2n=24,毛舞花姜2n=48,双翅舞花姜2n=48,澜沧舞花姜2n=32;(3)凹唇姜属1种:白斑凹唇姜2n=36。其中弯管姜、圆瓣姜、紫色姜、澜沧舞花姜、白斑凹唇姜的染色体数目为首次报道。     

Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope

Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K_d of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.     

Theoretical studies of the electrostatic stability of A,B, C,and Z-DNA conformers as a function of monovalent ion concentration and ion type

Theoretical calculations using a two-state model of counterion phosphate interactions have been performed in order to determine the electrostatic stability of DNA in various ionic environments. Comparison of the stabilities of different conformations leads to results consistent with the B→Z transitions observed in high-salt environments and suggest that at very low ionic strnegths the left-handed Z(II) form is an electrostatically preferred conformation. An ion-type dependence of the high-salt B→Z transition midpoint is predicted. Calculations of the energies of the A and C conformations are also discussed in relation to experimentally observed transitions.     

Genetic homogeneity of cystic fibrosis.

We studied large Amish/Mennonite/Hutterite kindreds that segregate cystic fibrosis (CF) for linkage between CF and the polymorphic DNA markers pJ3.11 and 7C22 located on chromosome 7. These inbred pedigrees consist of more than 300 members including 30 affected individuals. In these families, linkage between the CF locus and the chromosome 21 marker D21S5 and between CF and the marker at the met oncogene locus on chromosome 7 had been previously indicated. We now report linkage between CF and pJ3.11 (Z = 4.92, theta = 0) and between CF and 7C22 (Z = 3.42, theta = 0). Therefore, CF segregates in these large pedigrees in a manner consistent with data from smaller outbred families with respect to the markers on chromosome 7 closest to CF. These data are consistent with locus homogeneity for the defect causing CF in the populations that have been examined to date.     

Large-scale preparation of (9Z,12E)-[1-(13)C]-octadeca-9,12-dienoic acid, (9Z,12Z,15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid and their [1-(13)C] all-cis isomers

Several grams of labelled trans linoleic and linolenic acids with high chemical and isomeric purities (>97%) have been prepared for human metabolism studies. A total of 12.5 g of (9Z, 12E)-[1-(13)C]-octadeca-9,12-dienoic acid and 6.3 g of (9Z,12Z, 15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid were obtained in, respectively, seven steps (7.8% overall yield) and 11 steps (7% overall yield) from 7-bromo-heptan-1-ol. The trans bromo precursors used for the labelling were synthesised by using copper-catalysed couplings. The trans fatty acids were then obtained via the nitrile derivatives. A total of 23.5 g of (9Z,12Z)-[1-(13)C]-octadeca-9, 12-dienoic acid and 10.4 g of (9Z,12Z,15Z)-[1-(13)C]-octadeca-9,12, 15-trienoic acid were prepared in five steps in, respectively, 32 and 18% overall yield. Large quantities of bromo and chloro precursors were synthesised from the commercially available acid according to Barton's procedure. In all cases, the main impurities (>0.5%) of each labelled fatty acid have been characterised.     

B-Z conformational transition and hydration of poly (dC-dG).poly (dC-dG) in fibres.

The B-Z transition of poly(dC-dG).poly(dC-dG) has been studied by fibre X-ray diffraction and measurement of fibre dimensions. The polymorphism of the Z form is well observed as a function of variations of the r.h. (relative humidity). The Z to B transition is obtained at very high r.h. values. The cooperative transition from B to Z is associated with a disorganization of the fibre. Details about the hydration of the polynucleotide during conformational transitions are presented and it is shown that a nucleotide in Z form can be associated with up to 16 water molecules and up to 22 when in the B form.     

Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli.

Zymomonas mobilis ferments sugars to produce ethanol with two biochemically distinct isoenzymes of alcohol dehydrogenase. The adhA gene encoding alcohol dehydrogenase I has now been sequenced and compared with the adhB gene, which encodes the second isoenzyme. The deduced amino acid sequences for these gene products exhibited no apparent homology. Alcohol dehydrogenase I contained 337 amino acids, with a subunit molecular weight of 36,096. Based on comparisons of primary amino acid sequences, this enzyme belongs to the family of zinc alcohol dehydrogenases which have been described primarily in eucaryotes. Nearly all of the 22 strictly conserved amino acids in this group were also conserved in Z. mobilis alcohol dehydrogenase I. Alcohol dehydrogenase I is an abundant protein, although adhA lacked many of the features previously reported in four other highly expressed genes from Z. mobilis. Codon usage in adhA is not highly biased and includes many codons which were unused by pdc, adhB, gap, and pgk. The ribosomal binding region of adhA lacked the canonical Shine-Dalgarno sequence found in the other highly expressed genes from Z. mobilis. Although these features may facilitate the expression of high enzyme levels, they do not appear to be essential for the expression of Z. mobilis adhA.     

Molecular Structure,Conformation and Interactions of Antitumor Antibiotic Cyanonaphthridinomycin,a Covalent Binder of DNA

Abstract

X-ray, NMR and molecular modeling studies on cyanonaphthridinomycin (C₂₂ H₂₆N₄O₅), a DNA binding antibiotic, have been carried out to study the structure, conformation and interactions with DNA. The crystals belong to the space group P₂₁ with the cell dimensions of a = 5.934(1), b = 20.684(4), c = 16.866(3)A γ = 90.9° and Z = 4(two molecules/asymmetric unit). The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.087 for 4061 reflections. The conformation of the molecule is compared with that of naphthridinomycin. There are differences in the orientation of the methoxyl group and the saturated oxazole ring. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. Molecular mechanics studies were carried out to obtain the energy minimized structure and its comparison with X-ray and NMR results. Molecular modelling studies were performed to propose models for drug-DNA interactions. Both partial intercalation and groove-binding models have been proposed.     

The Z family, a group of transposed human immunoglobulin V kappa genes

A group of highly homologous transposed human V kappa I genes, which we call the Z family, was characterized. To date four members, ZI-ZIV, comprising about 230 kb, have been analyzed on cosmid clones. The largest region (ZI) has a length of 85 kb. The Z regions show extensive homology to each other according to restriction maps and hybridization data. In each Z region a solitary V kappa I gene was found. No V kappa genes of other subgroups were detected by hybridization. The nucleotide sequence of the ZI gene revealed a non-processed V kappa I pseudogene. Hybridization experiments with DNAs from rodent/human cell hybrids and other experimental data indicate that some and possibly all members of the Z family lie outside of the kappa locus which is located on chromosome 2; they have been transposed to other chromosomes. Because of their separation from the J kappa C kappa gene segment, the Z genes can be classified as pseudogenes independent of their sequences. We postulate that the Z family arose by amplification event(s). The Z regions can also be regarded as a small family of very long repetitive sequences.