recombination via restriction endonucleases and genetic translocation of an impicillin resistance gene, have been utilized for the construction of a series of cloning vehicles. These vectors have been derived by combining the essential replication properties of plasmid pMBl, a CoLEl type plasmid, with one, two or three resistance markers (tetracycline, ampicillin, chloramphenicol and colicin El production). During the construction of these vectors, the position of restriction sites used for cloning DNA fragments, relative to the antibiotic resistance genes and the origin of DNA replication, have been determined. The use of the cloning vectors pMB9, pBR313, pBR322, pBR324, pBR325 permits the molecular cloning and easy selection of RI, HI, II, II, III, I, I, I, I, I, II, I, I, I, l I, I, I, II, III, I, II, RII and virtually any blunt-ended generated DNA fragment. 相似文献
1H nuclear magnetic resonance spectra of 1 - (II) and 3-deazaadenosines (III) together with adenosine (I) in dimethylsulfoxide have been examined. Features of coupling constants indicate that the furanose rings of I, II, and III have similar conformational preferences and that conformations about the 4′-C–5′-C bond are preferentially . Nuclear Overhauser effect and spin-lattice relaxation-time measurements demonstrate that II predominantly adopts the -conformation similar to that of I, whereas that of III has a greater (freely rotating) component. The results suggest that the -conformation in II as well as I is stabilized presumably through a hydrogen bond between the 3-N and 5′-hydroxyl group. 相似文献
Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, and , showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of maize, but this activity was regenerated by the addition of any branching enzyme. 相似文献
Chenooxazoline3 (50–100 μM) inhibited (>50%) both 7α and 7β-dehydroxylase activities in whole cells and cell extracts of sp. V.P.I. 12708. Chenooxazoline (>50 μM) and methylchenooxazoline (>25 μM) but not lithooxazoline (≤100 μM) inhibited growing cultures of sp. V.P.I. 12708. Chenooxazoline (100 μM) also inhibited the growth of certain members of the genera , , and but not , , or the eucaryotic microorganism, (_< 400 μM). 相似文献
The binding of inhibitors to site I of rabbit muscle phsphorylase has beenstudied kinetically and thermodynamically for caffeine, adenine and adenosine. The effect of ligands on the tertiary structure has been investigated by studying the protection against 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) titration of the slow-reacting sulphydryl groups of the enzyme. Calorimetric and cysteinyl protection data taken together suggest that these inhibitors bind to both sites N and I even under conditions of saturation by glucose. Calorimetric results show that inhibitor binding to sites I and N at 25°C is driven enthalpically, although both and of interaction are significant. We conclude that attractive dispersion forces ought to be the main ones responsible for inhibitor binding to site I. AMP-activated phosphorylase is inhibited by both caffeine and adenine by cooperative and exclusive binding to the inactive conformation. The binding of the substrate (phosphate) and AMP when adenine is present was found to be exlusive to the active conformation, whereas non-exclusive binding of the activator was observed when caffeine was added. 相似文献
Methylated amino acids from ribosomal protein L33 of various strains (Q13, B and MRE600) were analyzed. It was found that while protein L33 from Q13 contains two methylated neutral amino acids (peaks I and II), only one methylated neutral amino acid (peak I) was found in protein L33 derived from both strains B and MRE600. The methylated amino acid present in peak I was identified as N-monomethylalanine by ion-exchange column chromatography, high-voltage paper electrophoresis and descending paper chromatography using different solvent systems. This marks the first time that N-monomethylalanine was found in any ribosomal protein. 相似文献
α-(1→2)--Fucosidase, β--galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β--Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor -acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by . 3) α-(1→2)--Fucosidase action is inhibited by or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action. 相似文献
The functional role of a chlorophyll complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, KP-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll complex and the other lacking the complex. KP-700 for the former preparation is half of that of the preparation which has the chlorophyll complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll complex. Experiments involving reconstitution of the chlorophyll complex with the antenna-depleted PS I preparation indicate a substantial recovery of the KP-700 rate. These results demonstrate that the chlorophyll complex functions as a light-harvesting antenna in PS I. 相似文献
Superior antitumor activity of 1-β-D-arabinofuranosylcytosine (ara-C) conjugates of prednisolone and prednisone against L1210 leukemic mice, based on ara-C content, has encouraged us to synthesize 5′-(cortisone-21-phosphoryl)-1-β-D-arabinofuranosylcytosine (I) and 5′-(cortisone-21-phosphoryl)-1-β-d-arabinofuranosylcytosine (II) by condensation of N4,2′,3′-triacetyl-1-β-d-arabinofuranosylcytosine 5′-monophosphate with cortisol and cortisone in the presence of N,N′-dicyclohexylcarbodiimide at room temperature followed by removing the acetyl groups in 2 N methanolic ammonia in 20% yield. The conjugates I and II inhibited the growth of L1210 by 50% (ED50) at 0.25 μM and 0.07 μM, respectively, while ara-C showed ED50 0.1 μM. However, the conjugates I and II exhibited 287% and 238% of at 50 mg/kg/day × 5 doses against L1210 leukemic mice, respectively, while ara-C at 25 mg and 50 mg/kg/day × 5 gave the respective 127% and 110% of . 相似文献
DNA polymerase activities in cell-free lysates of unfertilized eggs, larvae and immature ovaries of were compared to purified DNA polymerase I using several natural and synthetic templates. The templates were tested as the native and denatured forms of normal and DNase I treated molecules. Although the polymerases tended to prefer DNase I treated Xenopus DNA over the other templates tested, so did the polymerase I. In general, the template preferences of the polymerases studied depended in complex ways on both the form and the species of origin of the template. 相似文献
Recently, 1-β--arabinofuranosylcytosine-5′-diphosphate--1,2-dipalmitin (VIa) was reported to inhibit the growth of L51784 cells in mice and of human colon carcinoma HCT-15 cells, also in mice. This paper describes the synthesis of a single diastereomer by conversion of 1-β--arabinofuranosylcytosine 5′-monophosphate (II) to the nucleoside 5′-phosphomorpholidate (III), followed by reaction with -α-dipalmitoylphosphatidic acid (IV) to give 1-β--arabinofuranosylcytosine-5′-diphosphate--1,2-dipalmitin (V) in good yield. The separation of the product is described and its characterization by chromatography, elemental analysis, and spectroscopic methods. The lipophilic nature of V renders it insoluble in aqueous media and a method of sample preparation utilizing sonication techniques is described which provides a clear solution suitable for biological evaluation. In addition, the ability of V to inhibit the growth of L1210 cells and of mouse myeloma MPC 11 cells is desscribed and compared with 1-β--arabinofuranosylcytosine (I) and other lipophilic prodrugs of I. 相似文献
We show that the reductants present in the assay used to measure the formation of adenosylcobalamin from cob(III)alamin by cell-free extracts of human fibroblasts result in the non-enzymatic reduction of cob(III)alamin to cob(I)alamin. Hence, the assay uniquely estimates the activity of ATP:cob(I)alamin adenosyltransferase (EC 2.5.1.17). Based on additional studies with extracts of fibroblasts from patients in the class of human methylmalonic acidemia and from their parents, we conclude that this mutant class results from a specific deficiency of adenosyltransferase activity which is inherited as an autosomal recessive trait. 相似文献
First successful synthesis of functional photosynthetic phosphorylating membrane is reported. Etioplasts, highly enriched in cytoplasmic and plastid proteins, isolated from etiolated Cucumber cotyledons pretreated with kinetin and gibberellic acid, and illuminated in a cofactor fortified medium showed commencement of chlorophyll (Chl) synthesis immediately after illumination from exogenous δ-aminolevulinic acid, while photosystem I (PS I) activity commenced 15 min after the onset of illumination. When cotyledons pretreated with kinetin and gibberellic acid were illuminated directly, there was a lag phase of 30 min before the commencement of Chl synthesis and PS I activity developed after 1 h of illumination. In plastids developed both and , the electron flow from dichlorophenolindophenol to methyl-viologen was coupled to phosphorylation as observed by an increase in the electron transport rate on the addition of uncouplers. Analysis of polypeptide profiles of the greening plastids showed the disappearance of many higher molecular weight proteins during greening. Polypeptides of molecular weight 32, 20.5, 19.5 K absent in etioplasts appeared as distinct bands after 4 h of greening . 相似文献
The cleavage of the kDNA minicircles of by the restriction endonucleases I, II, I, I and II revealed that this kDNA is homogeneous in base sequence. This is in contrast with the kDNA of minicircles of the other species of trypanosomes so far studied. The 10 cleavage sites, obtained with these endonucleases, were ordered and a restriction cleavage map of the minicircles was thus drawn. 相似文献
Repeated Biogel P6 chromatography of the urine from a patient with fucosidosis yielded several fractions containing fucosyloligosaccharides and glycopeptides. Two of these were shown by 1H nuclear magnetic resonance (1H-n.m.r.) spectroscopy and permethylation analysis to have the following structures respectively: (I) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman () βman (1→4) glcNAc and (II) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman () βman (1→4) βglcNAc (1→4) [αfuc ()] βglcNAc-Asn. 相似文献
Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I () and X.Oxidation-reduction potential titrations confirmed that Centre A had , Centre B had . These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with mV, substantiating the identification of X as the primary electron acceptor of Photosystem I. 相似文献
The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as (78 residues), (39 residues), (21 residues) and (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure. 相似文献
A proteinase (called Proteinase I) present in myxamoebae of the cellular slime mold, , was labeled with [32P] by growth of cells on media containing [32P] orthophosphate. The labeled proteinase was purified to apparent homogeneity and characterized by dissociation chromatography and quantitative immune-precipitin analysis. Based upon the results of these studies it was concluded that phosphoryl moieties were tightly associated (presumably covalently bonded) with the polypeptide subunits of Proteinase I. 相似文献
A partially cleaved α1(I) chain, α1χ, has been isolated from earlier synthesized or older (acid-extracted) guinea pig skin collagen. The α1χ component is shown to be absent from the newly synthesized (neutral salt-extracted) collagen. This degradation is a result of specific proteolytic sission of α1(I) chain since the soluble collagen has no corresponding product from the α2 chain. The proteolytic cleavage is believed to result from processes related to natural physiological maturation of collagenous tissue. 相似文献
Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins. 相似文献