Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Addition chains”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1988

Limited fibrinogenolysis in stored whole blood and in fresh frozen plasma.

Measurement of the F-CB3 related antigen of the fibrinogen alpha-chain shows a time dependent increase of fibrino(geno)lysis in whole blood during storage from 70.7 +/- 16.4 pmol/ml (day 2) to 356.3 +/- 110 pmol/ml (day 39). Simultaneously PMN-elastase increases from 95 +/- 44 micrograms/l (day 2) to 3492 +/- 954 micrograms/l (day 39). The significant correlation between PMN-elastase and F-CB3 values (n = 180, r = 0.73, p less than 0.001) may reflect a relationship between the liberation of granulocyte enzymes and the extent of fibrino(geno)lysis. The isolation and separation of fibrinogen material on SDS-PAGE indicates a limited degradation of the parent fibrinogen molecule (340 kD). During storage the fraction of slightly degraded fibrinogen (300 kD) and pre-X fragment (280 kD) increases. The separation of the reduced fibrinogen material demonstrates that the limited fibrinogenolysis mainly took place in the A alpha-chain. Additional studies on fresh frozen plasma show that PMN-elastase values (98.6 +/- 41.8 micrograms/l), F-CB3 values (84.8 +/- 31.2 pmol/ml) and the SDS-PAGE pattern of the fibrinogen material are similar to the results in whole blood stored for two days.

Blood Preservation↗
PubMed1990

A protein structural change in aglycosylated IgG3 correlates with loss of huFc gamma R1 and huFc gamma R111 binding and/or activation.

Glycosylated chimeric mouse-human anti-NIP IgG3 antibody produced by growth of the J558L mouse B cell plasmacytoma is characterised with respect to the single carbohydrate chain at Asn-297 in the CH2 domain indicating that the mouse cell glycosyl transferases dictate the pattern of glycosylation rather than the human CH region of the heavy chain. Additionally, three unusual alpha-galactose-containing oligosaccharides are reported. Only the Fc region has detectable carbohydrate. Aglycosylated anti-NIP IgG3 antibody has been produced by cell growth in the presence of the antibiotic tunicamycin. Functionally, whilst the glycosylated intact IgG3 interacts with human Fc gamma R111 expressed on human killer (K) cells to trigger antibody-dependent cellular cytotoxicity the aglycosylated intact IgG3 fails to trigger cell lysis, localising the site on IgG for triggering human Fc gamma R111 mediated functions to the CH2 domain. The monomeric aglycosylated trypsin Fc fragment inhibits human Fc gamma R1 recognition by U937 cells 115-fold less well (K50 = 2 microM) than does glycosylated Fc (K50 = 17 nM), confirming that aglycosylation disrupts the site for human Fc gamma R1 within the CH2 domain and indicating that the trypsin Fc fragments reflect the functional properties of the intact IgG glycoforms. Structurally, 1H NMR shows that the absence of carbohydrate at Asn-297 results in a small and localised protein structural change in the vicinity of the reporter group His-268 within the CH2 domain. The site on IgG for triggering human Fc gamma R111 mediated functions is then localised to the vicinity of His-268. The profound impact of aglycosylation on human Fc gamma R1 recognition implies structural disruption of the proposed site for human Fc gamma R1 in the lower hinge region of IgG (residues 234-239), proximal to His-268.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1992

Cutaneous T-cell lymphoma: utility of antibodies to the variable regions of the human T-cell antigen receptor.

BACKGROUND: The clonotypic 90 kd Ti heterodimer of the human T-cell antigen receptor is composed of two distinct chains (alpha beta or rarely tau delta) that result from the recombination of variable (V), constant, joining, and, in the case of beta chains, additional diversity regions. OBJECTIVE: The variable region expression of human cutaneous T-cell lymphoma (CTCL) was studied. METHODS: Biopsy specimens from 13 patients with CTCL (7 plaque, 3 tumor stage, 3 Sézary syndrome) were examined immunohistochemically by a panel of seven commercially available monoclonal V-region antibodies. RESULTS: Two patients had significant anti-V-region staining. One patient with Sézary syndrome had two lesions, subjected to biopsy 4 months apart, that reacted with beta V5(a), a specificity previously documented by flow cytometry of leukemic cells. A patient with plaque-stage CTCL, negative for T-cell gene rearrangement by Southern blot, demonstrated reactivity with beta V5(c) largely limited to epidermotropic lymphocytes. CONCLUSION: Panels of V-region antibodies should be useful reagents for diagnosis and follow-up of CTCL.

Adult↗
PubMed1991

Penile detumescence: characterization of three phases.

In 22 dogs in which erection was induced by cavernous nerve stimulation, we analyzed the intracavernous pressure changes during detumescence without and with acute clamping of the aorta or electrostimulation of the lumbar sympathetic chains. Additionally, the degree of venous outflow obstruction was assessed by saline perfusion of the cavernous body during aortic occlusion. Detumescence had three distinct phases: an initial phase exhibiting a small pressure increase; a second phase showing a slow pressure decrease; and a third phase in which a fast decrease occurred. The first phase was abolished by aortic clamping, whereas the other phases were not significantly affected. Sympathetic stimulation abolished or prevented the second phase. Perfusion of the cavernous body during the second phase resulted in a pressure rise to off-scale values; however, when initiated during the terminal phase or in the nonstimulated penis, the pressure increase was slight. Our study indicates that the arterial flow rate influences the duration of the first phase of detumescence and that venous drainage is completely restored in the third phase. Furthermore, sympathetic stimulation causes an almost immediate full restoration of venous drainage, as cavernous perfusion initiated with an intracavernous pressure about twice as high as without sympathetic stimulation failed to increase pressure to off-scale values.

Animals↗
PubMed1995

The iron-binding protein ferritin is expressed in cells of the osteoblastic lineage in vitro and in vivo.

Ferritin, a metal-binding protein responsible for maintaining the bioavailability of iron, has been demonstrated in cells of the osteoblastic lineage. Messenger RNAs encoding the light and heavy chain subunits of ferritin were detected in ROS 17/2.8, ROS 25/1, and UMR106 rat osteosarcoma cell lines, in fetal rat calvaria, and in primary cultures of rat calvarial osteoblast-like cells. In vivo, the expression of ferritin light-chain mRNA was observed in both active osteoblasts and in osteocytes. A 450-kD iron-binding protein was immunoprecipitated from ROS 17/2.8 cells by an antiferritin antiserum. This protein comigrated with native ferritin, and could be dissociated into subunits comigrating with ferritin light and heavy chains. Addition of extracellular Fe59-transferrin to cultures of ROS 17/2.8 cells resulted in the sequestration of the iron in intracellular ferritin. These observations demonstrate that cells of the osteoblastic lineage possess a functional ferritin-based iron uptake and storage system capable of regulating metal homeostasis in bone.

Animals↗
PubMed1994

Rod outer segment retinol dehydrogenase: substrate specificity and role in phototransduction.

The reaction catalyzed by all-trans-retinol dehydrogenase of rod outer segments completes the quenching of photoactivated rhodopsin and initiates the cycle of reactions leading to regeneration of visual pigment. The goal of this study was to determine the kinetic parameters of the dehydrogenase at physiological levels of bleaching, to investigate its specificity, and to determine its possible role in modulating phototransduction. Reduction of all-trans-retinal could be measured after bleaching < 0.15% rhodopsin. Kinetic parameters for the forward reaction determined with endogenous all-trans-retinal were Km = 1.1 microM; Vmax = 7 nmol/min/mg rhodopsin. The low enzymatic activity suggests that at high bleach rates, all-trans-retinal could accumulate, increasing the steady state level of bleaching intermediates or promoting formation of pseudophotoproducts. Active pseudophotoproducts, which stimulate Gt activation and opsin phosphorylation by rhodopsin kinase, are formed with opsin and all-trans-retinal as well as retinal analogues lacking the 13 methyl or the terminal two carbons of the polyene chain. Addition of all-trans-retinol, NADP, and [32P]ATP to rod outer segments increased rhodopsin phosphorylation. Kinetic parameters for the reverse reaction determined with exogenous all-trans-retinol were Km = 10 microM; Vmax = 11 nmol/min/mg rhodopsin. Our results support the hypothesis that all-trans-retinol dehydrogenase could influence the phototransduction cascade, including activities of Gt, rhodopsin kinase, and binding of arrestin, by impeding the recycling of rhodopsin at high bleach levels.

Alcohol Oxidoreductases↗
PubMed1988

Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures.

Labeled heparan sulfate proteoglycans (HSPG) were isolated from wounded and confluent cultures of bovine aortic endothelial cells by nondegradative extraction with 4 M guanidine hydrochloride and detergent. HSPG were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography, and subclasses of different hydrodynamic size were isolated by gel filtration. Three major subclasses of HSPG were characterized structurally with respect to the presence and relative size of protein core, the presence and amount of nonsulfated oligosaccharide, and size and structure of heparan sulfate (HS) chains. The largest (600-800-kDa) HSPG subclass (I), isolated from cell layers and media of confluent cultures, bears 38-kDa HS chains on an apparently heterogeneous class of relatively large glycoprotein cores. HSPG II (150-200 kDa), isolated from cell layer or media, has 22-kDa HS chains and smaller core glycoproteins (less than 50 kDa). HSPG III, the subclass of smallest hydrodynamic size, has 13-kDa HS chains and a glycopeptide core of less than 15 kDa. All subclasses bear varying proportions of non-sulfated oligosaccharides of similar sizes. Comparisons of HS chain structure indicated that the different subclasses have similar proportions (49-55%) of N-sulfate, with both O-sulfate and highly N-sulfated blocks of disaccharide distributed similarly along HS chains. In addition, HS chains from subclasses II and III contain sequences that are insensitive to periodate oxidation or heparitinase digestion, suggesting that they contain increased proportions of iduronate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles.

The influence of electric surface charges on the polar headgroups and the hydrocarbon region of phospholipid membranes was studied by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with charged amphiphiles. A positive surface charge was generated with dialkyldimethylammonium salts and a negative surface charge with dialkyl phosphates. The POPC:amphiphile ratio and hence the surface charge density could be varied over a large range since stable liquid-crystalline bilayers were obtained even for the pure amphiphiles in water. POPC was selectively deuterated at both methylene segments of the choline moiety and at the cis double bond of the oleic acyl chain. Additional experiments were carried out with 1,2-dipalmitoyl-rac-glycero-3-phosphocholine labeled at the C-2 position of the glycerol backbone. Deuterium, phosphorus, and nitrogen-14 nuclear magnetic resonance (NMR) spectra were recorded for liquid-crystalline bilayers with varying concentrations of amphiphiles. Although the hydrocarbon region and the glycerol backbone were not significantly influenced by the addition of amphiphiles, very large perturbations of the phosphocholine headgroup were observed. Qualitatively, these results were similar to those observed previously with other cationic and anionic molecules and suggest that the electric surface charge is the essential driving force in changing the phospholipid headgroup orientation and conformation. While the P-N dipole is approximately parallel to the membrane surface in the pure phospholipid membrane, the addition of a positively charged amphiphile or the binding of cationic molecules moves the N+ end of the dipole toward the water phase, changing the orientation of the phosphate segment by more than 30 degrees at the highest amphiphile concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Ampholyte Mixtures↗
PubMed1980

Inhibition of ion permeability control properties of acetylcholine receptor from Torpedo californica by long-chain fatty acids.

The characteristics of fatty acid inhibition of acetylcholine receptor function were examined in membrane vesicles prepared from Torpedo californica electroplax. Inhibition of the carbamylcholine-induced increase in sodium ion permeability was correlated with the bulk melting point of exogenously incorporated fatty acids. Above its melting temperature, a fatty acid could inhibit the large increase in cation permeability normally elicited by agonist binding to receptor. Below its melting temperature, a fatty acid was ineffective. None of the fatty acids altered any of the ligand binding properties of the receptor. Inhibitory fatty acids did not induce changes in membrane fluidity, as determined by electron paramagnetic resonance using spin-labeled fatty acids. The spin-labeled fatty acids also acted as inhibitors, and the extent of inhibition depended largely on the position of the nitroxide group along the fatty acid chain. Addition of noninhibitory fatty acid to the vesicle membranes did not protect the receptor from inhibition by spin-labeled fatty acids. The effects of free fatty acids on acetylcholine receptor function are attributed to the disruptions of protein-lipid interactions.

Acetylcholine↗
PubMed1993

Unprecedented oxylipins from the marine green alga Acrosiphonia coalita.

The Oregon marine chlorophyte Acrosiphonia coalita produces an assortment of oxidized polyunsaturated fatty acids, or oxylipins. The smallest of these was a 10-carbon conjugated trienal 1 with antimicrobial properties. Related to 1 were three novel branched-chain conjugated trienals 4, 5, and 9 in which the aldehyde was present as a branch on a 17-carbon fatty acid chain. Additionally, two novel conjugated unbranched trienone octadecanoids 8 and 10 were also isolated and characterized. Finally, a family of related epoxy-alcohols (11, 12, 15, and 17) was obtained from A. coalita. Structures were determined by spectroscopic methods in combination with formation of various degradation products and derivatives. The absolute stereochemistry of several of these metabolites was determined by application of exciton chirality circular dichroic spectroscopy on benzoate derivatives.

Antifungal Agents↗
PubMed1997

CD44 in inflammation and metastasis.

CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered.

Animals↗
PubMed1989

Topography of glycosylation in yeast: characterization of GDPmannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles.

"Outer-chain" addition of mannose residues to yeast glycoproteins occurs in the Golgi compartment of the cell. Essential steps in this process are thought to include transport of GDPmannose from the cytoplasm into the lumen of Golgi vesicles, transfer of mannose to glycoprotein acceptors, hydrolysis of the resulting GDP to GMP, and return of GMP and inorganic phosphate to the cytoplasm. We report detection and characterization of a GDPmannose transport activity and a GDPase by yeast vesicles. The active transport of GDPmannose as well as the GDPase and another presumed Golgi enzyme, alpha 1,2-mannosyltransferase, are concentrated in a subcellular fraction that can be partially separated, by velocity sucrose gradient centrifugation, from a fraction enriched in an endoplasmic reticulum marker enzyme.

Biological Transport↗
PubMed1990

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed1997

cDNA cloning, genomic organization, and in vivo expression of rat N-syndecan.

The amino acid sequence of rat N-syndecan core protein was deduced from the cloned cDNA sequence. The sequence predicts a core protein of 442 amino acids with six structural domains: an NH2-terminal signal peptide, a membrane distal glycosaminoglycan attachment domain, a mucin homology domain, a membrane proximal glycosaminoglycan attachment domain, a single transmembrane domain, and a noncatalytic COOH-terminal cytoplasmic domain. Transfection of human 293 cells resulted in the expression of N-syndecan that was modified by heparan sulfate chain addition. Heparitinase digestion of the expressed proteoglycan produced a core protein that migrated on SDS-polyacrylamide gels at an apparent molecular weight of 120, 000, identical to N-syndecan synthesized by neonatal rat brain or Schwann cells. Rat genomic DNA coding for N-syndecan was isolated by hybridization screening. The rat N-syndecan gene is comprised of five exons. Each exon corresponds to a specific core protein structural domain, with the exception of the fifth exon, which contains the coding information for both the transmembrane and cytoplasmic domains as well as the 3'-untranslated region of the mRNA. The first intron is large, with a length of 22 kilobases. The expression of N-syndecan was investigated in late embryonic, neonatal, and adult rats by immunoblotting and Northern blotting analysis. Among the tissues and developmental stages studied, high levels of N-syndecan expression were restricted to the early postnatal nervous system. N-syndecan was expressed in all regions of the nervous system, including cortex, midbrain, spinal cord, and peripheral nerve. Immunohistochemical staining revealed high levels of N-syndecan expression in all brain regions and fiber tract areas.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of syndecan, an integral membrane proteoglycan.

We describe cDNA clones for a cell surface proteoglycan that bears both heparan sulfate and chondroitin sulfate and that links the cytoskeleton to the interstitial matrix. The cDNA encodes a unique core protein of 32,868 D that contains several structural features consistent with its role as a glycosamino-glycan-containing matrix anchor. The sequence shows discrete cytoplasmic, transmembrane, and NH2-terminal extracellular domains, indicating that the molecule is a type I integral membrane protein. The cytoplasmic domain is small and similar in size but not in sequence to that of the beta-chain of various integrins. The extracellular domain contains a single dibasic sequence adjacent to the extracellular face of the transmembrane domain, potentially serving as the protease-susceptible site involved in release of this domain from the cell surface. The extracellular domain contains two distinct types of putative glycosaminoglycan attachment sites; one type shows sequence characteristics of the sites previously described for chondroitin sulfate attachment (Bourdon, M. A., T. Krusius, S. Campbell, N. B. Schwartz, and E. Ruoslahti. 1987. Proc. Natl. Acad. Sci. USA. 84:3194-3198), but the other type has newly identified sequence characteristics that potentially correspond to heparan sulfate attachment sites. The single N-linked sugar recognition sequence is within the putative chondroitin sulfate attachment sequence, suggesting asparagine glycosylation as a mechanism for regulating chondroitin sulfate chain addition. Both 5' and 3' regions of this cDNA have sequences substantially identical to analogous regions of the human insulin receptor cDNA: a 99-bp region spanning the 5' untranslated and initial coding sequences is 67% identical and a 35-bp region in the 3' untranslated region is 81% identical in sequence. mRNA expression is tissue specific; various epithelial tissues show the same two sizes of mRNA (2.6 and 3.4 kb); in the same relative abundance (3:1), the cerebrum shows a single 4.5-kb mRNA. This core protein cDNA describes a new class of molecule, an integral membrane proteoglycan, that we propose to name syndecan (from the Greek syndein, to bind together).

Amino Acid Sequence↗
PubMed1996

Meibomian gland triglyceride fatty acid differences in chronic blepharitis patients.

In this study, we analyzed the triglyceride fraction of human meibomian gland secretions to determine whether specific fatty acids were significantly associated with specific chronic blepharitis disease groups. Triglycerides, isolated from the lipid component of human meibomian secretions by thin-layer chromatography, were transmethylated with sodium methoxide/methanol. Samples from individuals were then analyzed by gas chromatography-mass spectroscopy (GC-MS). Significant fatty acid differences were determined by nonparametric analyses, utilizing Kruskal-Wallis analysis of between group differences and Student-Newman-Keuls multiple comparisons. The analyses showed that the triglyceride samples contained the expected normal, iso, and anteiso fatty acids; disease group differences (from normals) in some of these fatty acid types were significant only with a Bonferroni adjustment. The triglycerides also contained previously unreported polyunsaturated fatty acids as well as two four-member families of fatty acids that had mass spectra and retention times consistent with dimethylated carbon chains. Additionally, some members of the meibomian keratoconjunctivitis (MKC) group contained an unusual pattern of normal 20-carbon fatty acids, including a higher level of saturated and lower levels of unsaturated fatty acids. An important exception was the presence of a monosaturated fatty acid whose GC retention time and mass spectrum were consistent with the uncommon cis-10-20:1 isomer. This pattern was associated with the presence of patient posterior hordeola. Thus, the association of triglyceride fatty acid composition with chronic blepharitis disease signs could be much more important than previously thought. Furthermore, this is the first example of the association of specific fatty acids with a specific chronic blepharitis disease sign.

Blepharitis↗
PubMed1981

Immunoglobulin expression in human lymphoblastoid cell lines with early B cell features.

Immunoglobulin expression was studied by direct immunofluorescence and by biosynthesis experiments in two human cell lines Raji and T 5.1. The basic phenotype of these cells was close to that of pre-B cells: large cells with intracytoplasmic IgM with a predominance of mu chains over light chains and no detectable surface immunoglobulins. The apparent molecular weight of heavy and light chains was abnormally large. Immunoglobulins were secreted at a low rate as pentameric IgM in the T 5.1 line and as subunits and free light chains in the Raji line. Spontaneous variations of this phenotype were observed: the cultured cells acquired mu and lambda chains, then additionally delta chains while they progressively lost detectable cytoplasmic mu chains, thus leading to a mature B cell phenotype. Subsequently, the cells had no detectable surface and cytoplasmic immunoglobulins and then they displayed a pre-B cell phenotype again. Attempts to induce further maturation using various potential inducers were unsuccessful.

B-Lymphocytes↗
PubMed1979

Yeast tRNA Leu UAG. Purification, properties and determination of the nucleotide sequence by radioactive derivative methods.

A second major species of leucine tRNA, tRNA Leu UAG (formerly designated tRNA Leu CUA) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNA Leu m5CAA (tRNA3 Leu). The primary structure of baker's yeast tRNA Leu UAG was elucidated by application of sensitive radioactive isotope derivative ("postlabeling") methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2'-O-Methylated guanosine was characterized using the [gamma-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the tRNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis. A different sequencing approach, consisting of a combination of "readout sequencing" with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNase T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units. Interesting structural features of tRNA Leu UAG are (a) the location of unique, leucine tRNA iso-acceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA Leu m5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

Adenine↗