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porcine willebrand factor: a population of multimers.purified porcine willebrand factor was analyzed by agarose-sodium dodso4 electrophoresis. multiple forms of the protein were found in a series of increasing molecular weights. a molecular mass calibration curve was constructed with fibrinogen (3.4 x 10(5) daltons), igm (1 x 10(6) daltons), and glutaraldehyde-crosslinked igm polymers (2, 3, and 4 x 10(6) daltons). as measured by this procedure, the apparent molecular weight of willebrand factor polymers ranged from 1.1 x 10(6) to 2.1 x 10(7). eac ...1978413873
polyribosome size analysis. measurement of number-average polyribosome sizes.the analysis of translational efficiencies of specific mrnas requires a determination of the polyribosome size. the appropriate value to use in such calculations is the number-average size. a method is described for accurately measuring the number-average size of total and of specific protein synthesizing polyribosomes using isokinetic sucrose density gradients and 125i-labeled antibodies. by this method, we demonstrated that albumin synthesizing polyribosomes from a serum albumin secreting mous ...1979534642
nuclear magnetic resonance investigation of the base-pairing structure of escherichia coli trnatyr monomer and dimer conformations.the structures of the escherichia coli tyrosine trna monomer and dimer have been investigated by high-resolution nuclear magnetic resonance (nmr). at 23 degrees c the monomer contains 26 +/- 2 base pairs and the low-field nmr spectrum (11.7-15 ppm) can be accounted for in terms of the cloverleaf structure (23 base pairs) and three additional resonances that are assigned to tertiary structure base pairs. assignments suggested for the various resonances are consistent with thermal denaturation stu ...1976782517
analysis of the two steps in polypeptide chain initiation inhibited by pactamycin.earlier work has shown that the inhibition by pactamycin (pm) of polypeptide chain initiation in reticulocyte extracts is associated with (1) a defect in the joining of the 60s subunit to the smaller initiation complex to form an 80s complex ("joining reaction") (kappen, l. s., suzuki, h., and goldberg, i. h. (1973), proc. natl. acad. sci. u.s.a. 70, 22) and (2) a block after the synthesis of the initial dipeptide (kappen, l. s., and goldberg, i. h. (1973), biochem. biophys. res. commun. 54, 108 ...19761247535
ubiquitin genes are differentially regulated in protoplast-derived cultures of nicotiana sylvestris and in response to various stresses.four ubiquitin mrna size classes were found to be differentially regulated in mesophyll protoplast-derived cultures of nicotiana sylvestris. three mrna families of 1.9, 1.6 and 1.35 kb were expressed as soon as protoplasts were isolated. the 1.9 and 1.6 kb size classes were transiently expressed during the first hours of culture, whereas the level of expression of the 1.35 kb size class was maintained as long as cells kept dividing. a 0.7 kb mrna size class started to be expressed just before th ...19921281439
inhibition of expression of a mouse alpha-globin gene by plasmids that include antisense oligonucleotides.plasmid-borne dnas, corresponding to 68-base oligodeoxynucleotides, synthesized in the antisense or sense configuration and based on the nucleotide sequences of various regions of the mouse alpha-globin mrna, were introduced with the gene for xanthine-guanine phosphoribosyl transferase from e. coli (ecogpt) into mouse erythroleukemia (mel) cells by protoplast fusion. specific inhibition of the synthesis of alpha-globin was observed only in the cells transformed with the plasmids with antisense 6 ...19921295698
enzymatic analysis of oligonucleotides containing cyclobutane pyrimidine photodimers with a cleaved intradimer phosphodiester linkage.our recent studies indicate that enzymatic hydrolysis of the intradimer phosphodiester linkage constitutes an early reaction in processing uv light-induced cis-syn-cyclobutane pyrimidine dimers in cultured human fibroblasts. before characterizing the resultant modified dimer sites in cellular dna, it is necessary to establish experimental conditions that can distinguish backbone-nicked from intact dimers. we thus constructed a model substrate, i.e. p(dt) 10 <> p(dt)10 containing a dimer with a r ...19921331055
consequences of 6-thioguanine incorporation into dna on polymerase, ligase, and endonuclease reactions.the incorporation of 6-thioguanine (s6g) in place of guanine proceeds readily in dna synthesis reactions catalyzed by mammalian and bacterial polymerases. this report summarizes the consequences of such incorporation studied to date. s6g was incorporated into one strand of a defined m13mp18 phage sequence in a (+)reaction catalyzed by the klenow fragment of escherichia coli dna polymerase i. after denaturation of the newly synthesized strand (containing s6g) and annealing with a reverse (-) 32p- ...19921331762
rapd analysis of campylobacter isolates: dna fingerprinting without the need to purify dna.a method was developed to obtain reproducible dna fingerprints from campylobacter by pcr-based amplification, without the need to isolate total dna. randomly amplified polymorphic dna (rapd) profiles were generated with three randomly designed 10-mers, using each separately as an amplification primer. a range of c. jejuni serotypes could be typed by rapd analysis. depending on the primer, the analysis of rapd profiles resulted in different levels of discrimination between the strains. clear corr ...19921368370
functional analysis of the mouse alpha-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures.we have compared the activities of mouse alpha-fetoprotein (afp) enhancers i, ii, and iii with their minimal enhancer fragments (mers) i, ii, and iii and with the entire 7-kilobase pair enhancer domain by transient expression assay in primary fetal mouse liver cells. the level of expression directed by the afp promoter [p(-1009)afpcat] alone is stimulated at least 10-fold by the entire afp enhancer domain (-1009 to -6983). enhancer i can drive the level of chloramphenicol acetyltransferase activ ...19921375227
antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit camp-activated but not calcium-activated chloride currents.phosphorylation of the cystic fibrosis transmembrane conductance regulator (cftr) by camp-dependent protein kinase leads to chloride flux in epithelial cells. is cftr also required for the calcium-dependent activation of chloride channels? we used antisense oligodeoxynucleotides to cftr to reduce the expression of cftr in colonic and tracheal epithelial cells. the antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of cftr mrna. sense and misantisense ...19921379720
template. phosphorothioate oligonucleotides duplexes as inhibitors of hiv-1 reverse transcriptase.we have investigated the interaction between a number of 14 mers phosphorothioate oligonucleotides and hiv-1 reverse transcriptase. two methods were used to measure the affinity of the analogs for the enzyme. in the first, the oligonucleotide or its duplex with poly(rl) were used as inhibitors of the enzyme using poly(ra).(dt)14 as template primer. in the second, the oligonucleotides or their duplexes were used to displace a fluorescent template primer complex of known affinity from its binding ...19921380799
differential mapping of fc gamma-binding and monoclonal antibody-reactive epitopes on ge, the fc gamma-binding glycoprotein of herpes simplex virus type 1.the entire 396 residue extracellular sequence of ge the hsv-1 fc gamma-binding glycoprotein has been studied to determine epitopes binding to two mab ii-481 and 88s previously demonstrated to react with ge at or near the fc gamma-binding regions. overlapping 7-mers constructed from the established sequence were tested with mab ii-481 and 88s along with their fab fragments. control mab of the same igg 2b subclass as well as whole rabbit and human igg and fc were also tested for binding to overlap ...19921382102
lipofectin enhances cellular uptake of antisense dna while inhibiting tumor cell growth.a natural dna oligomer (15-mer) was synthesized with a sequence complementary to the translation initiation codon region of the human tgf-alpha mrna and mixed with lipofectin to form unilamellar complexes. it was found that tumor cell growth was inhibited when hct116 cells were treated with lipofectin-dna oligomer complexes or with lipofectin alone. uptake of 32p-labeled 15-mers into colon tumor cells was compared in the presence and absence of lipofectin. the amount of labeled oligomer found in ...19921422086
opening of the replication origin of escherichia coli by dnaa protein with protein hu or ihf.opening of the three tandem repeats of a 13-mer in the replication origin (oric) of escherichia coli is a prime event in the replication in vitro of minichromosomes (bramhill, d., and kornberg, a. (1988) cell 54, 915-918). dnaa, the initiator protein, requires protein hu or ihf, along with a millimolar level of atp and negative superhelical density in the plasmid to open this region. the extent of opening, as judged by cleavage by a single-strand-specific endonuclease (i.e. p1 nuclease), correla ...19921429655
opposed actions of regulatory proteins, dnaa and icia, in opening the replication origin of escherichia coli.the opening of the three tandem 13-mers (iterons) in the replication origin (oric) of escherichia coli by dnaa protein, assisted by protein hu or ihf (hwang, d. s., and kornberg, a. (1992) j. biol. chem. 267, 23083-23086), represents an essential early stage in the initiation of chromosomal replication (bramhill, d., and kornberg, a. (1988) cell 54, 915-918). we now show by mutational alterations of the 13-mer region that oric function, both in vitro and in vivo, requires at-richness in the left ...19921429656
an oligomer complementary to the 5' end region of mdr1 gene decreases resistance to doxorubicin of human adenocarcinoma-resistant cells.acquired resistance to doxorubicin and other anti-cancer drugs is generally dependent on gene amplification of a specific nucleotide sequence, the mdr1 gene. verapamil, cyclosporin and other drugs have been used to circumvent the resistance in experimental models in vitro and/or in vivo. we have attempted to reverse the mdr phenotype by treating human adenocarcinoma resistant cells with 20 mers of synthetic unmodified oligodeoxynucleotide mdr1 antisenses. five odns towards different mrna regions ...19921444203
use of specific oligonucleotides for direct enumeration of listeria monocytogenes in food samples by colony hybridization and rapid detection by pcr.two 18-mer oligonucleotides derived from the sequence of hly, the gene coding for listeriolysin o, were shown to be specific for listeria monocytogenes in the genus listeria in colony hybridization tests. the oligonucleotides did not hybridize with any of the bacterial species found in food and co-isolated with listeria on selective media. they were used in colony hybridization tests for enumeration of l. monocytogenes present in food samples after direct plating on selective media plates. in ad ...19921448613
genotypic identification and characterization of species and strains within the genus candida by using random amplified polymorphic dna.random amplified polymorphic dna (rapd) was used to better characterize the genotypic relatedness among medically important candida species. by using short oligomer primers (10-mers) with arbitrarily chosen sequences in the polymerase chain reaction, distinctive and reproducible sets of polymerase chain reaction products were observed for isolates of c. albicans, c. lusitaniae, c. tropicalis, and torulopsis (candida) glabrata. the rapd analysis differentiated a physiologically homogeneous panel ...19921452710
specific inhibition of human immunodeficiency virus type 1 replication by antisense oligonucleotides: an in vitro model for treatment.we have developed a culture system, simulating in vivo conditions of human immunodeficiency virus type 1 (hiv-1) infection, to evaluate the long-term efficacy of antisense oligonucleotide treatment. five oligonucleotide phosphorothioates (28-mers), complementary to different regions of hiv-1 rna, blocked replication of the virus in a sequence-specific manner at 1 microm concentration. variations in antiviral activity were seen among the different oligonucleotides, revealing an effect of target s ...19921454800
studies on antisense inhibition of translation in vitro. anomalies and re-evaluation.experiments were carried out to better characterize antisense control of translation. results in an e. coli system confirmed specific inhibition of poly(u) translation. at low concentrations, certain homopolymers (including poly(ra)) stimulated translation. oligo(da(n)) was inhibitory at n less than or equal to 8. translation of globin mrna in reticulocyte lysates indicated that ssdna 15-mers targeted at beta-globin mrna inhibited both alpha- and beta-globin production. sequences targeted immedi ...19921516711
in vivo primary induction of virus-specific ctl by immunization with 9-mer synthetic peptides.a primary cytotoxic t lymphocyte (ctl) response in vivo requires antigen presentation by cytosolic processing and can not in general be obtained by vaccination with soluble proteins. in the present work we have found that vaccination of mice with pre-processed synthetic peptides, corresponding to endogenous 9-mers produced in influenza a virus-infected cells, resulted in strong primary ctl responses. the generated ctl efficiently killed virus-infected target cells with preference for viral strai ...19921517589
rna polymerases react differently at d(apg) and d(gpg) adducts in dna modified by cis-diamminedichloroplatinum(ii).two duplexes (20-mers) were constructed containing either a single cis-[pt(nh3)2[d(gpg)]] or cis-[pt(nh3)2[d(apg)]] intrastrand cross-link, the major dna adducts of the antitumor drug cis-diamminedichloroplatinum(ii). these synthetic duplexes were multimerized and the resultant polymers used as templates in single-step addition reactions of condensation of a single nucleoside triphosphate substrate to a dinucleotide primer (abortive elongation reaction) catalyzed by prokaryotic or eukaryotic rna ...19921536834
inhibition of t4 dna ligase activity by (+)-cc-1065: demonstration of the importance of the stiffening and winding effects of (+)-cc-1065 on dna.non-denaturing gel electrophoresis analysis demonstrates that the stiffening and winding effects of (+)-cc-1065 produce unusual proximal and distal inhibition of t4 dna ligase-catalysed ligation of covalently modified dna. (+)-cc-1065 is a potent antitumor antibiotic produced by streptomyces zelensis. this drug selectively bonds through n3 of adenine in dna and lies in the minor groove of dna, reacting in a highly sequence-selective manner. previous studies (lee et al., 1991) have shown that (+) ...19921543525
structure of the dnaa and dnaa-box region in the mycoplasma capricolum chromosome: conservation and variations in the course of evolution.we have previously shown that the dnaa gene and the dnaa-box region were conserved in bacteria representative of all three major branches of the eubacterial phylogenic tree: high g + c gram+, low-g + c gram+ and gram-. in the present work, we determined the structure of the dnaa region of mycoplasma capricolum and found that the dnaa gene and at least two other genes, rpmh and dnan, were conserved in this bacterium. an unusually high level of amino acid (aa) substitutions was observed in m. capr ...19921544573
effects of propofol, etomidate, midazolam, and fentanyl on motor evoked responses to transcranial electrical or magnetic stimulation in humans.the effects of propofol, etomidate, midazolam, and fentanyl on motor evoked responses to transcranial stimulation (tc-mers) were studied in five healthy human volunteers. each subject, in four separate sessions, received intravenous bolus doses of propofol 2 mg.kg-1, etomidate 0.3 mg.kg-1, midazolam 0.05 mg.kg-1, and fentanyl 3 micrograms.kg-1. electrical tc-mers (tce-mers) were elicited with anodal stimuli of 500-700 v. magnetic tc-mers (tcmag-mers) were elicited using a cadwell mes-10 magnetic ...19921550274
hla-a2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides.the mutant human cell line t2 is defective in antigen presentation in the context of class i major histocompatibility complex (mhc) molecules, and also in that transfected t2 cells show poor surface expression of exogenous human class i (hla) alleles. both defects are thought to lie in the transport of antigenic peptides derived from cytosolic proteins into the endoplasmic reticulum (er), as peptide-deficient class i molecules might be expected to be either unstable or retained in the er. the pr ...19921557127
a pcr-mediated gene synthesis strategy involving the assembly of oligonucleotides representing only one of the strands.a modification of pcr-mediated gene synthesis strategy is introduced. this modification enables the synthesis of a gene from oligonucleotides comprising only one of the two strands. bridging oligonucleotides (approximately 20-mers in length) complementary to the junctions of template strand oligonucleotides and two outer primers are also needed for pcr. a two-step pcr containing a first step of 10 cycles, followed by a second step of 20 cycles, differing only in the annealing conditions was used ...19921571150
a novel blocker-pcr method for detection of rare mutant alleles in the presence of an excess amount of normal dna.a novel polymerase chain reaction method was developed to preferentially amplify a segment of dna containing a base substitution mutation. this technique uses a pair of dideoxynucleotide-labeled oligonucleotides (18 mers) of normal sequences as blockers located between the two primers. by virtue of a subtle difference in the melting temperature between the blocker-normal dna and blocker-mutant dna hybrids, the method allows preferential amplification of the mutant dna. we used the human n-ras ge ...19921598207
identification of active peptide sequences in the carboxyl-terminal cell binding domain of human thrombospondin-1.thrombospondin (ts) mediates attachment, spreading, and motility of several cell types through at least four cell binding domains: the amino-terminal heparin binding domain, the type i repeats containing the csvtcg sequence, the rgda sequence in the last of the type iii calcium binding repeats and the carboxyl-terminal cell or platelet binding domain (cbd). the attachment of human melanoma cells (g361) to the cooh-terminal domain is independent of the rgda sequence and is inhibited by the monocl ...19921644809
identification of skeletal muscle protein-tyrosine phosphatases by amplification of conserved cdna sequences.specific protein-tyrosine phosphatase (ptpase) enzymes that regulate signal transduction by the insulin receptor in target tissues have not been identified. we evaluated the expression of ptpase homologs in skeletal muscle since this tissue is the major site of insulin-mediated glucose disposal in vivo. a rat skeletal muscle cdna pool was prepared with a set of degenerate oligonucleotide primers and ptpase cdna sequences were amplified using pairs of "guess-mers" that were deduced from highly co ...19911651716
intermediates in the chaperonin-assisted refolding of rhodanese are trapped at low temperature and show a small stoichiometry.in vitro refolding of the urea-unfolded, monomeric, mitochondrial enzyme rhodanese (thiosulfate sulfur-transferase; ec 2.8.1.1) is facilitated by the chaperonin proteins cpn60 and cpn10 from escherichia coli at 37 degrees c, but the refolding is strongly inhibited at 10 degrees c. in contrast, the unassisted refolding of rhodanese is efficient at 10 degrees c, but the refolding efficiency decreases as the temperature is raised. these observations provided two measures of the cpn60-rhodanese comp ...19911680127
inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by trna(lys).human immunodeficiency virus (hiv) reverse transcriptase (rt) uses host trna(lys) partially annealed to the primer binding site (pbs) as primer for the initiation of cdna synthesis. when assaying cdna synthesis with a template-primer complex formed by an rna fragment carrying the pbs site and bovine trna(lys) we noticed that an excess of primer trna inhibited strongly the dna polymerase activity of a recombinant hiv rt (p66-p51 heterodimeric form) produced in transformed yeast cells. the same in ...19901689823
identification and characterization of t helper cell epitopes of the major outer membrane protein of chlamydia trachomatis.chlamydia trachomatis serovars a, b, and c are the causative agents of trachoma, the world's leading cause of preventable blindness. immunoprophylaxis is a possible approach to control trachoma. the chlamydial major outer membrane protein (momp) is thought to play an important role in the development of protective immunity against chlamydial infection, and is therefore considered to be a promising candidate antigen in the development of a trachoma vaccine. much effort has been focused on the mol ...19901694217
herpes simplex virus type 1-specific immunity induced by peptides corresponding to an antigenic site of glycoprotein b.herpes simplex virus (hsv) envelope glycoproteins are the prime targets of adaptive antiviral immunity. previous investigation identified a protective, neutralizing, glycoprotein b1 (gb-1)-reactive monoclonal antibody (mab b6) and localized the linear epitope recognized by the mab to residue 84 of gb-1. three overlapping peptides (two 20-mers and one 18-mer), together spanning amino acids 63 to 110 of the wild-type sequence of gb-1, were synthesized and analyzed for their ability to stimulate im ...19901698994
identification of two t-cell epitopes on the candidate epstein-barr virus vaccine glycoprotein gp340 recognized by cd4+ t-cell clones.current efforts to develop an epstein-barr virus subunit vaccine are based on the major envelope glycoprotein gp340. given the central role of cd4+ t cells in regulating immune responses to subunit vaccine antigens, the present study has begun the work of identifying linear epitopes which are recognized by human cd4+ t cells within the 907-amino-acid sequence of gp340. a panel of gp340-specific cd4+ t-cell clones from an epstein-barr virus-immune donor were first assayed for their proliferative ...19911710291
a cluster of continuous antigenic structures in the transmembrane protein of hiv-1: individual patterns of reactivity in human sera.we investigated the antigenicity of a highly conserved region in the transmembrane protein of the human immunodeficiency virus type 1 (hiv-1). in order to identify antigenically important residues, amino-acid sequences of synthetic peptides representing this region were varied systematically: single residues were omitted from the sequence of hiv-env 583-599; threonines were substituted for pairs of residues in hiv-env 581-599; the sequences of heptadeca-peptides were shifted by single residues. ...19911713646
identification of an immunodominant b cell epitope on the hepatitis c virus nonstructural region defined by human monoclonal antibodies.several ebv-transformed b cell lines (bcl) were obtained from two patients with chronic hepatitis c virus (hcv) infection that secreted igg class antibodies to the hcv nonstructural ag c100-3. two cloned bcl, derived from the same parental line, generated stable cloned lines that secreted up to 20 mg/liter of specific igg1(kappa). supernatants from oligoclonal and cloned bcl were also analyzed by immunoblot and all strongly reacted with recombinant polypeptides derived from the putative ns4 regi ...19911717573
epitope mapping of snake venom phospholipases a2 with pseudexin monoclonal antibodies.fifteen different monoclonal antibodies, developed against a pseudexin a, b, and c mixture, were screened for linear epitope recognition. peptides (9-mers) spanning pseudexin b were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. four antibodies recognized linear epitopes of pseudexin a, pseudexin b, and also nonidentical sequences found in other phospholipases a2 (pla2s) as determined by enzyme-linked immunosorbent assays. three antibodies recognized ...19911718309
structure and assembly of the escherichia coli transcription termination factor rho and its interaction with rna. i. cryoelectron microscopic studies.cryoelectron microscopy has been used to visualize the escherichia coli transcription termination protein rho in a vitreously frozen state, without the use of strains, fixatives or other chemical perturbants. in the absence of rna cofactor, a variety of structures are observed, reflecting the heterogeneity of complexes formed by rho at protein concentrations near the physiological range (3 to 10 microm). one of the most common structural motifs we see is a six-membered ring of rho subunits (pres ...19911719215
identification of antibody epitopes within the cb-11 peptide of type ii collagen. ii. computer modelling studies of peptides and the interpretation of epitope scanning results.computer modelling techniques were used to investigate the structure of 8-mers from the cb-11 peptide of bovine type ii collagen which were recognised by sera from rats which had previously been injected with bovine type ii collage. it was discovered that all the hydrophobic peptides recognised by the rat sera were predicted to have collagenous-like secondary structures. the primary structure of the 8-mers which were recognised was also compared against the sequences in the owl protein sequence ...19911721847
icia protein, a specific inhibitor of initiation of escherichia coli chromosomal replication.specific binding of icia protein to the 13-mers in the origin of a minichromosome (oric) inhibits initiation of replication in vitro by blocking the opening of this region effected by the initiator dnaa protein (hwang, d.s., and kornberg, a. (1990) cell 63, 325-331). isolation of the icia gene (thöny, b., hwang, d.s., fradkin, l., and kornberg, a. (1991) proc. natl. acad. sci. u.s.a. 88, 4066-4070) has made possible the construction of an icia-overproducing strain, which in turn has simplified t ...19921733927
transcription in vivo within the replication origin of the escherichia coli chromosome: a mechanism for activating initiation of replication.within the replication origin, oric, of the escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by dnaa protein. in contrast, dnaa protein repressed the previously described ori-l leftward transcription. the former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers. the effects of transcription on the initiation of replication were also investigated by making constructs with ...19921736090
analysis of salmonella typhimurium hisd3052 revertants: the use of oligodeoxyribonucleotide colony hybridization, pcr, and direct sequencing in mutational analysis.a rapid method for determining the dna sequences of salmonella typhimurium hisd3052 revertants is presented. dna colony hybridization was used to analyze revertants previously studied by isono and yourno [proc natl acad sci usa 71:1612-1617, 1974]. synthetic oligodeoxyribonucleotide probes (18-mers) were able to distinguish sequences that differed by a single base pair. mutant his sequences not identified by probing analysis were amplified using polymerase chain reaction (pcr) and directly seque ...19911748083
[suppression of translation in vitro of the mrna of the m1 protein of influenza virus using antisense oligonucleotides].effect of antisense oligonucleotides on the in vitro translation of the influenza virus m1 protein mrna was investigated. the most efficient arrest of mrna translation was achieved by simultaneous action of two or three oligonucleotides (14-16-mers) complementary to the juxtaposed sequences in the 5'-terminus of the molecule around and upstream of the initiation codon.19911753959
oligonucleotides antisense to the interleukin 1 receptor mrna block the effects of interleukin 1 in cultured murine and human fibroblasts and in mice.phosphodiester and phosphorothioate oligodeoxynucleotides (18 mers) were constructed antisense to sequences of the recently cloned murine and human il-1 receptors. murine antisense oligonucleotides inhibited il-1-stimulated pge2 synthesis by murine fibroblasts in culture in a time (days) and concentration-dependent (3 microm-30 microm) fashion. murine sense oligonucleotide and an oligonucleotide antisense to human il-1 receptor were without effect. moreover, murine antisense oligonucleotides did ...19911833422
complementary directed modifications of nucleic acids and oncogene-directed mutagenesis in vivo.high reactivity of the polyalkylating ss oligomers that were sense or antisense 30-200-mers containing sequences complementary to e1 oncogenes of simian adenovirus sa7 and one alkylating residue -ch2ch2n(c2h5oh) (ch2)3n(ph-p-ch2oh)ch2ch2cl per each 25 bases of oligomers was demonstrated in vitro by alkylation of ss dna of recombinant m 13 mp8e1 and mp9e1 phages with inserted e1 sequences of adenovirus oncogene and then by followed complete and selective elimination of e1 sequences from recombina ...19911841269
analysis of the molecular mimicry between hla-b27 and a bacterial ompa protein using synthetic peptides.in spite of a lack of sequence 'homology' between hla-b27 and the bacterial ompa outer membrane proteins, they both react with the ye-2 monoclonal anti-hla-b27 antibody. the ye-2 antibody also reacted positively in elisa with a synthetic peptide derived from the segment spanning residues 63-84 of b*2705. the critical peptide residues were determined by testing first with overlapping peptides, followed by a replacement set made according to the determined epitope. the results were compared with t ...19911893633
a barbiturate-regulated protein binding to a common sequence in the cytochrome p450 genes of rodents and bacteria.analyses of the 5' regulatory sequences of genes encoding barbiturate-inducible cytochromes p450bm-1 and p450bm-3 from bacillus megaterium and of the 5' sequences of genes for barbiturate-inducible p450b and p450e of the rat revealed a string of 17 base pairs in each of the genes that shared a high degree of sequence identity. labeled oligonucleotide probes of each of these four sequences were tested in gel retardation assays with protein obtained from b. megaterium grown either in the presence ...19911902228
polymorphism in n-2-acetylaminofluorene induced dna structure as revealed by dnase i footprinting.in this paper, we have constructed double stranded helices (60-mers) containing a single n-2-acetylaminofluorene (-aaf) adduct covalently bound to one of the three guanine residues of the narl site (g1g2cg3cc). this sequence was identified as a strong frameshift mutation hot spot for many carcinogens that bind to the c8 position of guanine. using dnase i as a probe for dna conformation we show i) that the average size of the helix deformation extends over 3 to 5 base pairs in both directions fro ...19911945836
the properdin-like type i repeats of human thrombospondin contain a cell attachment site.thrombospondin (ts) is a modular adhesive glycoprotein that contains three domains previously implicated in the attachment of cells to ts. these include the amino-terminal heparin-binding domain, the carboxy terminal cell or platelet-binding domain, and an rgda sequence of ts. we have characterized a mab against human ts, designated a4.1, which inhibits the attachment of human melanoma cells (g361) to ts. the epitope for a4.1 lies within the amino terminal half of the central stalklike region of ...19911999454
ecdysterone regulatory elements function as both transcriptional activators and repressors.a synthetic, 23-bp ecdysterone regulatory element (ecre), derived from the upstream region of the drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (cat). hybrid constructs were transfected into drosophila s3 cells and assayed for ecdysterone-inducible cat expression. in the absence of ecdysterone a tandem pair of ecres repressed the high constitutive level of cat activit ...19912005885
icia, an escherichia coli gene encoding a specific inhibitor of chromosomal initiation of replication in vitro.the gene encoding the protein that binds the three 13-mers in the origin (oric) of escherichia coli to block initiation of replication in vitro has been cloned, sequenced, and overexpressed. the gene possesses an open reading frame for 297 amino acids (mass of 33,471 da). the protein has a motif for dna-binding (helix-turn-helix) and has homology to a diverse set of prokaryotic regulatory proteins, known as the lysr family. the protein, previously referred to as the 33-kda protein, has been name ...19912034653
[enzymatic ligation of dna fragments containing phosphoamide modification of the internucleotide bonds].dna fragments with the point amidophosphate (cyclohexylamido- or morpholido-) modification in the sugar-phosphate backbone were synthesized and separated into individual diastereoisomer. the isomers were separated by the reversed-phase hplc (rpc), and chirality at phosphorus was assigned by a stereochemical correlation scheme using phosphorothioate standards. the rpc-retention time values for rp-isomers were found to be lower than for sp-analogues. amidophosphate dna fragments were used as p- an ...19912064627
theoretical and experimental measures of dna helix stability and their relation to sequence specific repair of o6-ethylguanine lesions.recent work (breslauer et al. (1986) proc. natl. acad. sci. (u.s.a.), 83, 3746) has provided a method for calculating empirical thermodynamic quantities for helix to coil transitions from the base sequence of any oligomer. it is shown in this work that the dna helix binding energy, calculated with the amber force field, for 9-mers of the type 5'-gggxgeyggg-3', where x and y are any base and the central ge is o6-ethylguanine, correlates well with the empirical delta g for helix to strand transiti ...19912067552
large-scale economic synthesis of antisense phosphorothioate analogues of dna for preclinical investigations.the therapeutic potential of antisense oligonucleotides will heavily depend on a balance of two factors: pharmacologic effectiveness and cost of production. pharmacologic optimization will be achieved to a limited degree in in vitro systems, but substantial progress can only be made in the context of appropriate in vivo models. the quantities of synthetic oligonucleotides required for modest in vivo testing are several thousandfold greater than can be produced by conventional dna synthesis techn ...19902078018
oestrogen administration and the expression of the kallikrein gene family in the rat submandibular gland.using a series of oligonucleotide probes (18-21 mers) specific for members of the rat kallikrein/tonin (arginyl-esteropeptidase) gene family (ps, s1, s2, s3, k1, p1), we have shown by northern blot analysis that all six genes are expressed in the submandibular gland (smg), with ps (true kallikrein) the most abundant in both male and female rats. though female levels of ps mrna are similar to that in the male, levels of mrna from both the kallikrein-like (s1, k1, p1) and tonin (s2)/tonin-like (s3 ...19902155348
antisense oligodeoxyribonucleotides inhibit the expression of the gene for hepatitis b virus surface antigen.the effect of a series of antisense oligodeoxyribonucleotide [oligo(dn)] on the expression of the surface antigen (hbsag) gene of human hepatitis b virus (hbv) was examined using hepatocellular carcinoma cells that contain integrated hbv genomes. of a number of antisense oligo(dn)s tested, synthetic 15-mers directed at the cap site of mrna and regions of the translational initiation site of the hbsag gene were found to be highly effective and inhibited viral gene expression by as much as 96%. th ...19902177093
analysis of spontaneous and psoralen-induced salmonella typhimurium hisg46 revertants by oligodeoxyribonucleotide colony hybridization: use of psoralens to cross-link probes to target sequences.an improved dna colony-hybridization method for the rapid characterization of salmonella typhimurium hisg46 revertants is described. oligodeoxyribonucleotides (15-mers) complementary to each of 6 possible transition or transversion mutations and an extragenic suppressor mutation, underlying the his+ phenotype, were prepared. optimal sequence discrimination was achieved by hybridizing 15-mers at the apparent dissociation temperature (td) for 2 h with chromosomal dna of revertant colonies affixed ...19902179713
genetic reconstruction and characterization of the recombinant transacylase (e2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex. implication of histidine 391 as an active site residue.genetically altered transacylase (e2b) proteins of the bovine branched-chain alpha-keto acid dehydrogenase complex were overexpressed in escherichia coli and characterized. deletion by psti or bal31 digestion of the amino-terminal region of the inner-core domain (residues 175-421) beyond residue 209 resulted in a complete loss of transacylase activity. the enzyme assay was carried out using [1-14c]isovaleryl-coa and exogenous dihydrolipoamide as substrates. the removal of 4 residues (thr-ile-pro ...19902198287
application of long synthetic oligonucleotides for gene analysis: effect of probe length and stringency conditions on hybridization specificity.two different lengths of long unique synthetic oligonucleotide probes (37- and 48-mers) specific for human major histocompatibility complex (mhc) class ii beta genes were synthesized. these oligonucleotides were utilized to examine factors influencing hybridization specificity. both probe length and stringency of washing conditions were found to be crucial factors for sequence-specific hybridization.19902206600
a novel protein binds a key origin sequence to block replication of an e. coli minichromosome.a sequence of three tandem repeats of a 13-mer in the replication origin (oric) of e. coli is the highly conserved site of opening of the duplex for initiation of dna synthesis. a protein that binds this sequence has been discovered in e. coli and purified to homogeneity. this novel 33 kd polypeptide behaves as a dimer. binding to the 13-mers is specific and limited to this region. at a ratio of 10-20 monomers per oric plasmid, the binding blocks initiation by preventing the opening of the 13-me ...19902208289
recombinant hnrnp protein a1 and its n-terminal domain show preferential affinity for oligodeoxynucleotides homologous to intron/exon acceptor sites.the reported binding preference of human hnrnp protein a1 for the 3'-splice site of some introns (swanson and dreyfuss (1988) embo j. 7, 3519-3529; mayrand and pederson (1990) nucleic acids res. 18, 3307-3318) was tested by assaying in vitro the binding of purified recombinant a1 protein (expressed in bacteria) to synthetic oligodeoxynucleotides (21-mers) of suitable sequence. in such a minimal system we find preferential binding of protein a1 to oligodeoxynucleotide sequences corresponding to t ...19902251120
reca protein self-assembly. ii. analytical equilibrium ultracentrifugation studies of the entropy-driven self-association of reca.we have investigated the self-association of reca protein from escherichia coli by equilibrium ultracentrifugation. monomeric reca (mr = 37,842) was observed in reversible equilibrium with trimers, hexamers and dodecamers in the presence of 1.5 m-kcl, 5 mm-hepes, 1 mm-edta, 2 mm-atp (ph 7.0) at 1 degrees c. the equilibrium was strongly temperature-dependent, with polymerization being favored as the temperature was raised from 1 degrees c 21 degrees c, and was reversible with respect to temperatu ...19902266565
amplification of a long sequence that includes a processed pseudogene for elongation factor 2 in the mouse.quantitative southern blotting analysis has demonstrated that mouse cells contain about 70 copies per haploid genome of a dna sequence related to the gene for elongation factor 2. the restriction maps of seven cosmids that each carry one copy of the ef2-related sequence (mer) and nucleotide sequences of mers were highly conserved among the cosmids. data obtained by such analyses suggest that mers were produced by the integration of one copy of mer derived from poly(a)+ mrna for ef2 into a specif ...19902303263
dynamics of dna polymerase iii holoenzyme of escherichia coli in replication of a multiprimed template.movements of dna polymerase iii holoenzyme (holoenzyme) in replicating a template multiprimed with synthetic pentadecadeoxynucleotides (15-mers) annealed at known positions on a single-stranded circular or linear dna have been analyzed. after extension of one 15-mer on a multiprimed template, holoenzyme moves downstream in the direction of chain elongation to the next primer. holoenzyme readily traverses a duplex, even 400 base pairs long, to exploit its 3'-hydroxyl end as the next available pri ...19852413035
antisense oligodeoxynucleotides provide insight into mechanism of translation initiation of two sendai virus mrnas.translation of sendai virus nucleocapsid protein (np) and phosphoprotein (p/c) mrnas in rabbit reticulocyte lysates in the presence of antisense oligodeoxynucleotides (15-20-mers) showed that oligonucleotides having complementarity within the 5' noncoding region of the mrnas blocked translation, oligonucleotides having complementarity within 20 nucleotides upstream from the initiator codon blocked translation only partially, and oligonucleotides complementary to the coding region of mrna had no ...19872438279
evaluation of n-ras oncogene anti-sense, sense and nonsense sequence methylphosphonate oligonucleotide analogues.we have investigated the potential for using anti-sense non-ionic methylphosphonate oligonucleotide analogues to study the relationship between oncogene expression and maintenance of the transformed phenotype in malignant cells. our results confirmed that the methylphosphonates are resistant to biochemical degradation and are devoid of non-specific toxicity towards cultured human ht29 cells. at low temperature (less than 5 degrees c) both n-ras anti-sense and nonsense analogue 9-mers formed 1:1 ...19882457379
sequence-specific cleavage of rna using chimeric dna splints and rnase h.to cleave rna molecules using e. coli rnase h in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-o-methyl)ribonucleotide(s) was designed to be used as a dna splint. our model experiments with ribooligomer the splint duplexes (9 mers) and rnase h demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short dna-containing splint directs a unique cleavage of rna by rnase h. the method could be applied to lon ...19882465538
[in vitro anti-hiv activity of phosphorothioate alpha-anomeric oligodeoxynucleotides].oligonucleotide analogs consisting exclusively of alpha-anomeric deoxynucleoside units bridged with phosphorothioate linkages have been synthesized and tested in vitro as antiviral agents against human immunodeficiency virus (hiv) in human t cells. two 28-mers, an homopolymer alpha-s-dc28 and an oligomer alpha-s-anti-rev complementary to the initiation site of the regulatory viral gene rev exhibited antiviral activities comparable to those reported for the corresponding beta-anomeric phosphoroth ...19892516764
the dnaa initiator protein binds separate domains in the replication origin of escherichia coli.after binding to its four 9-mer boxes in the 245-base pair escherichia coli replication origin (oric), dnaa protein effects the formation of an "open complex" in an adjacent region made up of three 13-mers (bramhill, d., and kornberg, a. (1988) cell 52, 743-755). this open complex formation requires the atp form of dnaa protein assisted by hu protein (sekimizu, k., bramhill, d., and kornberg, a. (1987) cell 50, 259-265). we now provide direct evidence that dnaa protein binds the 13-mers, sequenc ...19892539372
amplification of human papillomavirus dna sequences by using conserved primers.the polymerase chain reaction has potential for use in the detection of small amounts of human papillomavirus (hpv) viral nucleic acids present in clinical specimens. however, new hpv types for which no probes exist would remain undetected by using type-specific primers for the polymerase chain reaction before hybridization. primers corresponding to highly conserved hpv sequences may be useful for detecting low amounts of known hpv dna as well as new hpv types. here we analyze a pair of primers ...19892556429
sequence analysis of rsk, a portion of the 95-kilobase plasmid of salmonella typhimurium associated with resistance to the bactericidal activity of serum.increased sensitivity to killing by human serum complement occurs in salmonella typhimurium strains in which the 95-kilobase virulence plasmid is integrated into the chromosome. this phenotypic change appears to be due to alterations in plasmid gene expression and is reversed by the presence of an autonomous plasmid bearing a cloned region of the virulence plasmid. accordingly, this region has been termed rsk for reduced serum killing. sequence analysis of the region reveals that rsk is composed ...19892645213
comparative inhibition of ras p21 protein synthesis with phosphorus-modified antisense oligonucleotides.a rabbit reticulocyte lysate translation assay was used to quantitatively compare a series of antisense oligodeoxyribonucleotides (11-mers) having different internucleoside linkages and various degrees of complementarity (100-80%) with the start codon and downstream 8 bases of balb-ras p21 mrna. the oligomers had either contiguous phosphodiester, or alternating methylphosphonate-phosphodiester, or contiguous methylphosphonate, or contiguous phosphorothioate linkages. under the conditions used fo ...19892679622
sequencing of megabase plus dna by hybridization: theory of the method.a mismatch-free hybridization of oligonucleotides containing from 11 to 20 monomers to unknown dna represents, in essence, a sequencing of a complementary target. realizing this, we have used probability calculations and, in part, computer simulations to estimate the types and numbers of oligonucleotides that would have to be synthesized in order to sequence a megabase plus segment of dna. we estimate that 95,000 specific mixes of 11-mers, mainly of the 5'(a,t,c,g)(a,t,c,g)n8(a,t,c,g)3' type, hy ...19892737674
chemical synthesis of the 24 rna fragments corresponding to hop stunt viroid.a general and practical synthetic method of oligoribonucleotides (10-20 mers) by using the cyanoethyl phosphoramidite approach was described. in this experiment 9-phenylxanthen-9-yl (pix) and 9-(4-methoxy)phenylxanthen-9-yl (mox) groups were employed for the 5'-hydroxyls and tetrahydropyranyl (thp) group was used for the 2'-hydroxyl protecting groups. in addition, suitable acyl groups were introduced for the protection of the lactam functions of guanine and uracil moieties.19892813059
the yeast uasg is a transcriptional enhancer in human hela cells in the presence of the gal4 trans-activator.the yeast trans-activator protein gal4, when expressed in hela cells, stimulates transcription from several class b (ii) eukaryotic promoters containing gal4 binding sites either as the full uasg or as synthetic 17-mers. the characteristics of this activation are indistinguishable from those of the sv40 enhancer. transcription was similarly stimulated from either complex promoter regions containing multiple upstream elements or from a simple promoter region composed of only a tata box. addition ...19882830022
comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system.antisense oligonucleotides containing either anionic diester or neutral methylphosphonate internucleoside linkages were prepared by automated synthesis, and were compared for their ability to arrest translation of human dihydrofolate reductase (dhfr) mrna in a nuclease treated rabbit reticulocyte lysate. in the case of oligodeoxyribonucleotides, tandem targeting of three 14-mers resulted in synergistic and complete selective inhibition of dhfr synthesis at a total oligomer concentration of 25 mi ...19882836793
enzymatic amplification of myosin heavy-chain mrna sequences in vitro.we have developed a procedure that detects the presence of mrna coding for human beta-myosin heavy chain in small amounts of total, unfractionated rna isolated from heart or skeletal muscle. the protocol is based on the enzymatic amplification in vitro of a selected 106-bp myosin isotype-specific subregion of this mrna. the method, which is a modification of the so-called "polymerase chain reaction," requires two synthetic oligonucleotide primers (20-mers), reverse transcriptase, and dna polymer ...19882840250
a model for initiation at origins of dna replication.many prokaryotic origins resemble e. coli oric in possessing essential at-rich sequences, tandemly repeated. the role of these repeats may be in the initial opening of the duplex by the initiator protein, as has been found for the 13-mers in oric and is implied for the 11-mers of the lambda origin. regulatory influences on the effective action of the initiator protein of e. coli (dnaa protein) include transcriptional activation of the origin, nucleotide binding and membrane attachment of the pro ...19882843291
in situ hybridization of putative somatostatin mrna within hypothalamus of the rat using synthetic oligonucleotide probes.the distribution of mrna with high sequence homology to somatostatin mrna within the periventricular hypothalamus of rat was assessed using in situ hybridization techniques with synthetic oligodeoxyribonucleotide probes, complementary to the 3' coding region of rat somatostatin mrna. the probes (22- and 24-mers) were 5'-end labeled using t4 polynucleotide kinase and gamma-32p-atp. they were used either individually or after ligation with t4 dna ligase to form a 46-mer. serial tissue sections (le ...19852860116
cc/gg contacts facilitate the b to a transition of dna in solution.self-complementary decadeoxynucleotides, ccgatatcgg, ccagatctgg, ccctgcaggg, gggggccccc, were designed and synthesized to estimate the a-philic free energy of cc/gg contacts. first, regions of temperature-stability of the double-stranded conformation were determined for each 10-mer. then, circular dichroism spectra were recorded for the b-family forms at different temperatures, counter-ion concentrations and trifluoroethanol contents. a cooperative change typical of the b-a transition is observe ...19862908426
substructure of human von willebrand factor.using electron microscopy, we have visualized the substructure of human von willebrand factor (vwf) purified by two different approaches. vwf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 x 6.5 nm) connected to ...19852932468
comparison of the relative mutagenicities of o-alkylthymines site-specifically incorporated into phi x174 dna.the relative mutagenicities of o-alkylthymine-dna adducts were analyzed in vivo by site-specific mutagenesis. purified dna polymerases were used to incorporate o4-methyl (me)-, o4-ethyl (et)-, o4-isopropyl (ipr)-, or o2-me-dttp onto the 3' terminus of a synthetic oligonucleotide (15-mer) hybridized to phi x174 am3 dna. the product oligonucleotides were further extended in the presence of unmodified dntps to yield 21-mers containing single o-alkylthymine adducts opposite the adenine residue of th ...19872958453
polyomavirus enhancer contains multiple redundant sequence elements that activate both dna replication and gene expression.sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral dna replication (tyndall et al., nucleic acids res. 9:6231-6250, 1981). we have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. one approach, recently described by de villiers et al. (nature [london], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptiona ...19852985964
[general approach to the engineering of synthetic dna].a useful and efficient approach to the synthesis of dna duplexes of practically unlimited length has been developed. the proposed methodology is based on the use of temporary restriction sites for subcloning and assembling the segments of the desired dna. it allows the utilization of chemically synthesized oligonucleotides of various length (from 10- to 100-mers) for the duplex construction. the application of this approach to the synthesis of a gene for the functionally active bacteriorhodopsin ...19853004509
isolation of a cdna clone for the human lysosomal proteinase cathepsin b.the cysteine proteinase cathepsin b is one member of the lysosomal acid hydrolases. based on the peptide sequence of rat liver cathepsin b, an oligonucleotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cdna libraries. a recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and dna sequence analysis confirmed that this clone encodes human cathepsin b. the clone, designated pcb-1, has sequences ...19863010323
xrep, a plasmid-stimulating x chromosomal sequence bearing similarities to the bk virus replication origin and viral enhancers.the human x chromosome-linked fragment, "xrep," was sequenced because it exerts a positive effect on plasmid growth in both e. coli and saccharomyces cerevisiae. the sequence revealed three features similar to the human bk virus replication origin: xrep has a true palindrome, cctcc(t)3cctcc, which is similar to "true" palindrome-like sequences found at the replication origins of polyoma [cctc(t/c)10ctcc], bk [cctc(a/g)8cctcc] and sv40 [cctcc(a)6gcctcc] viruses. twenty nucleotides away from the t ...19863025813
cytosine methylation does not affect binding of transcription factor sp1.dna methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. we used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor sp1 to its target sequence (a g + c-rich sequence known as a "gc box"). concatemers of double-stranded 14-mers containing a gc box successfully competed with the human metallothionein iia promoter for binding to sp1 in dnase i protection experiments. the prese ...19883281160
optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene dna probes.we present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions (temperature and monovalent cation concentration). target dna was immobilized to the nitrocellulose filter with ...19883291638
functional mapping of the genome of the b19 (human) parvovirus by in vitro translation after negative hybrid selection.we have analyzed the coding capacity of b19 parvovirus transcripts by in vitro translation using the negative hybrid selection technique. five different antisense oligonucleotides (18-mers) corresponding to different portions of the b19 genome were hybridized to rna samples extracted from human erythroid bone marrow cells infected with b19 parvovirus in vitro, and rnase h was added to cleave specific b19 rna molecules at selected sites. b19-specific translation products of these rna samples were ...19883373576
[an improved chemico-enzymatic method of synthesizing long dna segments].a simple and economy method of the biochemical assembling of long double-stranded dna segments is described. a single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates. this single-stranded polynucleotide was converted, in the presence of dna polymerase 1 and a 12-meric primer, in to the full-length double-stranded dna (th ...19883382431
an acridine-linked oligodeoxynucleotide targeted to the common 5' end of trypanosome mrnas kills cultured parasites.anti-messenger oligodeoxynucleotides covalently linked to an intercalating agent were tested for their ability to inhibit translation of trypanosoma brucei mrnas in a cell-free system. the sequence of these oligodeoxynucleotides was complementary to part of the 35-nucleotide (nt) sequence which is present at the 5' end of all trypanosome mrnas (the so-called mini-exon sequence). in a rabbit reticulocyte lysate, a nonadeoxynucleotide linked to an acridine derivative, specifically inhibited protei ...19873446576
processivity and kinetics of the reaction of exonuclease i from escherichia coli with polydeoxyribonucleotides.the enzyme exonuclease i from escherichia coli hydrolyzes successive nucleotides from the 3'-termini of single-stranded deoxyribonucleotide homopolymers. when the reaction is stopped after partial hydrolysis, only intact starting material and small oligomers can be isolated. the distribution of oligomeric products varies with the base composition of the polymer but the largest oligomer that can be isolated from the reaction of exonuclease i with homopolymers of deoxyadenylate, deoxythymidylate, ...19863519606
a study of side reactions occurring during synthesis of oligodeoxynucleotides containing o6-alkyldeoxyguanosine residues at preselected sites.as part of our studies on the molecular mechanisms of mutation by carcinogens we have synthesized 12 oligonucleotides (15-mers) containing an o6-alkylguanine residue at a preselected position for use as primers in the enzymatic synthesis of biologically active dna. ten of these oligonucleotides are derived from a minus strand sequence carrying the modified nucleotide in the third codon of gene g of bacteriophage phi x174 dna. two others are derived from plus strand sequences carrying the modific ...19873607028
oligonucleotide probes for bacterial acylcarrier protein genes.using a recently-introduced rapid manual method, we have synthesized a family of thirty six individual oligonucleotides of unique sequence (18-mers), which correspond to the conserved amino acid sequence, gadsld, found at the 4'-phosphopantetheine-binding site of the acylcarrier component of bacterial and plant fatty acid synthases. hybridisation of each of these oligonucleotides to southern blots of restricted streptomyces erythreus dna under stringent conditions showed that (i) only two probes ...19854084606
isolation and characterization of a cdna clone for the amino-terminal portion of the pro-alpha 1(ii) chain of cartilage collagen.we have isolated a cdna clone (prcol 2) which is complementary to the 5'-terminal portion of the rat pro-alpha 1(ii) chain mrna. a synthetic oligonucleotide was used both as a primer for cdna synthesis and as a probe for screening a cdna library. the probe was a mixture of sixteen 14-mers deduced from an amino acid sequence present in the amino-terminal telopeptide of the rat cartilage alpha 1(ii) chain. this primer was chosen so that the resulting cdna would contain the sequence of the 5' end o ...19846094525
use of complementary dna oligomers to probe trp leader transcript secondary structures involved in transcription pausing and termination.dna oligomers were synthesized that are perfectly complementary to different segments of the tryptophan (trp) operon leader transcript. these 15 nucleotide long oligomers were used as probes of the involvement of transcript secondary structures in two processes: transcription pausing at the pause site located near base pair 90 in the leader region, and transcription termination at the attenuator. the 15-mers were complementary to the four segments of the trp leader transcript which have been sho ...19846201827
use of synthetic oligonucleotide probes complementary to genes for human hla-dr alpha and beta as extension primers for the isolation of 5'-specific genomic clones.we have synthesized 175-nucleotide-long probes for the dna of human histocompatibility antigens hla-dr alpha and beta by extending on poly(a)+ mrna from b-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the nh2 terminus of both polypeptides. the synthesis of the probe for the alpha-chain dna was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted ...19836403940
isolation of polysomes from nostoc sp. mac and translation of messenger rna in a heterologous cell-free system.a method has been established which isolated polysomes from the lysozyme/edta-shocked cyanobacterium, nostoc sp. mac. in a typical preparation the total recovery of rna as polysomes was 83%, in which 77% of the polysome fraction was present at sizes greater than 5-mers and 23% as 2-4-mers. messenger rna isolated from such a preparation of polysomes produced a 10-fold stimulation in the incorporation of [35s]methionine into polypeptides by a cell-free system of escherichia coli. the in vitro-synt ...19836415228
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